A 20-year review of pediatric pancreatic tumors. Pancreatic tumors are rare surgical problems in infants and children. A 20-year audit (1971 to 1991) of this institution showed six patients ranging in age from 3 weeks to 16 years who were operated on for pancreatic neoplasms. Five of these tumors were malignant, bringing the reported experience to 71 cases. This series of malignancies included three solid cystic tumors, one insulin-secreting tumor, and one pancreatoblastoma. The clinical presentations varied: three had abdominal pain, one developed hypoglycemia, and one had an abdominal mass with jaundice. In five of the six patients pancreatic pathology was suspected preoperatively. All were treated primarily with pancreatic resection including one pancreatoduodenectomy. No radiotherapy or chemotherapy was used. The perioperative mortality was 0% with a morbidity of 50%. The long-term results are encouraging, with all patients alive after a mean follow-up of 7.8 years. These data suggest that aggressive surgical therapy is warranted in the management of pediatric pancreatic tumors.


Treatment of bilateral Wilms' tumor: comparison of initial biopsy and chemotherapy to initial surgical resection in the preservation of renal mass and function. The National Wilms' Tumor Study 3 (NWTS-3) recommended treatment of bilateral Wilms' tumor with initial biopsy followed by chemotherapy with subsequent operation to resect the remaining tumor. This study was performed to determine if this approach preserves renal mass and function when compared with initial surgical resection followed by chemotherapy. Over a 20-year period (1970 to 1990), 15 patients with synchronous bilateral Wilms' tumor were treated at the Childrens Hospital of Los Angeles. Eight patients in the surgical group underwent initial unilateral nephrectomy with contralateral biopsy, wedge resection, or partial nephrectomy and subsequent chemotherapy. The seven patients in the chemotherapy group underwent bilateral tumor biopsy, followed by chemotherapy and subsequent tumor resection. Patients were assigned to each group in a nonrandomized manner according to the preference of the attending oncologist and surgeon. Comparison of the two groups showed no significant differences in sex distribution, initial renal function, tumor histology, dose and field of radiotherapy, presence or absence of positive surgical margins, and local recurrence rates. Patients in the surgery group were slightly older than those in the chemotherapy group: 3.6 +/- 2.2 versus 2.3 +/- 2.2 years. The percent of renal mass involved by tumor for the surgery group was 52 +/- 12 versus 73 +/- 16 for the chemotherapy group (P = .03). The percent of renal mass preserved following all operations, local recurrence rates, incidence of metastases, and survival was nearly identical between the two groups. There were three cases of renal failure in the surgery group and one case of renal failure in the chemotherapy group.(ABSTRACT TRUNCATED AT 250 WORDS)


Freezing behaviour of microencapsulated water. The freezing behaviour of water in polyurea microcapsules was studied through DSC (differential scanning calorimetry) and ESR (electron spin resonance) measurements under a non-equilibrium condition to show that supercooling of water becomes more noticeable with decreasing droplet size of the liquid. Thermodynamics of small systems was found applicable to analyse the experimental findings, even though the process of water freezing in the microcapsules was not of an equilibrium nature.


Chemotherapy can convert unresectable hepatoblastoma. The surgical evaluation and management of children with hepatoblastoma has changed with recent advances in imaging modalities and preoperative chemotherapy. Pediatric Oncology Group (POG) Study no. 8697 has followed 63 patients with hepatoblastoma from 1986 to 1991. Twenty-six patients underwent primary tumor resection followed by chemotherapy consisting of cisplatin, vincristine, and 5-fluorouracil (group I). Thirty-seven patients with "unresectable" tumors received preoperative chemotherapy. Twenty-nine of these patients responded to chemotherapy and 26 underwent delayed surgical resection (group II). Eight patients had an inadequate response to chemotherapy; two have had successful liver transplantation and six are dead of disease progression. "Unresectable tumor" involved both liver lobes (25 patients), encased the inferior vena cava (2), involved adjacent tissues (1), involved the hepatic veins (2), or was deemed too large for safe resection (7). Two patients had distant metastases. The reason for an unresectable designation was not reported in five patients. The determination for an unresectable designation included exploratory laparotomy in 14 patients, angiogram in 7, computed tomography scan in 20, and magnetic resonance imaging in 3 patients. Operative times and transfusion requirements were similar in both groups. Perioperative complications were higher in patients in group II. There was no mortality and only minor morbidity associated with chemotherapy in each group. In both groups 77% of the patients are in complete remission after 13 to 54 months. Preoperative chemotherapy can allow successful resection of initially "unresectable" hepatoblastoma. Primary resection that may result in exsanguination should be postponed and chemotherapy given.


Treatment of acute pulmonary failure with extracorporeal support: 100% survival in a pediatric population. Since February 1990, five children, aged 10 days to 6.5 years, were treated with extracorporeal lung support at our hospital for acute, unrelenting pulmonary failure. Two had viral pneumonia: one with respiratory syncytial virus (RSV) bronchiolitis, and one with herpes simplex virus pneumonia, encephalitis, and disseminated intravascular coagulation. One presented with a febrile illness followed by a pulmonary hemorrhage. Two patients had adult respiratory distress syndrome (ARDS) complicating severe systemic illnesses, toxic epidermal necrolysis in one and cat scratch disease with encephalitis in the other. All children had diffuse parenchymal lung disease by chest x-ray. On maximum medical management all patients were developing carbon dioxide retention and progressive hypoxemia, exceeding previously established NIH study criteria for extracorporeal treatment. Three children (10 days, 2 months, 13 months) were placed on venoarterial support and two children (20 months and 6.5 years) were placed on venovenous extracorporeal support (ECCO2R). Three of the five had open lung biopsies performed, which showed findings consistent with a moderate to severe cellular phase of ARDS. No viral inclusions were found in the patient with RSV infection. One hundred percent immediate survival was achieved in this patient population. Average duration of support was 330 hours (range, 89 to 840). Following completion of extracorporeal support, all children were successfully weaned from the ventilator with an average time to extubation of 23.2 days (range, 2 to 58 days). One child died of congestive heart failure following palliative surgery for a complex noncyanotic congenital cardiac lesion 35 days after successfully weaning from extracorporeal support for an acute febrile illness and pulmonary hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)


Autoregulation of neuroblastoma growth by vasoactive intestinal peptide. Elevated serum levels of vasoactive intestinal peptide (VIP) are associated with some cases of neuroblastoma and correlate with a favorable prognosis. VIP has previously been shown in our laboratory to cause the in vitro growth inhibition and morphological differentiation of the human neuroblastoma cell line, LA-N-5. It is now shown that LA-N-5 cells express immunoreactive VIP and bear specific VIP receptors. Antagonism of endogenous VIP, either by competitive inhibition or receptor blockade, increased cell proliferation, suggesting that VIP is operative in normal growth regulation. Intracellular and extracellular levels of VIP were also shown to increase significantly during the retinoic acid-induced differentiation of these cells. Furthermore, a concomitant marked increase in VIP receptor expression was demonstrated with cellular differentiation. These receptors remain functional as evidenced by a matching increase in the level of detectable cAMP generated in response to exogenous VIP. It is concluded that VIP is a normal autoregulator of neuroblastoma cell growth and differentiation, and that retinoic acid-mediated differentiation may be, in part, due to endogenous VIP.


Neutrophil modulation by Actinobacillus actinomycetemcomitans. II. Phagocytosis and development of respiratory burst. Compromised neutrophil function has been found in a number of patients with localized juvenile periodontitis (LJP), although the pathogenic mechanism is unknown. Since infection with Actinobacillus actinomycetemcomitans is frequently found in patients with LJP, we have evaluated in vitro the effect of a bacterial extract of A. actinomycetemcomitans on the development of the respiratory burst by neutrophils. Pre-incubation of neutrophils with bacterial extract increased H2O2 induced by FMLP and zymosan in a dose-dependent fashion. Substitution of FMLP for bacterial extract produced similar results. Moreover, FMLP and bacterial extract had an additive effect on superoxide production following phagocytosis of zymosan. In contrast, bacterial extract significantly decreased PMA-stimulated H2O2, but pre-incubation with FMLP instead of bacterial extract failed to decrease PMA-stimulated H2O2. Bacterial extract did not change the percentage of cells activated by FMLP, opsonized zymosan, or PMA. Heat-treated bacterial extract induced effects similar to non-treated extract. Bacterial extract treated with proteinase K or phenol extraction increased FMLP or zymosan stimulated H2O2 equivalent to non-treated bacterial extract. In contrast, proteinase K or phenol extraction abolished the inhibitory effect of bacterial extract on PMA-stimulated H2O2 production. The bacterial extract component(s) that inhibits PMA-stimulated H2O2 is therefore a protein(s), resistant to 56 degrees C, and is not endotoxin. The partially activated state of PMNs exposed to A. actinomycetemcomitans extract, combined with their reduced ability to respond to a protein kinase C-dependent stimulus, may partially explain the abnormalities noted in LJP patients.


Five parameters of gingival crevicular fluid from eight surfaces in periodontal health and disease. Volume and amounts of myeloperoxidase (MPO), lactoferrin (LF), aryl sulfatase (AS) and lactate dehydrogenase (LDH) were measured in gingival crevicular fluid (GCF) collected from the mesial and distal proximal surfaces of the premolars and first and second molars of 3 subject groups. Group assignment was based on subject mean gingival index (GI) and probing depth (PD) of sampled sites as follows: healthy, GI less than or equal to 0.5, PD less than or equal to 3.0; disease 1, GI greater than or equal to 1.0, PD greater than or equal to 3.0 mm; disease 2, PD greater than or equal to 4.0 mm. Attachment loss (ATL) of most sites in the 3 groups was: healthy, 0-1 mm; disease 1, 1-2 mm; and disease 2, 4-9 mm. GCF volume differed among surfaces and teeth in each of the 3 groups. The greater amount of GCF collected from posterior locations was not related to the GI and PD. Differences with sampling location in amounts of GCF constituents were restricted to MPO and LF. Most of these differences (greater amounts at posterior sites) were associated with more severe disease. Variability in amount and composition of GCF collected from different sites, therefore, should be considered in experiments which include quantitation of GCF parameters. The ratio of MPO in disease group 2 to disease group 1 was greater than similar ratios for GCF volume and LF, AS and LDH. The quantity of MPO was the only measure which differed between the 2 disease groups at all surfaces. MPO thus appears to have the greatest potential, among the measured parameters, to serve as a marker for advanced periodontal disease.


