Somatostatin receptor elevation in rat striatum after diisopropylfluorophosphate administration. The acute and chronic administration of diisopropylfluorophosphate (DFP), an inhibitor of acetylcholinesterase or of atropine, a blocker of muscarinic cholinergic receptors, did not affect somatostatin-like immunoreactivity (SLI) content in the striatum of rats. Acute and chronic DFP administration increased the number of specific 125I-Tyr11-somatostatin (125I-Tyr11-SS) receptors in cells dissociated from the striatum without changing the affinity constant. Although the increase could be blocked by pretreatment with atropine, it was not due to a direct effect by DFP on somatostatin (SS) receptors, because no rise in 125I-Tyr11-SS binding was produced by high concentrations of DFP (10(-5) M) when added in vitro. The acute administration of atropine alone had no observable effect on the number of SS receptors. However, repeated atropine administration produced a significant decrease in the 125I-Tyr11-SS binding in cells dissociated from the striatum, although the affinity constant was unchanged. The results suggest that interactions between somatostatinergic and cholinergic receptors may be of importance in the rat striatum.


Bidirectional effect of beta-carboline agonists at the benzodiazepine-GABAA receptor chloride ionophore complex on GABA-stimulated 36Cl- uptake. beta-Carboline agonists produced a left shift of the GABA concentration-chloride uptake curve or a reduction in the maximal increase in GABA-stimulated 36Cl- uptake depending on their concentration. The enhancement of the GABA effect occurs only at lower beta-carboline and GABA concentrations and is smaller for the partial agonist ZK 9126 compared to the full agonist ZK 93423. The opposite effect, inhibition of GABA-stimulated chloride conductance, is observed only at higher concentrations of beta-carboline agonists and GABA. The reduction of the GABA maximal response by the partial agonist ZK 91296 is greater than by the full agonist ZK 93423. The transformation of GABA-stimulated 36Cl- uptake data to specific chloride influx (36Cl- uptake per nM of GABA) reveals that the GABA concentration-response curve consists of three parts characterized by differences in the molar effectiveness of GABA relative to the GABA concentration. The molar effectiveness of GABA is a measure of the sensitivity of the GABAA receptor chloride ionophore complex and shows adaptive changes by this complex to increasing concentrations of GABA and/or beta-carboline. We conclude from our data that the change from GABA-sensitive to GABA-insensitive conformation of the GABAA receptor occurs with increasing concentrations of GABA and/or beta-carboline. Both conformations maintain positive heterotropic cooperativity with beta-carboline binding sites, one responsible for positive and the other responsible for negative effects of beta-carboline agonists on chloride uptake.


Jaw muscle activity, the nucleus accumbens, and dopaminergic agonists: a new approach. A method was developed to analyze electromyographic (EMG) signals in terms of power, viz., a measure for overall muscle activity, and number of seconds marked by distinct frequency ranges. With the help of this method, the effects of intraaccumbens administration of distilled water, the D1 receptor agonist SK&F 38393 (SKF; 5 micrograms), the D2 receptor agonist LY 171555 (LY; 10 micrograms), and their combination upon the EMG signals of the masseter and the digastric muscle were analysed in freely moving rats. Only the combined treatment affected the power: The noted increase was limited to the digastric muscle. The time/frequency analysis was limited to frequency ranges 3-4 Hz (class A), 4-5 and 5-6 Hz (class B), and 6-7, 7-8, ..., 12-13, and 13-14 Hz (class C). Apart from a small effect of SKF alone and of SKF in combination with LY on class B of the masseter muscle, neither SKF nor LY affected class A or B. SKF and LY increased and decreased, respectively, class C in both muscles. The data suggest that SKF and LY elicited both opposite and synergistic effects. The method is a new tool to analyze EMG signals in freely moving rats.