Comparative study of subgingival microbiological sampling techniques. The presence of specific bacteria in subgingival plaque has been used as an indicator of active periodontal disease. The technique of subgingival sampling may conjecturally influence the identification and enumeration of microorganisms reported. In this study, paper point sampling and scaler sampling are compared. Subgingival samples using both methods were taken from three surfaces in each of 12 patients at the following time points: at each of two appointments one week apart before treatment and at each of two appointments 12 and 13 weeks following treatment. Microbiological analyses were undertaken to determine the total number of colony forming units, the proportions of suspected periodontal pathogens, and the number of spirochetes using phase contrast microscopy. Significantly higher numbers of colony forming units and spirochetes were found for paper point sampling both before and after treatment.


Longitudinal monitoring for disease progression of localized juvenile periodontitis. The present study was performed to investigate the factors associated with disease progression in localized juvenile periodontitis (LJP) patients by longitudinal monitoring of microbiological changes. Following a 9-month period, 9 LJP patients were divided into 2 groups based upon attachment loss, progressing and non-progressing. Both groups received scaling, root planing, and modified Widman flaps. Clinical and microbiological data were obtained at baseline, following the observation period, and at 6 and 12 months post-treatment. At 6 and 12 months post-treatment significantly more cocci were persistent in the non-progressing group than in the progressing group. Actinobacillus actinomycetemcomitans was also more frequently isolated in the progressing group than in the non-progressing group initially and following the 9-month observation period. Also after treatment, A. actinomycetemcomitans recolonized earlier in the progressing group than in the non-progressing group. These studies suggest that A. actinomycetemcomitans may play a role in disease progression in LJP; however, they do not eliminate the possibility that other organisms may also play a role, since A. actinomycetemcomitans was not detected in all of the patients in whom disease progressed.


Effects of a bisphosphonate on experimental periodontitis in monkeys. Bisphosphonates have been shown to increase bone mass in estrogen-deficient patients by inhibiting osteoclast activity. The purpose of this study was to measure clinical and radiographic effects of a bisphosphonate on periodontitis development in monkeys. Twenty-seven (27) adult cynomolgus monkeys were studied. After quarantine, baseline data were obtained including plaque index, gingival index, clinical probing depth measurements, and intraoral radiographs. Standardized radiographs were analyzed for quantitative changes in bone density using a computer assisted densitometric (CADIA) system. Animals were divided into 3 groups to receive 1 of the 3 treatment agents; these agents consisted of two levels of the test drug (alendronate) and a saline placebo. Agents were injected in the saphenous vein of the lower leg every 2 weeks for 16 weeks. One week after the initiation of treatment agent injections, mandibular right molars and premolars were ligated with 3-0 silk sutures to induce periodontitis. Ligated teeth were also inoculated with Porphyromonas gingivalis to insure a significant etiologic challenge. Nonligated homologous teeth served as controls. Clinical measurements and radiographs were repeated at 8 and 16 weeks after ligation. The bisphosphonate at a concentration of 0.05 mg/kg significantly retarded the progression of periodontitis as measured by bone density changes. The higher level dose of the test drug did not differ from placebo with respect to loss of bone density. Clinical indices were not affected significantly by the test drugs. Drugs that alter bone metabolism may offer a new approach to the treatment of periodontal disease.


Phospholipid metabolism in platelets from stroke-prone spontaneously hypertensive rats and Wistar Kyoto rats. Platelets from stroke-prone spontaneously hypertensive rats (SHRSP) show severe hypofunctions accompanied by defective protein (P47) phosphorylation. To examine the mechanism of platelet hypofunctions, phospholipid metabolism in SHRSP was compared with that in Wistar Kyoto rats (WKY). Phosphatidylinositol (PI) content was 20% less in SHRSP than in WKY, but no changes were observed in other phospholipids. Incorporation of [3H]-arachidonic acid (AA) into PI and phosphatidylethanolamine (PE) was 12% and 11% lower, and that into phosphatidylcholine (PC) was 6% higher in SHRSP than in WKY. Thrombin-induced diacylglycerol and phosphatidic acid formation were similar in both groups of platelets. Thrombin-induced release of [14C]-AA from the labeled platelets and its metabolism to eicosanoids occurred at similar rates. These results suggest that reduced formation of diacylglycerol, an activator of protein kinase C (PKC), does not cause defective phosphorylation of P47, a substrate of PKC, in SHRSP. However it remains unclear how the lower PI content and the altered distribution of AA in PC and PE is related to SHRSP platelet hypofunctions.


High affinity specific binding of 2-[125I]iodomelatonin by spleen membrane preparations of chicken. The binding sites for 2-[125I]iodomelatonin in chicken spleens were characterized. The binding was rapid, stable, saturable, reversible, and of high affinity. Both melatonin and 6-chloromelatonin strongly inhibited the binding. The dissociation constant (Kd) obtained from the Scatchard analysis was 31.4 +/- 5.19 pmol/l (3-weeks old, n = 4), which was in good agreement with the Kd (50.6 pmol/l) calculated from the kinetic study. The maximum number of binding sites (Bmax) was 1.09 +/- 0.11 fmol/mg protein (3-weeks old, n = 4). Twelve 11-week-old chicks were killed in two groups at mid-light or mid-dark. Saturation studies indicated no significant difference (P greater than 0.05) in the Kd between mid-light (42.1 +/- 3.9 pmol/l) and mid-dark (31.6 +/- 4.9 pmol/l). The maximum number of binding sites (Bmax) at mid-light and mid-dark were 1.52 +/- 0.16 and 1.35 +/- 0.08 fmol/mg protein, respectively, with no significant variation (P greater than 0.05) recorded. However, when the whole spleen was taken into consideration, the Bmax per spleen protein of the mid-light samples (253 +/- 36 fmol/spleen protein) was significantly greater than that (129 +/- 16 fmol/spleen protein) of the mid-dark samples (P less than 0.05). This indicated that in our study a diurnal rhythm of the total number of 2-[125I]iodomelatonin binding sites might exist in the chicken spleen.


[Genetical responses of neuronal cells to synaptic transmission]. To understand the cellular mechanisms that lead to the generation of synaptic plasticity of neuronal cells, it is important to understand the intracellular responses of neuronal cells stimulated via synaptic transmission. The stimulation of mouse cerebellar granule cells via NMDA (N-methyl-D-aspartate) receptors caused an increase in deoxyribonucleic acid (DNA)-binding activities to TRE (12-O-tetradecanoylphorbol-13-acetate-responsive element) and CRE (adenosine 3',5'-cyclic monophosphate-responsive element) motifs, depending upon the presence of extracellular Ca2+. The increases in TRE- and CRE-binding activities were also detected with the stimulation of non-NMDA receptors by kainate. The increases in TRE- and CRE-binding activities were both mediated by the same DNA-binding complexes whose binding affinity to CRE was about three-fold higher than that to TRE. On the other hand, the stimulation of neuroblastoma x glioma hybrid NG108-15 via muscarinic acetylcholine receptors, alpha 2-adrenergic receptors and bradykinin receptors caused a rapid induction of zif/268. An additive effect on the induction of zif/268 was observed when the different stimuli were simultaneously added. Thus, it is extremely likely that signals transduced via synaptic transmission are transferred to the level of gene expression and evoke some events which might contribute to the generation of synaptic plasticity in neuronal cells. In addition, we have found that the direct injection of plasmid DNAs into mouse skeletal muscle with fructose, glucose or NaCl solution led to a long-term expression of the introduced gene in muscle cells.


Distribution and toxicity of 5-fluorouracil after intraperitoneal and anal submucosal administration. The correlation of 5-fluorouracil (5-FU) distribution and its toxicity had been investigated in Albino rats. H3-5-FU was administrated either by intraperitoneal (I.P.) or anal submucosal (A.sm.) route. 5-FU was promptly distributed in different organs with marked accumulation in the pelvic area after A.sm. and in liver and kidney after I.P. administration. Acute toxicity (L.D. 50) was stronger after I.P. (63 mg/kg compared with 80 mg/kg after A.sm.). Side effects expressed in elevation of transaminase and alkaline phosphatase and increase in liver tissue glucose-6-phosphatase and total white blood count were much pronounced after I.P. administration. The results suggest the possibility of using A.sm. route for administration.


Enhancement of percutaneous absorption by laurocapram. The in vitro treatment of shed snake skin and hairless rat skin with laurocapram resulted in dramatic decreases in the amounts of cholesterol, phospholipids, and ceramides but not triglycerides in the skins. Scanning electron microscopic observations of hairless rat skin treated with laurocapram indicated looseness and cell separation of the stratum corneum probably caused by the extensive extraction of the intercellular lipids. An ESR study demonstrated the increased fluidity of the corneum lipids after laurocapram treatment. The apparent rotational correlation time of 16-doxyl-stearic acid was decreased by 1.6-2 times after treatment with laurocapram. No penetration of laurocapram itself through shed snake skin and hairless rat skin was detected in vitro, except when the reservoir solvent was 60% ethanol or propylene glycol. The enhancer was hardly metabolized during a 48-h incubation with skin homogenate. Pretreatment of shed snake skin with laurocapram increased significantly the penetration of sulfanilamide and indomethacin through the skin. These results indicate that laurocapram penetrating into the stratum corneum interacts with structured lipids in the intercellular channels and releases them, thereby enhancing the penetration of hydrophilic drugs through the channels. Additionally, laurocapram penetrating into the intracellular matrix of the corneum fluidizes the intracellular lipids and causes the reduction of diffusional resistance.


[New naphthalenic ligands of melatoninergic receptors]. Twenty three naphthalenic bio-isosteres of melatonin have been synthesized. The main structural variations concerned the acylamino substituents of the side chain and the alkoxy group on the 7-position of the naphthalene. Some of these compounds show greater affinity than melatonin itself for the melatonin receptor. The results of this study provide new informations on the structure affinity relationships and on the mode of interaction at the melatonin binding site.