Presynaptic dopaminergic inhibition of the spinal reflex in rats. Dopaminergic influence on spinal monosynaptic transmission was examined in rats. Monosynaptic mass reflex (MMR) was recorded from the ventral root L6 following supramaximal stimulation (0.2 Hz; 0.1 ms) to the ipsilateral dorsal root L6 in spinalized rat under pentobarbitone sodium (40 mg/kg, i.p.) anaesthesia. MMR was inhibited by intravenous administration of the dopaminergic agonist, apomorphine (50-200 ug/kg) in a dose-dependent manner. The attenuatory effect of apomorphine (200 ug/kg i.v.) on the reflex could be reversed by the dopaminergic antagonist haloperidol (0.5 mg/kg, i.v.). Under tetanic stimulation (200 Hz; 15s), the pretetanic relative inhibition induced by apomorphine (200 ug/kg, i.v.) was increased only for a short period immediately after the cessation of tetanic stimulation. The results indicate existence of presynaptic dopamine receptors on the afferent terminals converging on the motoneurone which may functionally modulate the spinal motor output.


[High level of tumor necrosis factor alpha activity of plasma and serum from acute phase hemorrhagic fever with renal syndrome]. The level of TNF-alpha activity of plasma and serum from HFRS acute phase patients was investigated. The level of TNF (tumor necrosis factor) activity of plasma and serum from acute phase patients was higher than that of plasma and serum from convalescent phase patients or from normal group. The higher TNF activity of plasma or serum from acute phase patients was due to TNF-alpha, because the TNF activity was almost completely inhibited by polyclonal mouse serum against rHuTNF-alpha. Also, the increase in TNF-alpha positive ratio of PBMC from acute phase HFRS patients was found as compared to convalescent phase patients or to normal group. All these results strongly suggest that high level of TNF-alpha activity of plasma and serum may be involved in the development of clinic symptoms and pathological lesions during the course of HFRS disease.


Immunohistochemical identification of P-glycoprotein in previously untreated, diffuse large cell and immunoblastic lymphomas. Expression of P-glycoprotein has been linked to multidrug resistance in cancer cell lines and human tumors. We investigated the frequency and clinical significance of P-glycoprotein immunoreactivity in 57 previously untreated diffuse large cell and immunoblastic lymphomas. Banked frozen tissue, which had been obtained prior to chemotherapy, was tested for reactivity with 2 monoclonal antibodies (MRK16 and C219) that recognize different domains of P-glycoprotein, using an immunoperoxidase technique. Thirteen of 57 lymphomas (23%) showed strong staining of greater than 50% of neoplastic cells; 15 of 57 (26%) showed labeling of a minority (11-50%) of neoplastic lymphocytes; 14 of 57 (25%) yielded equivocal results (reactivity in less than 10% of cells); and 15 of 57 (26%) were negative for P-glycoprotein. The 2 monoclonal antibodies were comparable in reactivity. Expression of MDR-1 mRNA was determined in 6 cases with sufficient available tissue, and did not correlate well with the percentages of cells reactive for P-glycoprotein by immunohistochemistry. Thirty-nine of our 57 patients completed multiagent chemotherapy. Contrary to our expectations, we found that P-glycoprotein immunoreactivity did not decrease the likelihood of response to induction chemotherapy. Median survival also was not adversely affected.


Taxol blocks processes essential for prostate tumor cell (PC-3 ML) invasion and metastases. We have examined the antimetastatic effects of taxol on a PC-3 human prostatic tumor variant (PC-3 ML) which metastasizes to the lumbar vertebrae in severe combined immunodeficiency-carrying (SCID) mice. Immunofluorescence labeling indicated that taxol (0.5 to 1.0 microM for 6 h) produced an abnormal bundling of microtubules in a dosage-dependent manner. Slot blotting and gelatinase assays revealed that taxol inhibited secretion of the M(r) 72,000 and M(r) 92,000 type IV collagenases plus a M(r) 57,000 gelatinase. Radioimmunoprecipitation measurements confirmed that the drug inhibited both the secretion and the synthesis of the M(r) 72,000 collagenase. Taxol also blocked total protein secretion but did not influence total protein synthesis or turnover. Boyden chamber chemotactic studies further showed that taxol (0.5 to 1.0 microM) inhibited invasion of Matrigel. More importantly, studies in SCID mice demonstrated that taxol (50 to 250 mg/m2/day) blocked the establishment, growth, and long-term survival of PC-3 ML cells.


Atherosclerosis in internal mammary arteries selected for coronary artery bypass grafting. Atheromas are infrequent in the internal mammary artery (IMA) which is now the conduit of choice for coronary artery bypass grafting (CABG). Described are two cases of significant atherosclerotic stenosis near the distal ends of IMAs selected for CABG.


Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil. Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.


Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt, Triturus alpestris (Urodela). Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA- and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, originating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.


Gastrulation in the mouse: the role of the homeobox gene goosecoid. Mouse goosecoid is a homeobox gene expressed briefly during early gastrulation. Its mRNA accumulates as a patch on the side of the epiblast at the site where the primitive streak is first formed. goosecoid-expressing cells are then found at the anterior end of the developing primitive streak, and finally in the anteriormost mesoderm at the tip of the early mouse gastrula, a region that gives rise to the head process. Treatment of early mouse embryos with activin results in goosecoid mRNA accumulation in the entire epiblast, suggesting that a localized signal induces goosecoid expression during development. Transplantation experiments indicate that the tip of the murine early gastrula is the equivalent of the organizer of the amphibian gastrula.


Structure and expression of a plastid-encoded groEL homologous heat-shock gene in a thermophilic unicellular red alga. A gene homologous to the E. coli groEL locus was identified on the plastid genome of the unicellular red alga Cyanidium caldarium strain 14-1-1 (synonym: Galdieria sulphuraria). The complete nucleotide sequence was determined and compared to bacterial- and nuclear-encoded counterparts of higher plants. At the amino-acid level the C. caldarium gene shows 70% homology to the corresponding gene of the cyanobacterium Synechococcus and 52% homology to nuclear-encoded counterparts of higher plants, respectively. Northern and Western blot experiments were used to investigate the dependence of the transcript- and protein-level on culture temperature and heat shock.


Gonadotrophin secretion in vitro by cells of a pituitary tumour from a patient with multiple endocrine neoplasia type I. The aim was to investigate the hormone secretory products of a pituitary tumour from a patient with multiple endocrine neoplasia type I (MEN I) utilizing cell culture and immunoassay techniques. Adenoma tissue was enzymically dispersed and established in cell culture. Medium was collected for hormone measurement after 2 days, and also after 24-hour periods during long-term culture. In addition, tissue fixed at surgery was analysed by immunocytochemistry and electron microscopy. The subject was a 59-year-old male with a clinical history characteristic of familial MEN I syndrome. Pituitary hormones in serum and culture medium were measured by fully characterized radioimmunoassays. Preoperative serum LH and FSH levels were normal, or slightly elevated, and there was a progressively blunted gonadotrophin response to GnRH throughout the 8 years prior to adenomectomy. TRH induced a small, paradoxical increase in serum gonadotrophin levels 2 weeks preoperatively. Post-operative pituitary hormone responses to standard stimulation tests showed an active normal pituitary. In vitro, the pituitary tumour cells secreted only gonadotrophins and glycoprotein hormone alpha-subunit. The fixed tumour tissue immunostained for alpha-subunit alone, and electron microscopy confirmed the presence of secretory granules with diameters of 100-280 nm. Gonadotrophin secretion continued throughout 77 days in long-term culture, but whilst LH was released at a steady rate, that of FSH transiently increased between days 29 and 48 in vitro. These data demonstrate that a pituitary tumour associated with the MEN I syndrome secreted gonadotrophins in vitro.


The use of psychotropic medications in clinical dermatology. In this article, the use of selected psychopharmacologic medications was explained in detail. If a patient who is obviously suffering from a psychiatric component of his or her disorder that can be responsive to psychopharmacotherapy refuses to accept a referral to a psychiatrist or other mental health professional, the dermatologist must decide whether to use these medications. If he or she decides to use these medications, it should always be remembered that the choice of psychopharmacologic treatment depends on the nature of the underlying psychopathology-anxiety, psychosis, depression, or compulsion-rather than on the dermatologic label such as neurotic excoriations. It is the hope of the author that more patients who fall into this interface between psychiatry and dermatology will be helped if dermatologists become more familiar with the availability and use of these medications.