Identification of the major degradation products of 4-methoxy-2-(3-phenyl-2-propynyl)phenol formed by exposure to air and light. Exposure of 4-methoxy-2-(3-phenyl-2-propynyl)phenol (CO/1828) to air and light (accelerated by temperature) yields 1-(2-hydroxy-5- methoxyphenyl)-3-phenylpropynone as the major degradation product. With extraction, this product rapidly degrades to 5-methoxyaurone and 6-methoxyflavone. In addition, a mixture of dimeric and heterodimeric compounds that are not fully identified was observed. These results indicate the formation of a reactive ortho-quinone methide as an unstable intermediate. This hypothesis is supported by evidence that the aurone slowly isomerizes into the flavone in control samples. Identification of compounds was accomplished with mass spectrometry, nuclear magnetic resonance spectrometry, UV-high-performance liquid chromatography, and comparison with authentic samples.


Gradient polyacrylamide gel electrophoresis for determination of molecular weights of heparin preparations and low-molecular-weight heparin derivatives. The M(r) values of pharmaceutical heparins and low-molecular-weight (LMW) heparin derivatives were examined as part of a collaborative study to develop methods for their characterization. Standard methods of M(r) determination rely on gel permeation high-performance liquid chromatography (HPLC). We report the use of gradient polyacrylamide gel electrophoresis (PAGE) to determine the M(r) values of pharmaceutical heparins and LMW heparin derivatives. This approach offers certain advantages over the HPLC method. Gradient PAGE analysis was performed in parallel, on multiple samples, with the same standard curve. HPLC was performed serially. Gradient PAGE gave higher resolution than HPLC, and thus, a mixture of easily obtained standards was used in place of individual standards for the construction of a standard curve. Heparin and various LMW heparin samples were analyzed by both gradient PAGE and conventional gel permeation HPLC methods. The number-average M(r), weight-average M(r), and polydispersity were examined by both techniques and found to be similar. This study demonstrates that gradient PAGE analysis is a sensitive method for the determination of the M(r) values of heparin and LMW heparin.


Modulation of mu-mediated antinociception in the mouse involves opioid delta-2 receptors. Recently, subtypes of the opioid delta receptor have been identified. It is not known, however, if a subtype of opioid delta receptor can be associated with the known modulatory action of delta agonists on mu-mediated antinociception. Thus, the present study has used the delta subtype-selective antagonists, [D-Ala2,Leu5,Cys6]enkephalin (DALCE) (delta 1 antagonist) and naltrindole-5'-isothiocyanate (5'-NTII) (delta 2 antagonist) in an effort to determine whether the positive and negative modulation of morphine antinociception produced by opioid delta agonists was the result of activity at specific subtypes of opioid delta receptors. Intracerebroventricular morphine produced a dose-related antinociceptive effect which was not antagonized by coadministration of the delta antagonist, ICI 174,864, or by pretreatment 24 hr before testing with the DALCE or 5'-NTII. Coadministration with morphine of a nonantinociceptive dose of DPDPE or [D-Ala2,Glu4]deltorphin resulted in a leftward displacement of the morphine dose-effect curve (i.e., positive modulation), whereas coadministration of a nonantinociceptive dose of [Met5]enkephalin with morphine resulted in a rightward displacement of the morphine dose-effect curve (i.e., negative modulation). Both the positive and the negative modulatory actions were antagonized when the experiment was conducted in the presence of the delta antagonist, ICI 174,864, or when the mice were pretreated with the delta 2 antagonist, 5'-NTII. In contrast, pretreatment with the delta 1 antagonist, DALCE, failed to affect either the positive or the negative modulatory actions of these delta agonists on morphine antinociception. The data suggest the involvement of an opioid delta 2 receptor in the modulation of morphine antinociception.


Enhanced blood pressure and renal hemodynamic effect of chronic versus acute lisinopril administration in the rabbit. This study was aimed at evaluating the factors responsible for the marked renal hemodynamic effect of 6-day treatment with lisinopril. Blood pressure (BP) and renal blood flow (RBF) were monitored in six groups of rabbits. Animals treated with lisinopril for 6 days (Group I) had lower BP (77 +/- 3 mm Hg) than normal controls (Groups II/III, 106 +/- 3 mm Hg, P < .05) or those given lisinopril acutely (Group IV, 93 +/- 8 mm Hg, P < .05). In addition, RBF was higher in Group I (81 +/- 2 ml/min) than in Groups II/III (54 +/- 5 ml/min, P < .05) or Group IV (66 +/- 8 ml/min, P < .05). Intrarenal arterial infusion of a B2 bradykinin receptor antagonist, D-Arg-O-[Hyp-3-Thi-5,8-D-Phe-7]bradykinin, had no effect on either BP or RBF in Group I. Administration of lisinopril for 6 days also resulted in attenuation of the vasoconstrictor responses to renal nerve stimulation (Group V). Intravenous infusion of D-Arg-O-[Hyp-3-Thi-5,8-D-Phe-7]bradykinin had no effect on the responses to nerve stimulation in lisinopril-treated rabbits (Group V) or their controls (Group VI). Moreover, D-Arg-O-[Hyp-3-Thi-5,8-D-Phe-7]bradykinin given i.v. did not alter the BP or RBF in Groups V and VI. The results indicate that angiotensin converting enzyme inhibition over a 6-day period is more effective than acute inhibition in lowering BP and dilating the renal vascular bed. The use of bradykinin antagonists did not indicate kinin involvement in the long-term effect of lisinopril on BP and RBF.


Methylene blue inhibits nitrovasodilator- and endothelium-derived relaxing factor-induced cyclic GMP accumulation in cultured pulmonary arterial smooth muscle cells via generation of superoxide anion. The mechanism of modulation of cyclic guanosine monophosphate (cGMP) accumulation by methylene blue (MB), a putative inhibitor of soluble guanylate cyclase, was investigated in cultured rabbit pulmonary arterial smooth muscle cells (RPASM). Control or MB-pretreated RPASM were stimulated with sodium nitroprusside (SNP), nitrosothiols or endothelium-derived relaxing factor (EDRF) released basally from bovine pulmonary arterial endothelial cells, in short-term co-cultures. The putative EDRF, S-nitroso-L-cysteine (CYSNO), a stable deaminated analog of CYSNO, S-nitroso-3-mercaptoproprionic acid (MPANO) and SNP produced concentration-dependent (1-100 microM) increase (1.5- to 12-fold) in RPASM cGMP levels. MB pretreatment inhibited CYSNO and SNP-induced cGMP accumulation by 51% to 100%, but MPANO-mediated responses were not altered by MB. The inhibition profile of MB on nitrovasodilator-induced cGMP accumulation was quantitatively reproduced by extracellular generation of superoxide anion with xanthine (100 microM) and xanthine oxidase (5 mU). Similarly to MB pretreatment, superoxide anion generation had no effects on base-line cGMP levels or cGMP responses elicited by MPANO. Furthermore, MB induced a dose- and time-dependent generation of superoxide anion from RPASM, as evidenced from spectrophotometric determination of cytochrome c reduction. Inhibition of cGMP accumulation in response to CYSNO and SNP by MB was completely prevented by superoxide dismutase but not catalase. Selective pretreatment of endothelial cells with MB before co-culture with untreated RPASM produced a reduction in RPASM cGMP levels of a magnitude comparable with that seen in co-cultures of MB-pretreated RPASM with untreated endothelial cells, and which was partially prevented by superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)


On the mechanism of papaverine inhibition of the voltage-dependent Ca++ current in isolated smooth muscle cells from the guinea pig trachea. The effects of papaverine, a smooth muscle relaxant agent, on the voltage-dependent Ca++ current were examined in isolated smooth muscle cells from the guinea pig trachea. The tight-seal whole cell voltage clamp technique was used. Papaverine (1-100 microM) inhibited the Ba++ inward current (IBa) through the voltage-dependent L-type Ca++ channel in a concentration-dependent fashion. The inhibitory effect of papaverine on IBa appeared to have both tonic and use-dependent components. In addition to the reduction of the maximal conductance of IBa, papaverine (20 microM) shifted the quasi-steady-state inactivation curve of IBa to more negative membrane potentials by approximately 10 mV. These effects of papaverine on IBa were completely reversible. Although it has been suggested that papaverine inhibited phosphodiesterase to increase intracellular cyclic AMP, phosphodiesterase inhibitors (theophylline, 500 microM, and 3-isobutyl-1-methylxanthine, 500 microM), isoproterenol (2 microM) and dibutyryl cyclic AMP (1 mM) did not affect IBa. Thus, papaverine inhibits IBa in a way independent of intracellular cyclic AMP. Papaverine also had inhibitory effects on other membrane currents (i.e., the voltage-dependent transient outward K+ current and the Ca(++)-activated oscillatory K+ current), which may result in an enhancement of the excitability of the cells. These results suggest that inhibition of the voltage-dependent L-type Ca++ channel is involved in the papaverine-induced relaxation of the tracheal smooth muscle.