Predischarge exercise echocardiography in patients with unstable angina who respond to medical treatment. The diagnostic and prognostic value of predischarge exercise echocardiography (echo) was assessed prospectively in 36 patients with unstable angina soon after stabilization on medical treatment. Two-dimensional echo was performed at rest and immediately after a symptom-limited exercise test. Patients with previous myocardial infarction, coronary revascularization, left bundle-branch block and dilated cardiomyopathy were excluded. Left ventricular regional wall motion was analyzed visually and a wall motion score index (WMSI) was derived. Patients were followed prospectively for an average period of 26 months (range 16-34 months). The study end points were a new cardiac event defined as acute myocardial infarction or a need for coronary revascularization because of a recurrence of severe medically refractory angina. Sixteen patients (44%) had positive exercise electrocardiography (ECG), while exercise echo was positive in 22 patients (61%). Of 28 patients undergoing coronary angiography, 23 had significant coronary artery disease (CAD). The sensitivity of exercise ECG in detecting CAD was 61% while the corresponding result was 83% for exercise echo. Cardiac events occurred in 21 patients (58%). Exercise ECG was positive in 12 of these patients (57%), while a positive exercise echo was found in 17 patients (81%). There were significantly more patients with positive exercise echo among patients experiencing cardiac events than among those without cardiac events (p less than 0.01). In patients with CAD, WMSI decreased significantly after exercise (p less than 0.05). Exercise WMSI was also significantly lower in patients with CAD than in those without CAD (p less than 0.02). Exercise WMSI also discriminated patients with cardiac events from those without such events (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


What's new in tocolytics.

What's new in tocolytics. Tocolytic therapy represents the final obstetric end point in the prevention of premature births. For the past two decades, betamimetic therapy has been the mainstay of tocolytic therapy. Newer agents, indomethacin and nifedipine, offer new hope in the treatment of premature labor. With the introduction of these newer agents, combination drug therapy appears to be on the horizon.


Somatostatin in gastric juice in normal subjects and patients with duodenal ulcer. Somatostatin in gastric juice was determined in normal subjects and patients with duodenal ulcer. Gel exclusion chromatography of gastric juice revealed that the main immunoreactivity existed at the position of somatostatin-14. A large amount of somatostatin was present in gastric juice, and the quantity increased following tetragastrin stimulation. Furthermore, there was a good inverse correlation between somatostatin concentration and acidity of gastric juice; however, there was no difference between normal subjects and patients with duodenal ulcer in the amount of somatostatin released into gastric juice.


Effects of oleic acid and endogenous bile on duodenal secretion of somatostatin in man. We studied the effects of intraduodenal oleic acid on the release of somatostatin to plasma and the correlation between endogenous bile output and plasma somatostatin. In five normal persons infusion of 0, 5, 10, 20, and 40 mM oleic acid dose-dependently increased the levels of somatostatin during as well as after gallbladder emptying. The difference between somatostatin concentration during and after gallbladder emptying was not significant. The amylase secretion also was significantly correlated to the dose of fat, whereas the output of bile salts was the same for all fat doses used. Our observations indicate that intraduodenal oleic acid--and not bile salts--releases somatostatin from the gut.


Effect of portacaval anastomosis on glutamine synthetase activities in liver, brain, and skeletal muscle. Glutamine synthetase is responsible for the ATP-dependent amidation of glutamate to glutamine. In liver the enzyme is highly localized in perivenous hepatocytes; in brain the enzyme is localized in astrocytes. Portacaval anastomosis resulted in liver atrophy, hyperammonemia, and up to 90% loss of glutamine synthetase activity in liver homogenates. This effect, which appears to be irreversible, probably reflects the selective loss of perivenous hepatocytes following portacaval anastomosis. Glutamine synthetase activities in brain were unaffected by portacaval anastomosis of up to 12 weeks' duration. Enzyme activities in homogenates of skeletal muscle, on the other hand, were significantly increased at one and four weeks after shunt surgery. These effects were not the result of decreased food intake in shunted animals. These findings suggest fundamentally different regulatory mechanisms for glutamine synthetase in these tissues. Skeletal muscle may thus provide an important alternative site for ammonia detoxification after portal-systemic shunting.


Influence of P-4502E1 induction on benzene metabolism in rat hepatocytes and on biliary metabolite excretion. The influence of P-4502E1 induction on the metabolite pattern of benzene was studied in hepatocytes in vitro and in bile in vivo, and compared with that obtained with phenol (the major benzene metabolite). Eight metabolites from benzene and four from phenol (including conjugates) represented over 90% of total metabolites. Benzene metabolism (0.1 mM) in hepatocytes from isopropanol-treated rats (2.5 ml/kg, orally) was 3-fold higher than in corresponding cells from control rats, primarily because of increased formation of hydroquinone and phenylglutathione. Immunoblotting of microsomes revealed a parallel induction of P-4502E1 in hepatocytes from isopropanol-treated rats. In contrast, treatment with 3-methylcholanthrene or phenobarbital caused a decrease of P-4502E1, together with reduced benzene metabolism at 0.01 mM benzene. Addition of isoniazid (5 mM) resulted in a strong inhibition of benzene and phenol metabolism. Benzene metabolites were determined in bile following intraperitoneal administration of benzene (2.5 and 150 mg/kg). Biliary benzene metabolites were increased 2- to 3-fold after isopropanol treatment. Hydroquinone sulfate was identified as a major biliary metabolite of phenol. The results suggest that treatment with inducers of P-4502E1 leads, even at low benzene exposure, to an increased release of potentially myelotoxic metabolites from liver into the systemic circulation.


Identification of the antibiotic hops component, colupulone, as an inducer of hepatic cytochrome P-4503A in the mouse. A higher level of cytochrome P-450 (P450)-dependent ethylmorphine (EM) N-demethylase activity was observed in hepatic microsomes from mice fed a natural-ingredient diet ("crude diet") than in those from mice fed a semi-purified diet ("purified diet"). This led to the testing of individual ingredients of the crude diet as inducers of the P-450 system. Brewers yeast proved to be the most significant inductive component of the crude diet. Further investigation revealed that hop components (lupulones) absorbed on yeast during the brewing process were responsible for the induction of the P-450 system. The induction of P-450 and several P-450-dependent monooxygenase activities (EM N-demethylation, aniline hydroxylation, benzo[a]pyrene hydroxylation) by colupulone with respect to dose and time course were investigated. The very large increase in EM N-demethylase activity elicited by colupulone suggested that P-4503A had been induced. Western blot technology verified this speculation. Western blot analysis of microsomal protein from mice fed hops, brewers yeast, or the residue of a hexane extract of hops supported the conclusion that all of these substances induced P-4503A. These substances were also relatively good inducers of P-4502B, but not as inductive of this isozyme as the crude diet. This is interpreted to mean that not all of the inductive properties of the crude diet are due to hop components. These studies question the use of crude commercial diets in studies of P-450 systems. They may also challenge some current definitions of "constitutive" and "induced" P-450s.


Disposition and pharmacokinetics of [14C]finasteride after oral administration in humans. The disposition of [14C]finasteride, a competitive inhibitor of steroid 5 alpha-reductase, was investigated after oral administration of 38.1 mg (18.4 microCi) of drug in six healthy volunteers. Plasma, urine, and feces were collected for 7 days and assayed for total radioactivity. Concentrations of finasteride and its neutral metabolite, omega-hydroxyfinasteride (monohydroxylated on the t-butyl side chain), in plasma and urine were determined by HPLC assay. Mean excretion of radioactivity equivalents in urine and feces equaled 39.1 +/- 4.7% and 56.8 +/- 5.0% of the dose, respectively. The mean peak plasma concentrations reached for total radioactivity (ng equivalents), finasteride, and omega-hydroxyfinasteride were 596.5 +/- 88.3, 313.8 +/- 99.4, and 73.7 +/- 11.8 ng/ml, respectively, at approximately 2 hr; the mean terminal half-life for drug and metabolite was 5.9 +/- 1.3 and 8.4 +/- 1.7 hr, respectively. Of the 24-hr plasma radioactivity, 40.9% was finasteride, 11.8% was the neutral metabolite, and 26.7% was characterized as an acidic fraction of radioactivity. Binding of [14C]finasteride to plasma protein was extensive (91.3 to 89.8%), with a trend suggesting concentration dependency (range 0.02 to 2 micrograms/ml). Little of the dose was excreted in urine as parent (0.04%) or omega-hydroxyfinasteride (0.4%). Urinary excretion of radioactivity was largely in the form of acidic metabolite(s)--18.4 +/- 1.7% of the dose was eliminated as the omega-monocarboxylic acid metabolite. Finasteride was scarcely excreted unchanged in feces. In humans, finasteride is extensively metabolized through oxidative pathways.(ABSTRACT TRUNCATED AT 250 WORDS)