Opioid receptor antagonists discriminate between presynaptic mu and delta receptors and the adenylate cyclase-coupled opioid receptor complex in the brain. The present study addressed the question as to whether or not' interacting mu and delta opioid receptors, which may constitute an opioid receptor complex-inhibitory coupled to adenylate cyclase in rat neostriatum, display different antagonistic properties than the classical (noncomplexed) mu and delta receptors. In concentrations that antagonized the presynaptic inhibitory effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) on [3H]norepinephrine release from rat neocortical slices, the cyclic somatostatin-related mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 did not affect the inhibition of dopamine-sensitive adenylate cyclase caused by DAMGO in neostriatal slices. The delta opioid receptor antagonist naltrindole appeared to be about 200-fold more effective as an antagonist against inhibitory effect of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 on [14C]acetylcholine release from neostriatal slices than against the inhibitory effect of DAMGO on [3H]norepinephrine release from neocortical slices, in agreement with the involvement of presynaptic delta and mu receptors, respectively. However, regarding the inhibitory effect of DAMGO and [D-Ser2(O-tert-butyl),Leu5] enkephalyl-Thr6 on adenylate cyclase activity in neostriatal slices, naltrindole not only displayed a very low affinity but also only 10-fold delta-selectivity. In striking contrast to D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 and naltrindole, naloxone did not discriminate between the neurotransmitter release-and adenylate cyclase-inhibitory effects of DAMGO and [D-Ser2(O-tert-butyl), Leu5]enkephalyl-Thr6.(ABSTRACT TRUNCATED AT 250 WORDS)


Specific inhibition of isoproterenol-stimulated cyclic AMP accumulation by M2 muscarinic receptors in rat intestinal smooth muscle. The ability of oxotremorine-M to inhibit cyclic AMP accumulation in the presence of a variety of adenylate cyclase activators was studied in slices from the longitudinal muscle of the rat ileum. Oxotremorine-M was found to inhibit forskolin- and isoproterenol-stimulated cyclic AMP accumulation maximally by 17 and 32%, respectively, but not the stimulation due to other activators of adenylate cyclase. Inhibition of cyclic AMP accumulation by oxotremorine-M was unaffected by tetrodotoxin and was completely reversed by atropine. AF-DX 116 (11[[2-[(diethylamino)methyl]-1- piperidynyl]acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one) an M2-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant (KB) of 0.20 microM, consistent with the dissociation constants for binding at the M2 muscarinic receptor site (KD = 0.092 microM) and inhibition of adenylate cyclase activity (KB = 0.13 microM). Hexahydrosiladifenidol, an M3-selective antagonist, shifted the oxotremorine-M dose-response curve to the right with a dissociation constant of 0.67 microM, again consistent with the dissociation constant for binding at the M2 site (KD = 0.83 microM). The agreement between the estimates of the dissociation constants of muscarinic antagonists for binding and for inhibition of cyclic AMP accumulation suggest that oxotremorine-M inhibition of isoproterenol-stimulated cyclic AMP accumulation in slices of rat intestinal smooth muscle is mediated by the M2 receptor.


Pharmacological profile of HS-142-1, a novel nonpeptide atrial natriuretic peptide (ANP) antagonist of microbial origin. II. Restoration by HS-142-1 of ANP-induced inhibition of aldosterone production in adrenal glomerulosa cells. Atrial natriuretic peptide (ANP) is known to inhibit the aldosterone production by adrenal glomerulosa cells stimulated by angiotensin II. However, the mechanism of the inhibitory action is still somewhat uncertain. In this study we used HS-142-1, a novel nonpeptide antagonist for ANP receptor, to examine the role of cyclic GMP in the inhibition of aldosterone production by ANP. Aldosterone production by isolated bovine adrenal glomerulosa cells was stimulated by angiotensin II at a concentration of 10(-8) M. The angiotensin II-stimulated aldosterone production was inhibited by rat ANP in a dose-dependent manner at concentrations ranging from 10(-9) to 10(-7) M. HS-142-1, at concentrations of 0.1 to 100 micrograms/ml, reversed the inhibition by ANP of the angiotensin II-stimulated aldosterone production. On the other hand, intracellular concentration of cyclic GMP increased rapidly as early as 1 min after the exposure of the cells to 10(-8) M ANP in the presence of angiotensin II. This increase of intracellular cyclic GMP level was again reduced by HS-142-1 at concentrations similar to those that reversed the inhibition by ANP of the aldosterone production. These results suggest that ANP inhibits the aldosterone production through a guanylyl cyclase-coupled pathway in adrenal glomerulosa cells.


Involvement of supraspinal epsilon and mu opioid receptors in inhibition of the tail-flick response induced by etorphine in the mouse. Etorphine, a potent opioid agonist, has been reported to bind to both mu and epsilon opioid receptors. The present studies were designed to determine what types of opioid receptors and neurotransmitters for descending pain control systems were involved in antinociception induced by etorphine in mice. Morphine, a typical mu opioid receptor agonist, and beta-endorphin, an epsilon opioid receptor agonist, were used for comparison. Antinociceptive response induced by etorphine (20 ng) given i.c.v was blocked by i.c.v administration of D-Phe-Cys-Tyr-D-Tyr-Orn-Thr-Pen-Thr-NH2 (CTOP, 25 ng) and beta-endorphin-(1-27) [beta-EP-(1-27)] (6 micrograms), but not ICI 174,864 (ICI, 5 micrograms) or norbinaltorphimine (N-BNI, 5 micrograms). The antinociception induced by i.c.v. etorphine was also antagonized by the i.c.v. pretreatment of beta-funaltrexamine (beta-FNA, 50 ng, 24 hr). Intracerebroventricular administration of beta-EP-(1-27) (3 micrograms) caused a further attenuation of the i.c.v. etorphine-induced antinociception in mice pretreated with beta-FNA. The antinociceptive response induced by morphine (2 micrograms) given i.c.v. was blocked by i.c.v. administration of CTOP (25 ng) or beta-FNA (50 ng), but not beta-EP-(1-27) (6 micrograms), ICI (5 micrograms) or N-BNI (5 micrograms). These results indicate that the antinociception induced by etorphine given i.c.v. is mediated by the stimulation of both mu and epsilon opioid receptors whereas the antinociception induced by morphine given i.c.v. is mediated by the stimulation of mu, but not epsilon opioid receptors at supraspinal sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Isobolographic and dose-response analyses of the interaction between intrathecal mu and delta agonists: effects of naltrindole and its benzofuran analog (NTB). Intrathecal administration of DPDPE, PL017, DAMGO, morphine and DADLE produced a dose-dependent increase in hot plate response latency, with the order of potency (ED50 nmol) being: DAMGO (0.17) > DADLE (0.70) > or = PL017 (1.2) > morphine (15) > DPDPE (130). Characteristics of the spinal mu and delta interaction were determined independently by two methods. 1) In the presence of a fixed dose of DPDPE (150 nmol), there was a left shift in the dose-response curve of the mu agonist, with the magnitude of the shifts being greater than those anticipated from a simple additive interaction: PL017 (31-fold) > or = DAMGO (20-fold) > morphine (6.5-fold) > 4-fold (theoretical additive shift). 2) With an isobolographic analysis, a statistically significant nonlinearity was observed, suggesting a multiplicative interaction upon coadministration of the delta-selective ligand DPDPE together with all the tested mu-selective agonists. Examining antagonist activity, mu agonists were antagonized in a dose-dependent fashion by naloxone and naltrindole-benzofuran analog, whereas DPDPE was reversed by all three antagonists used, naltrindole, naltrindole-benzofuran analog and naloxone. The synergic effect produced by the coadministration of PL017 and DPDPE, was reversed in a dose-dependent fashion by all three antagonists, suggesting that the interaction requires the concurrent agonist occupancy of mu and delta receptors.


Neuromedin B binds with high affinity, elevates cytosolic calcium and stimulates the growth of small-cell lung cancer cell lines. Previously, gastrin-releasing peptide (GRP) receptors were identified on small-cell lung cancer (SCLC) cells and GRP functioned as a SCLC autocrine growth factor. Here the effects of neuromedin B (NMB) on SCLC cells were investigated. [125I-Tyr0]NMB bound with high affinity to three of seven SCLC cell lines examined. [125I-Tyr0]NMB bound to SCLC cell line NCI-H209 and NCI-H345 in a time-dependent and reversible manner. [125I-Tyr0]NMB bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 800/cell). Specific [125I-Tyr0]NMB binding was inhibited with high affinity by NMB (IC50 = 1 nM) and moderate affinity by bombesin, GRP and [D-Arg1, D-Pro2, D-Trp7,9, Leu11]substance P ([APTTL]SP) but not GRP1-16 (IC50 = 50, 100, 1,000 and > 10,000 nM, respectively). In Fura 2 AM loaded NCI-H345 cells, NMB elevated cytosolic calcium in a concentration-dependent manner. NMB (10 nM) elevated the cytosolic calcium from 150 to 180 nM and calcium was released from intracellular pools. The increase in cytosolic calcium caused by 10 nM NMB was reversed by 1 microM [APTTL]SP but not 1 microM [D-Phe6]bombesin6-13methylester, a GRP receptor antagonist. Also, NMB stimulated the clonal growth of NCI-H209 and NCI-H345 in a concentration-dependent manner. The increase in the clonal growth caused by NMB was reversed by 1 microM [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of some SCLC cells.


Ca(++)-activated DNA fragmentation and dimethylnitrosamine-induced hepatic necrosis: effects of Ca(++)-endonuclease and poly(ADP-ribose) polymerase inhibitors in mice. Several hepatotoxic agents damage Ca++ regulation and produce toxic cell death in a manner consistent with a cause-and-effect relationship; however, vital targets of Ca++ remain unidentified. Recent results show that DNA may be the chief Ca++ target during apoptosis, a form of cell death considered distinct from toxic cell death or necrosis. The present studies explored whether nuclear Ca++ regulation is lost before dimethylnitrosamine-induced necrosis, whether DNA is attacked by Ca(++)-dependent endonucleases and whether inhibitors of Ca(++)-endonuclease activity and the DNA repair enzyme poly(ADP-ribose)polymerase affect necrosis. Adult male ICR mice received 100 mg/kg of dimethylnitrosamine i.p. By 2 to 4 hr, total nuclear Ca++ reached 150 to 180% of control and DNA fragmentation was 140 to 170% of control. Electrophoresis of DNA revealed a sharp decline in genomic DNA with the appearance of DNA fragments in a ladder-like pattern. Ca++ elevation and DNA fragmentation preceded toxic cell death by 4 hr or more and reached peak values at 18 to 24 hr, coincident with maximal alanine aminotransferase leakage. Aurintricarboxylic acid, a Ca(++)-endonuclease inhibitor, reduced toxicity 67%. 3-Aminobenzamide, nicotinamide adenine dinucleotide and theophylline, inhibitors of poly(ADP-ribose)polymerase-mediated DNA repair, potentiated liver damage 2-fold. These results support the hypothesis that DNA fragmentation plays a contributing role in toxic cell death induced by dimethylnitrosamine. Furthermore, the findings suggest that new opportunities may exist to moderate the toxicity of alkylating hepatotoxins by altering DNA regulation.


Inhibition of human cardiac cyclic AMP-phosphodiesterases by R 80122, a new selective cyclic AMP-phosphodiesterase III inhibitor: a comparison with other cardiotonic compounds. Four cyclic AMP (cAMP)-phosphodiesterases (PDE) belonging to families I, II, III and IV were identified in homogenates from human failing hearts. On fractionation of cardiac membranes, the cyclic GMP (cGMP)-inhibitable cAMP-PDE III copurified with the sarcoplasmic reticulum. cAMP-PDE activities were separated from the soluble fraction by DEAE-ion exchange chromatography and identified as belonging to the four different families of cAMP-PDEs. Various cAMP-PDE inhibitors, mostly cardiotonic compounds, were tested for their inhibitory potency on the different cAMP-PDEs and their selectivity for the type III isoenzyme was determined. Isobutylmethylxanthine, papaverine, theophylline and dipyridamole inhibited PDE activity in a weak and nonselective manner. Milrinone, enoximone, adibendan, pimobendan, bemoridan and the newly synthesized 1,2,3,5-tetrahydro-2-oxoimidazo[2,1-b]quinazoline derivatives, R 81267 and R 80122 were selective PDE III inhibitors. However, the IC50 values on this enzyme varied from 10 microM for enoximone to 0.036 microM for R 80122. The selectivity of the drugs for PDE III was calculated by division of the IC50 value for PDE I, II or IV by the IC50 value for PDE III. PDE I/PDE III ratio ranged from 95 for enoximone to near 28,000 for R 80122; the PDE II/PDE III ratios ranged from 95 for enoximone to 3,500 for R 80122. Although there was strong variation between the drugs, most of them showed a high selectivity for PDE III in comparison to PDE I and to PDE II. In contrast, PDE IV appeared to be more sensitive to these substances.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of beta-endorphin on mu and delta opioid receptor-coupled G-protein activity: low-Km GTPase studies. Human beta-endorphin 1-31 (beta-END) stimulated low-Km GTPase activity in a concentration-dependent and saturable manner in membranes prepared from the delta opioid receptor-containing hybrid cell line NG108-15 and from the mu opioid receptor-enriched human neuroblastoma cell line SK-N-SH. Naloxone and the delta-selective antagonist, ICI 174,864, blocked the stimulation of the GTPase activity produced by beta-END in NG108-15 cell membranes, whereas only naloxone inhibited the beta-END-induced stimulation in SK-N-SH cell membranes, suggesting that beta-END was acting through both mu and delta opioid receptors. Treatment of the cells with Bordetella pertussis toxin before the preparation of membranes blocked the stimulation of low-Km GTPase by beta-END in both cell lines. Activation of NG108-15 and SK-N-SH low-Km GTPase by beta-END was sodium-dependent, and lithium and potassium were poor promoters of this activation. These results demonstrate that beta-END stimulates the interaction of both mu and delta opioid receptors with B. pertussis toxin-sensitive G-proteins in SK-N-SH and NG108-15 cell membranes, respectively.


Association of cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers in bovine retinal rod outer segment plasma membranes. 1. Cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers from bovine photoreceptors were examined by investigation of the Ca2+ fluxes from vesicles of rod outer segment (ROS) membranes and from proteoliposomes obtained by solubilization of the ROS membrane proteins and reconstitution in soy bean L-alpha-phosphatidylcholine (PC). 2. Whereas vesicles obtained by mild sonication of ROS membranes in a Ca(2+)-containing buffer yielded a maximal cyclic GMP-induced Ca2+ release of about 2.5% and a maximal Na(+)-induced Ca2+ release of about 7%, freezing and thawing of ROS membranes prior to sonication elevated these maximal Ca2+ releases to about 17% for cyclic GMP, and to about 34% for Na+. These observations are in agreement with the view that cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers are localized only in the plasma membrane of the photoreceptors (which in bovine ROS makes up about 6% of the total membrane), whereas freezing and thawing results in fusion of disc and plasma membranes, thus leading to a distribution of these proteins over a much larger membrane area. 3. For fused ROS membranes, the cyclic GMP-releasable fraction of Ca2+ of 17% is an upper bound; assuming that the cyclic GMP-gated channels are randomly distributed we estimate that about 37% of the vesicles contain at least one cyclic GMP-gated channel. The mean diameter of the vesicles prepared by sonication was determined to be 0.12 +/- 0.04 micron, and therefore the fused ROS membranes contain about sixteen cyclic GMP-gated channels/microns 2. If all cyclic GMP-gated channels originated from the plasma membrane, we estimate that the plasma membrane contains about 270 cyclic GMP-gated channels/microns 2. 4. In vesicles prepared from fused ROS membranes, Na(+)-Ca2+ exchange after activation of the cyclic GMP-gated channels. On the other hand, after an exhaustive Na(+)-Ca2+ exchange, only little, if any, Ca2+ was released upon addition of cyclic GMP, demonstrating that cyclic GMP-gated channels and Na(+)-Ca(2+)-K+ exchangers occur on the same vesicle fraction. This observation suggests that Na(+)-Ca(2+)-K+ exchangers do not distribute independently of the cyclic GMP-gated channels upon membrane fusion but are apparently associated with the cyclic GMP-gated channels.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium current activation and charge movement in denervated mammalian skeletal muscle fibres. 1. Calcium current (ICa) activation was studied in denervated extensor digitorum longus muscle fibres of the rat. Denervation was performed by surgically removing 6-8 mm of the sciatic nerve at the sciatic notch. Controls were normal fibres from non-operated rats. Electrical recordings were carried out using the double Vaseline-gap technique. 2. Current-voltage (I-V) curves showed that the ICa amplitude increased during the first 4-6 days after denervation and subsequently decreased during the second week. Between days 4 and 6 after denervation, the peak ICa amplitude (at 0 mV) was -5.9 +/- 0.5 microA/microF (mean +/- S.E.M.) as compared with -4.8 +/- 0.3 microA/microF in normal fibres. Between days 14 and 15 after denervation, the ICa amplitude was -2.9 +/- 0.4 microA/microF. 3. The time constant of ICa activation (tau a) was significantly increased by denervation. At 0 mV, tau a in normal fibres was 44.8 +/- 1.4 ms. Between 4 and 6 days after denervation tau a was 58.1 +/- 4.8 ms, and between 14 and 15 days after denervation, 55.8 +/- 3.8 ms. 4. The time constant of deactivation (tau d) decreased after denervation. At -10 mV, the tau d in normal fibres was 103.4 +/- 14 ms. The value decreased to 74.5 +/- 8.6 and 74.0 +/- 17 ms between days 4 and 6 and days 14 and 15 of denervation respectively. 5. Charge movement (Qon) was reduced progressively without major changes in the steepness (k) and position on the voltage axis of the Qon-Vm relationship. The fitted parameters under control were Qmax = 15.4 nC/microF, mid-point potential Vq1/2 = -25.2 mV and k = 11.9 mV. Between days 14 and 15 of denervation, the values for Qmax, Vq1/2 and k were 6.7 nC/microF, -36.8 mV and 11.3 mV respectively. 6. Calcium permeability (PCa) in normal and denervated fibres at stages during denervation was calculated according to the Hodgkin-Huxley model. At 0 mV PCa was 1.24 x 10(-5) cm/s in normal fibres, and 7.43 x 10(-6) cm/s after 2 weeks of denervation. 7. The m infinity-Vm relationship was shifted to more positive potentials after denervation without significant changes in the steepness factor k. The V1/2 value in normal fibres was -4.4 mV, and 5.8 mV after two weeks of denervation. 8. The ICa sensitivity to nifedipine was not modified in the different groups of denervated fibres studied. With 10 microM-nifedipine, the 1-(ICa in nifedipine/ICa control) relationships were 0.74 +/- 0.03 in normal fibres and 0.76 +/- 0.12, 14 days after denervation.


Novel isoform of Ca2+ channel in rat fetal cardiomyocytes. 1. Single cardiomyocytes of 18-day-old rat fetuses were isolated to characterize the cardiac Ca2+ channels in the fetal period, using whole-cell voltage clamp (Na+, K(+)-free external solution and K(+)-free internal solution), and depolarizing test pulses from a holding potential (HP) of -87 mV were applied. 2. The Ca2+ current was completely blocked by 2 mM-CO2+, but not completely blocked by the dihydropyridine (DHP) Ca2+ antagonist nifedipine. Nifedipine (3 microM) decreased the amplitude of the current (at -7 mV) by 65.9 +/- 3.4% (n = 20). At a HP of -47 mV, nifedipine decreased the Ca2+ current to about the same degree. Diltiazem (1 microM) did not block the nifedipine-resistant current which remained. 3. Nitrendipine, another DHP Ca2+ antagonist, had effects on the Ca2+ current similar to those of nifedipine. 4. The DHP-resistant current was not blocked by T-type channel blockers (Ni2+, tetramethrine) or an N-type blocker (omega-conotoxin). 5. In conclusion, rat fetal cardiomyocytes may have a unique type of Ca2+ channel (ICa(fe)), which decreases in amplitude and becomes less prominent during subsequent development.


Membrane hyperpolarization inhibits agonist-induced synthesis of inositol 1,4,5-trisphosphate in rabbit mesenteric artery. 1. Effects of membrane hyperpolarization induced by pinacidil on Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), isometric tension, membrane potential and production of inositol 1,4,5-trisphosphate (IP3) in smooth muscle cells of the rabbit mesenteric artery. 2. Pinacidil (0.1-10 microM) concentration dependently hyperpolarized the smooth muscle membrane with a reduction in membrane resistance. Glibenclamide (1 microM) blocked the membrane hyperpolarization induced by 1 microM-pinacidil. NA (10 microM) depolarized the smooth muscle membrane with associated oscillations. Pinacidil (1 microM) inhibited this response and glibenclamide (1 microM) prevented the action of pinacidil on both the NA-induced events. 3. In thin smooth muscle strips, 10 microM-NA produced a large phasic and a subsequent small tonic increase in [Ca2+]i with associated oscillations. These changes in [Ca2+]i seemed to be coincident with phasic, tonic and oscillatory contractions, respectively. Pinacidil (0.1-1 microM) inhibited the increases in [Ca2+]i and in tension induced by NA, but not by 128 mM-K+. Glibenclamide inhibited these actions of pinacidil. Pinacidil (1 microM) also inhibited the contraction induced by 10 microM-NA in strips treated with A23187 (which functionally removes cellular Ca2+ storage sites), suggesting that membrane hyperpolarization inhibits Ca2+ influxes activated by NA. 4. In Ca2(+)-free solution containing 2 mM-EGTA, NA (10 microM) transiently increased [Ca2+]i, tension and synthesis of IP3. Pinacidil (over 0.1 microM) inhibited the increases in [Ca2+]i, tension and synthesis of IP3 induced by 10 microM-NA in Ca2(+)-free solution containing 5.9 mM-K+, but not in a similar solution containing 40 or 128 mM-K+. Glibenclamide (1 microM) inhibited these actions of pinacidil. These inhibitory actions of pinacidil were still observed in solutions containing low Na+ or low Cl-. These results suggest that pinacidil inhibits NA-induced Ca2+ release from storage sites through an inhibition of IP3 synthesis resulting from its membrane hyperpolarizing action. 5. In beta-escin-treated skinned strips, NA (10 microM) or IP3 (20 microM) increased Ca2+ in Ca2(+)-free solution containing 50 microM-EGTA and 3 microM-guanosine triphosphate (GTP) after brief application of 0.3 microM-Ca2+, suggesting Ca2+ is released from intracellular storage sites. Heparin (500 micrograms/ml, an inhibitor of the IP3 receptor), but not pinacidil (1 microM) or glibenclamide (1 microM), inhibited the Ca2+ release from storage sites induced by NA or IP3. These results suggest that membrane hyperpolarization is essential for the inhibitory action of pinacidil on the NA-induced Ca2(+)-releasing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)


Comparison of the actions of baclofen at pre- and postsynaptic receptors in the rat hippocampus in vitro. 1. Intracellular microelectrode recordings were used to study the cellular location, pharmacology, and mechanism of action of gamma-aminobutyric acidB (GABAB) receptors on pyramidal cells and presynaptic axonal endings in area CA3 of organotypic hippocampal slice cultures. 2. Baclofen (bath applied at 10 microM) caused a 10-15 mV hyperpolarization of CA3 cells and a 75-100% decrease in the amplitude of excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs). Baclofen reduced the amplitude of monosynaptic IPSPs elicited in the presence of excitatory amino acid receptor antagonists, as well as the amplitude of EPSPs elicited after blocking GABAA receptors and reducing subsequent epileptic bursts with excitatory amino acid receptor antagonists. These data indicate that GABAB receptors are located on both excitatory and inhibitory presynaptic elements. 3. The GABAB receptor antagonist CGP 35 348 blocked the postsynaptic action of baclofen, the late IPSP, and the reduction of EPSPs and monosynaptic IPSPs by baclofen. 3-Aminopropylphosphinic acid (3-APA) mimicked all the pre- and postsynaptic actions of baclofen, and its effects were fully antagonized by CGP 35 348. 4. Incubation of cultures with pertussis toxin (500 ng/ml for 48 h) prevented both the postsynaptic hyperpolarization and the block of monosynaptic IPSPs induced by baclofen. The action of baclofen on isolated EPSPs, however, was not affected by pertussis toxin treatment. Stimulation of protein kinase C with phorbol ester (phorbol 12, 13 dibutyrate, 1 microM for 10 min) reduced all pre- and postsynaptic effects of GABAB receptor activation. 5. Barium (bath applied at 1 mM) prevented both the baclofen-induced hyperpolarization of pyramidal cells and the block of monosynaptic IPSPs by baclofen. In the presence of barium, however, baclofen was fully capable of blocking EPSPs. 6. We conclude that pre- and postsynaptic GABAB receptors are pharmacologically indistinguishable, at present, and that all actions of GABAB receptors are inhibited by stimulation of protein kinase C. Both the postsynaptic action of baclofen and the block of GABA release from interneurons are mediated by pertussis toxin-sensitive G proteins which can be inactivated by stimulation of protein kinase C. Baclofen acts at postsynaptic sites and on the axon terminals of inhibitory interneurons by activating the same barium-sensitive K+ conductance. GABAB receptors on excitatory axons must, however, work through some other mechanism.


On the inhibitory actions of baclofen and gamma-aminobutyric acid in rat ventral midbrain culture. 1. Whole-cell voltage-clamp recordings were used to study the effects of (-)-baclofen and of gamma-aminobutyric acid (GABA) on neurones cultured from the ventral midbrain of embryonic rats. 2. Baclofen induced an outward current (IBac) at a holding potential of -60 mV. The maximal current was 80 pA, and half-maximal current was evoked by 5 microM-baclofen. The proportion of cells affected by baclofen was greater in 25-day-old cultures than in 14-day-old cultures. 3. IBac was blocked by barium (1 mM), and it reversed polarity at a potential that changed according to the Nernst equation when the extracellular potassium concentration was changed. The reversal potential was not different when recording electrodes contained caesium instead of potassium. 4. GABA (10-20 microM), in the presence of picrotoxin (50 microM) and bicuculline (50 microM), also evoked a small potassium current at -60 mV. There was no correlation between the amplitude of the potassium current caused by GABA and that caused by baclofen measured in the same neurones. 5. Spontaneous synaptic currents (up to hundreds of picoamps) were observed that were blocked by picrotoxin (20 microM; IPSCs) or by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM; EPSCs); the amplitude and frequency were strongly reduced by baclofen and by GABA. 6. Spontaneous synaptic currents of lower amplitudes (up to 60 pA) remained in the presence of tetrodotoxin. IPSCs (blocked by picrotoxin, reversal at -50 mV) and EPSCs (blocked by CNQX, reversal at 0 mV) were reduced in frequency by baclofen. GABA, in the presence of bicuculline and picrotoxin, had a similar effect on the EPSCs. This action of baclofen persisted in barium (1 mM), and was observed as readily in cells cultured for 14 days as those cultured for 25 days. 7. Some spontaneous synaptic currents remained in the presence of tetrodotoxin and cadmium (100 microM). Their frequency was reduced by baclofen. The effectiveness of baclofen was greater on cells that had been longer in culture. 8. It is concluded that activation of GABAB receptors has two main effects on neurones cultured from rat ventral midbrain. These are potassium conductance increase, and inhibition of the spontaneous release of GABA and excitatory amino acids; both effects can be observed in tetrodotoxin and cadmium.


Film thickness of new adhesive luting agents. This study determined and compared the film thicknesses of new adhesive luting agents. The method was in compliance with American National Standards Institution/American Dental Association (ADA) Specification No. 8 for zinc phosphate cement. Each of the 20 materials tested was manipulated exactly as described in the manufacturers' instructions. An electronic gauge with an accuracy of 0.5 micron was recalibrated after each recording, and each luting agent was measured 10 times. The mean film thickness and standard deviation were calculated for each luting agent; an analysis of variance and a multiple comparison test were also performed. Nine materials satisfied the ADA type I specification for film thicknesses less than 25 microns, and these included a hydroxyapatite cement, glass ionomer cements, zinc phosphate cements, and polycarboxylate cements. Five other materials met the ADA type II specification for film thicknesses of less than 40 microns, and these included a glass ionomer cement, a resinous cement, a zinc phosphate cement, and glass ionomer-resinous hybrids. Six resinous cements recorded film thicknesses greater than 40 microns, and suggestions were made regarding future development and research.


Measurement of protein phosphatase activity in biological samples using synthetic phosphopeptides. A method has been developed for measuring specific protein phosphatase activity in biological samples using synthetic, phospho-Kemptide and phospho-GS-peptide. This method uses ion-exchange chromatography to determine phosphatase activity by quantifying the release of [32P]phosphate directly. The method was used to measure phosphatase activity of rat kidney, adrenals, heart, and liver cytosol and the activity of purified alkaline phosphatases, protein phosphatase 1, and protein phosphatase 2A. Ion-exchange chromatography was also used for the preparation of the radiolabeled phosphopeptide substrates. This method results in high recovery and specific activity of the labeled peptides. These techniques should be useful in isolating and characterizing specific protein phosphatases found in cells.


Extracellular recordings from the motor nervous system of the nematode, Ascaris suum. 1. The close association of muscle and neurons in Ascaris suum makes it difficult to determine whether spikes recorded from nerve cords originate in muscle or neurons. We have developed criteria that distinguish muscle and neuronal activity. There are two categories of extracellular spikes. 2. The first category consists of spikes with a wide range of amplitudes, marked by large spikes. These spikes, which can be recorded over lateral muscle and over the dorsal and ventral nerve cords, are abolished when muscle is disrupted or removed, or when curare is applied. Large spikes are relatively infrequent, are correlated with intracellularly recorded muscle events, and respond to polarizations of motor neurons, implying that they originate in muscle. 3. The second spike category, small amplitude spikes, is exclusive to the ventral nerve cord, occurs more frequently than large spikes and displays patterned firing. Small spikes are not affected by muscle removal or by curare, and are correlated with motor neuronal post-synaptic potentials, but not with intracellularly recorded muscle events. We infer that they originate in neurons. 4. Low level activity recorded extracellularly over nerve cords may represent muscle activity due to tonic motor neuronal synaptic transmission. It responds to motor neuronal polarization and is suppressed by curare or muscle removal.


Interactions of membrane excitability mutations affecting potassium and sodium currents in the flight and giant fiber escape systems of Drosophila. We have studied the influence of the K(+)-current mutations eag and Sh and the Na(+)-current mutation napts upon two well-defined neural circuits that underlie flight and an escape response in Drosophila, recording from dorsal longitudinal and tergotrochanteral muscles. Mutations of Sh and eag affected refractory period and following frequency, but not latency, of the jump-and-flight escape response. The napts mutation altered these 3 physiological parameters of the "jump" (TTM), but not the "flight" (DLM), branch, suggesting differences in the vulnerability of different circuit components to the mutation. In contrast to their interaction in some other systems, napts did not counteract the effects of eag and Sh upon these physiological parameters in eag Sh; nap triple mutants. In eag Sh double mutants, in which multiple K+ currents may be diminished, flight muscles showed abnormal rhythmic activity not associated with flight, and some flies also had an abnormal wings-down posture. The low-frequency spikes probably originated in the flight muscle motoneurons, but the coordination between muscle fibers during this "non-flight activity" was distinct from flight. Nevertheless, in spite of the presence of this non-flight activity in resting eag Sh flies, those animals with normal wing posture were also able to fly, with a normal pattern of muscle activity. This suggests that in these mutants, the DLM motoneuron circuit is able to switch between two patterns of output, non-flight activity and flight.(ABSTRACT TRUNCATED AT 250 WORDS)


Complications of superficial parotidectomy versus extracapsular lumpectomy in the treatment of benign parotid lesions. A total of 46 patients with benign parotid gland tumours have been operated upon between 1979 and 1989 in the Department of Otolaryngology at Leicester Royal Infirmary. Thirty-one were pleomorphic adenomas, six Warthin's tumours and nine miscellaneous tumours or tumour-like lesions. Extracapsular lumpectomy without facial nerve preparation was used in 31 cases (67%) and superficial parotidectomy in 15 cases (33%). One case of permanent partial facial palsy and one recurrence occurred in patients who underwent superficial parotidectomy. Frey's syndrome occurred in 40% of patients undergoing superficial parotidectomy. No permanent palsy, recurrence or Frey's syndrome occurred after extracapsular lumpectomy. These results suggest that extracapsular lumpectomy may reduce the morbidity rate in carefully selected patients.


Genotoxic effects of linear alkyl benzene sulfonate, sodium pentachlorophenate and dichromate on Tetrahymena pyriformis. DNA in macro- and micronuclei of Tetrahymena pyriformis treated with linear alkyl benzene sulfonate (LAS) and sodium pentachlorophenate (PCP-Na) were determined by microspectrophotometry. The effects on rate of formation of macronuclear DNA extrusion bodies were also studied. We found DNA content of micronuclei in 0.14 ppm LAS and 0.9 ppb PCP-Na was lower than in that of the control, and LAS was able to increase the formation rate of macronuclear DNA extrusion bodies (the formation rate was 54% in 11.3 ppm LAS and 25.6% in 16.7 ppm dichromate). We concluded that 0.14 ppm LAS (below the maximum acceptable toxicant concentration) was genotoxic, whereas 0.014 ppm LAS was not. Dichromate 0.05 ppm and 0.9 ppb PCP-Na, equal to and below the maximum acceptable toxicant concentration, respectively, were potentially genotoxic.


Fibrous histiocytomas in the tropics.

Fibrous histiocytomas in the tropics. Sixty patients with fibrous histiocytomas - malignant fibrous histiocytomas and benign histiocytomas - presenting at University College Hospital, Ibadan over a 10-year period have been evaluated retrospectively. A malignant:benign tumour ratio of 2.8:1, a male:female ratio of 1:1, and a predilection for malignant histiocytomas to occur early (16-25 years) in contrast to benign histiocytomas (26-35 years) were noted. While the trunk accounted for one-third of cases, the extremities accounted for two-thirds of all patients. A long history of slow growth (from 6 months to 11 years) of a large bossellated tumour anywhere in the body, with a tendency to recurrence after excision and potential for metastasis, constitute the main characteristics of this tumour.


Evidence for a role for a uterine renin-angiotensin system in decidualization in rats. Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.


Induction of meiotic maturation of follicle-enclosed oocytes of rabbits by a transient increase followed by an abrupt decrease in cyclic AMP concentration. The involvement of cyclic adenosine monophosphate (cAMP) in mammalian oocyte maturation was assessed using cultures of rabbit cumulus-oocyte complexes and perfused rabbit ovaries. Rabbit cumulus-oocyte complexes were cultured in Brackett's medium with or without forskolin at 10(-4), 10(-5) or 10(-6) mol l-1 for 3-6 h. At 3 or 4 h spontaneous meiotic maturation was significantly (P < 0.05) inhibited by forskolin at 10(-4) mol l-1. With prolonged incubation, spontaneous maturation progressed despite exposure to forskolin. In the second experiment ovaries were perfused for 12 h with forskolin (10(-4), 10(-5) or 10(-6) mol l-1) or medium alone. Neither ovulation nor degeneration of follicular oocytes occurred in any perfused ovary. The percentage of follicular oocytes achieving germinal vesicle breakdown was significantly (P < 0.001) increased in response to forskolin in a dose-related manner. In an additional experiment, ovaries were perfused with forskolin at 10(-4) mol l-1. A significant increase in the cAMP content in the follicle was observed within 30 min, but the ability to produce cAMP in response to forskolin decreased as the duration of perfusion was increased. Intraoocyte cAMP increased significantly within 30 min and reached its maximum 2 h after exposure to forskolin. Thereafter, cAMP levels in the oocytes decreased abruptly. This drop in intraoocyte cAMP concentration was followed by the resumption of meiosis. The alterations of intraoocyte cAMP contents following exposure to hCG in vivo paralleled those observed in the ovaries perfused with forskolin. These data suggest that a transient, but not continuous, increase in cAMP concentration after the gonadotrophin surge may be required to initiate oocyte maturation.


Increase in concentration of uterine oxytocin receptors and decrease in response to 13,14-dihydro-15-keto prostaglandin F2 alpha in ewes after withdrawal of exogenous progesterone. Ovariectomized ewes were treated with progesterone and oestradiol to induce oestrus (day of expected oestrus = day 0) and with progesterone on days 1 to 12. The concentrations of endometrial oxytocin receptors and the 13,14-dihydro-15-keto prostaglandin F2 alpha (PGFM) response induced by oxytocin were measured on days 12, 14, 16 and 18 after the cessation of progesterone treatment on day 12, by a receptor binding assay and direct radioimmunoassay, respectively. During the period of treatment, the concentrations of plasma progesterone were high and remained above 2 ng ml-1 until day 13 when they dropped rapidly to less than 0.5 ng ml-1 by day 14. The concentrations of oxytocin receptors in endometrium of control ewes were high (820.7 +/- 91.7 (SEM) fmol mg-1 protein). Treatment with progesterone significantly (P < 0.01) reduced the concentrations of the receptors on days 12 and 14 (144.1 +/- 65.0 and 200.4 +/- 45.4 fmol mg-1 protein, respectively). The receptor concentrations then increased to relatively high values on day 16 (1021.4 +/- 216.6 fmol mg-1 protein) and remained high until day 18 (677.7 +/- 103.4 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of a single dose of methotrexate on 5- and 12-lipoxygenase products in patients with rheumatoid arthritis. The inhibition of 5-lipoxygenase could be involved in the mechanism of action of methotrexate (MTX). We studied 8 patients with active rheumatoid arthritis (RA) immediately before and the day after the first dose of MTX (10 mg intramuscularly). Leukotriene B4 (LTB4) formation by polymorphonuclear leukocytes was significantly decreased (32%, p less than 0.01). This essentially involved released LTB4. A slight decrease was also obtained in omega-oxidation products. Similar results were obtained for plasma LTB4 (30%, p less than 0.02). A non-significant decrease in 5-HETE was noted. Conversely, 12-HETE was not modified. Our results suggest MTX has an effect on the 5-lipoxygenase pathway, particularly at the LTA4 epoxide hydrolase step, since 5-HETE and 6-trans-LTB4 isomers are not involved.


Peripheral neuropathy in mixed cryoglobulinemia: clinical and electrophysiologic investigations. Peripheral neuropathy has been described in different rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus and systemic vasculitis, but usually in limited numbers of patients. Nerve injury is more frequently reported in mixed cryoglobulinemia. In earlier studies generally performed in small series of patients, prevalence of peripheral neuropathy varied widely. We evaluated prevalence of peripheral neuropathy in 33 unselected patients with mixed cryoglobulinemia (25 women, 8 men, aged from 45-71 years). Neurologic involvement was detected using a complete clinical and electrophysiologic assessment, including sensory motor conduction velocities, F wave and H reflex. Neurologic examination revealed a neuropathy in 48% of subjects, while electrophysiologic variables were altered in 82%; a percentage similar to that of subjective symptoms (91%). Among electrophysiologic investigations, F wave was altered in 22/33 subjects (67%); therefore, this variable seems to be the most reliable for the detection of neurologic involvement. Cryocrit levels were significantly higher in patients with peripheral neuropathy: abnormal examination (p less than 0.01), sensory motor conduction (p less than 0.04), and F wave alterations (p less than 0.008). In addition, hemorheological abnormalities seem to contribute to the pathogenesis of nerve injury. Our results indicate that peripheral neuropathy, to a variable degree, is present in the majority of patients with mixed cryoglobulinemia, and a complete clinical and electrophysiologic investigation can be useful for an early and correct diagnosis.


Sleep, Epstein-Barr virus infection, musculoskeletal pain, and depressive symptoms in chronic fatigue syndrome. Sleep physiology, viral serology and symptoms of 14 patients with chronic fatigue syndrome (CFS) were compared with 12 healthy controls. All patients described unrefreshing sleep and showed a prominent alpha electroencephalographic nonrapid eye movement (7.5-11.0 Hz) sleep anomaly (p less than or equal to 0.001), but had no physiologic daytime sleepiness. There were no group differences in Epstein-Barr virus (EBV) antibody titers. The patient group had more fibrositis tender points (p less than 0.0001), described more somatic complaints (p less than 0.0001), and more depressive symptoms (p less than 0.0001). Patients with CFS do not show evidence for a specific chronic EBV infection, but show altered sleep physiology, numerous tender points, diffuse pain, and depressive symptoms. These features are similar to those found in fibromyalgia syndrome.


Cytokine regulation of prolyl 4-hydroxylase production in skin fibroblast cultures from patients with systemic sclerosis: contribution to collagen synthesis and fibrosis. Prolyl 4-hydroxylase (PH) is an enzyme that catalyzes an essential step in procollagen synthesis. PH production, as measured by the new ELISA method, was significantly higher in fibroblast cultures from patients with systemic sclerosis (SSc; N = 7) than in cultures from healthy controls (NC; N = 5) (p less than 0.01). Transforming growth factor-beta and tumor necrosis factor-alpha significantly increased PH production, but recombinant interferon-gamma had a significant negative effect on PH production. Since the production of PH is relevant to collagen synthesis by fibroblasts of patients with SSc, regulation of PH production may have therapeutic value.


Physical structure and chemical composition of abomasal phytobezoars of goats and sheep. The diameters of fibres from phytobezoars varied between 2 and 30 microns, with an average 58% measuring less than < 15 microns, 36% between 15 and 23 microns, and 6% greater than 23 microns. These diameters were very similar to those of the pappus hairs surrounding the seeds of the Karoo bushes Chrysocoma ciliata, Eriocephalus ericoides and Gnidia polycephala (means of 6.06 microns, 16.67 microns and 22.73 microns respectively). The microscopic structure and ultrastructure of these pappus hairs moreover closely resembled the fibres of bezoars but were quite distinct from that of hair or wool. Of particular interest was the presence of annular thickenings as well as terminal hooks on both bezoar fibres and pappus hairs, both of which structures are believed to be involved in the formation of bezoars. This close similarity between the fibres in bezoars and those of pappus hairs in the 3 plant species studied, leads to the conclusion that the bezoars consisted largely of pappus hairs of these and possibly other species. The chemical composition of bezoars found in goats and sheep, resembled that of pappus hairs and stems of the Karoo bushes Eriocephalus ericoides and Gnidia polycephala more closely than that of mohair. Ash, nitrogen, phosphorus and zinc concentrations in both bezoars and plant material were similar, but very different to that of mohair; however, the calcium, magnesium, potassium and manganese levels of bezoars were more similar to mohair than plant matter. This may have been due to a leaching effect.


Experimental reproduction of phytobezoars. In a series of in virtro experiments (n = 64) using the mature flowers or seeds and pappus hairs of Karoo bushes Chrysocoma ciliata, Dimorphotheca cuneata, Eriocephalus ericoides, Gazania krebsiana and Gnidia polycephala, small phytobezoars were formed from material of E. ericoides, G. krebsiana and G. polycephala either alone or in combinations. The ratio of fibre to liquid used was found to be optimal around 1: 7 and milling of material enhanced bezoar formation. Time and pH did not appear to significantly affect the formation of bezoars. Other factors were either standardised or not studied. In a small in vivo trial, 4 groups of one sheep and one goat each were fed 10% of either C. ciliata, E. ericoides, G. polycephala seeds and pappus hairs, or a mixture of all 3, added to a basal diet of milled lucerne fed for up to 31 d. Small phytobezoars were formed in the goats but not the sheep receiving C. ciliata, and G. polycephala material. It was concluded that phytobezoars could form in goats or sheep eating large quantities of mature flowers or seeds of several Karoo bushes.


Modeling of G-protein-coupled receptors: application to dopamine, adrenaline, serotonin, acetylcholine, and mammalian opsin receptors. Hydropathicity analysis of 39 G-protein-coupled receptors (GPCR) reveals seven hydrophobic stretches corresponding to membrane spanning alpha-helices. The alignment of the primary sequences shows a high degree of homology in the GPCR transmembrane regions. 3D models of 39 GPCRs were generated using the refined model of bacteriorhodopsin as a template. Five cationic neurotransmitter receptors (serotonergic 5-HT2, dopaminergic D2, muscarinic m2, adrenergic alpha 2 and beta 2 receptors) were taken as prototypes and studied in detail. The 3D models of the cationic neurotransmitter receptors, together with their primary structure comparison, indicate that the agonist binding site is located near the extracellular face of the receptor and involves residues of the membrane-spanning helices 3, 4, 5, 6, and 7. The binding site consists of a negatively-charged Asp located at the middle of transmembrane helix 3 and a hydrophobic pocket containing conserved aromatic residues on helices 4, 5, 6, and 7. To define the precise receptor-ligand interactions, the natural neurotransmitters were docked into the binding sites. Residues responsible for the affinity, selectivity, and eventually stereospecificity of dopamine, adrenaline, noradrenaline, serotonin, and acetylcholine for their receptors were identified. The ligands are involved in electrostatic interactions as well as hydrogen bonds and specific hydrophobic aromatic interactions. All the GPCRs possess invariant hinge residues, which might be responsible for a conformational change during agonist binding and therefore influence dissociation and association of G-proteins to the receptors. The role of hydrophobic interactions and hydrogen bonds in the conformational change of the receptors, modulating the coupling to the G-protein, is discussed with regard to these residues. The models are in agreement with published data obtained from mutagenesis and labeling studies and represent important working hypotheses to direct future mutagenesis studies. They also enable structure-activity relationship studies and more rational drug design. The 3D models of other G-protein-coupled receptors have been generated in a similar way.


Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells. The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M3 subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [3H]quinuclidinyl benzilate ([3H]QNB) was most sensitive to atropine and the M3-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M2 antagonist AFDX116. This, and the binding characteristics of [3H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M3 subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 microM) reduced Bmax of [3H]4-DAMP binding from 27 +/- 4 to 13 +/- 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolin-induced cAMP formation in SMG cells with an EC50 of 200 nM, but paraoxon was greater than 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 microM atropine, that by paraoxon was unaffected by up to 100 microM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect beta-adrenoreceptor function in the preparation, since it did not affect the 10 microM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 microM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells.(ABSTRACT TRUNCATED AT 250 WORDS)


The impact of physician denial upon patient autonomy and well-being. It is now widely accepted that a patient's ability to engage in autonomous decision-making can be seriously threatened when she denies significant aspects of her medical condition. In this paper I use a true case to reveal the harmful effects of physician denial upon patient autonomy and well-being. I suggest further that such physician denial may be more common than is generally acknowledged, since aspects of the contemporary medical ethos likely serve to reinforce rather than to undercut such denial.


Evidence for an alpha-herpesvirus indigenous to mountain gorillas. Sera from wild mountain gorillas were screened for antibodies reactive with primate alpha-herpesviruses. Four of seven individuals tested (58%) were positive. In all four sera the highest titers were to HSV-2 followed by HSV-1 and SA8. Immunoblot analysis confirmed a preferential reactivity with HSV-2 antigen. Further analysis by competition ELISA indicated that these gorillas had experienced infection with a virus antigenically similar but not identical to HSV-2. These results represent the first evidence for an alpha-herpesvirus indigenous in a free ranging, nonhuman anthropoid species.


Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction. The Ca2+ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m)[125I]iodophenyl) diazirine and then isolated and proteolysed with calpain II, the [125I] was traced from the 565 kDa parent to Mr 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa fragment was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin.


Modulation of inwardly rectifying Na(+)-K+ channels by serotonin and cyclic nucleotides in salivary gland cells of the leech, Haementeria. The electrically excitable salivary cells of the giant Amazon leech, Haementeria, display a time-dependent inward rectification. Under voltage clamp, hyperpolarizing steps to membrane potentials negative to about -70 mV were associated with the activation of a slow inward current (Ih) which showed no inactivation with time. The time course of activation of Ih was described by a single-exponential function and was strongly voltage dependent. The activation curve of Ih ranged from -72 to -118 mV, with half-activation occurring at -100 mV. Ion-substitution experiments indicated that Ih is carried by both Na+ and K+ ions. 5-Hydroxytryptamine (5-HT) increased the amplitude of Ih and its rate of activation. It also produced a positive shift of the activation curve of the conductance underlying Ih (Gh) without altering the slope factor, thus indicating that the voltage dependence of Ih was modulated by 5-HT. Cs+ blocked both Ih and the 5-HT-potentiated current in a voltage-independent manner, whereas Ba2+ had little effect. It is concluded that 5-HT increases Ih by modulating the inwardly rectifying Na(+)-K+ channels in the salivary cells. The effect of 5-HT may be mediated by an increase in adenylate cyclase activity since Ih was increased by 8-bromo-cyclic AMP and by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. In contrast, Ih was reduced by 8-bromo-cyclic GMP and by zaprinast (an inhibitor of cyclic GMP-sensitive phosphodiesterase). Cyclic GMP itself also reduced Ih, and the effect was specific to the 3',5' form; 2',3'-cyclic GMP was inactive. The results suggest that the inward-rectifier channel may be modulated in opposite directions by cyclic AMP and cyclic GMP.


Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle. Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels.


Slow voltage inactivation of Ca2+ currents and bursting mechanisms for the mouse pancreatic beta-cell. Recent whole-cell electrophysiological data concerning the properties of the Ca2+ currents in mouse beta-cells are fitted by a two-current model of Ca2+ channel kinetics. When the beta-cell K+ currents are added to this model, only large modifications of the measured Ca2+ currents will reproduce the bursting pattern normally observed in mouse islets. However, when the measured Ca2+ currents are modified only slightly and used in conjunction with a K+ conductance that can be modulated dynamically by ATP concentration, reasonable bursting is obtained. Under these conditions it is the K-ATP conductance, rather than the slow voltage inactivation of the Ca2+ current, that determines the interburst interval. We find that this latter model can be reconciled with experiments that limit the possible periodic variation of the K-ATP conductance and with recent observations of intracellular Ca2+ bursting in islets.


Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication. Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.


Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia. Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na+ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nM tetrodotoxin, while the other type was insensitive to 10 microM tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na+ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na+ current. The slow inactivation of the tetrodotoxin-insensitive Na+ current was kinetically distinct from the ordinary short-time "steady-state" inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxin-insensitive Na+ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.


Regulation of a potassium-selective current in rabbit corneal epithelium by cyclic GMP, carbachol and diltiazem. The effects of cyclic GMP (cGMP), carbachol and diltiazem on a potassium-selective, delayed-rectifier current in freshly dissociated rabbit corneal epithelial cells were studied using a modified perforated-patch-clamp technique. The current was stimulated by both 500 microM cGMP (2.3-4.5-fold, mean = 2.9) and 250 nM carbachol, a muscarinic agonist (1.12-7.04-fold, mean = 3.8), and the stimulated current was totally blocked by diltiazem (10 microM). The effects of cGMP appeared to be, at least in part, different from those of carbachol as they required the presence of external calcium. Single-channel data suggest that cGMP and carbachol activate the potassium current by increasing the open probability of the channel via a second-messenger system and that the action of diltiazem is probably through a direct blocking effect on the open channel.