Identification of biliary metabolites of m-dichlorobenzene in rats. At least 12 metabolites were observed in the bile of rats administered with m-dichlorobenzene in the preliminary experiments by HPLC. Some of them were assumed to be conjugates containing phenyl and dihydro-hydroxyphenyl moieties with glutathione or cysteine by enzymatic and thermal reactions. To isolate these metabolites, the bile collected from 20 rats administered with 500 mg/kg of m-dichlorobenzene and 300 mg/kg of cysteine was separated into five fractions by HPLC. Eighteen metabolites were isolated from these fractions by TLC and HPLC. The chemical structure of the major metabolite excreted was determined as trans-2,4-dichloro-6-(glutathion-S-yl)cyclohexa-2,4-dien+ ++-1-ol by 13C-NMR, 1H-NMR, FAB-MS, and some reaction experiments. The metabolite excreted in the secondary largest amount was identified as its positional isomer, trans-3,5-dichloro-6-(glutathion-S-yl)cyclohexa-2,4-dien+ ++-1-ol. Their diastereomers were also observed in the bile. trans-2,4-Dichloro-6-(cystein-S-yl)cyclohexa-2,4-dien- 1-ol and its positional isomer, which were possibly derived from glutathione conjugates above, were also identified. 3,5-Dichlorophenyl conjugates with glutathione or cysteine and 3,5-dichlorophenyl mercapturic acid, and their 2,4-dichlorophenyl isomers, were excreted. Three monochlorophenol conjugates, of which chemical structures were still not established, were present in the bile.


Methacrylonitrile: in vivo metabolism to cyanide in rats, mice, and gerbils. Methacrylonitrile (MeAN), a widely used industrial chemical, is metabolized to cyanide in rats, mice, and gerbils. Cyanide levels following oral administration of 0.5 or 1 LD50 dose of MeAN were determined in blood and organs of treated animals. Male Mongolian gerbils were 50-fold more sensitive to MeAN than Sprague-Dawley rats and about 5-fold more sensitive than were Albino-Swiss mice. The signs of MeAN toxicity were typically those of cyanide-related central nervous system poisoning in all the three species. The difference was in the time required for the onset of these signs. Mice and gerbils developed the toxicity signs faster than the rats. All the three species showed a dose-response relationship in metabolism of MeAN to cyanide. However, in mice and gerbils, the peak blood concentration of cyanide occurred 1 hr following MeAN administration, whereas it took 3 hr for rats to reach peak blood cyanide levels. In the rats treated with phenobarbital or those starved for 18 hr, the metabolism of MeAN to cyanide increased significantly (159-178% and 181-201% of control, respectively), and the treatment of rats with CoCl2 resulted in a significant decrease in MeAN metabolism to cyanide (60-68% of control). These studies indicate a distinct species difference in the toxicity and metabolism of MeAN.


Glucuronidation of the dopamine D-1 receptor antagonists NNC 0756 and NNC 0772 in liver microsomes. Glucuronidation of the two enantiomeric dopamine D-1 antagonists, NNC 0756 ([(+)-8-chloro-7-hydroxy-5-(2,3-dihydrobenzofuran-7-yl)-3-methyl- 2,3,4,5,tetrahydro-1H-3-benzazepine, acetate]) and NNC 0772 [(-)-8-chloro-7-hydroxy-5-(2,3-dihydrobenzofuran-7-yl)-3-methyl-2,3,4,5- tetrahydro-1H-3-benzazepine, HCl], was studied in rat and human liver microsomes. In rats, the reaction exhibited biphasic kinetics for both enantiomers as shown by Eadie-Hofstee plots. Both the high- and low-affinity reactions showed a high degree of stereoselectivity, primarily because of the large differences in Km values. For the high- and low-affinity reactions, the (-)-enantiomer, NNC 0772, had a 4- and 6-fold higher Km value, respectively. The difference in Vmax values were less significant, with 3.0- and 1.1-fold higher values for the (-)-enantiomer. Treatment of rats with known inducers of UDP-glucuronosyltransferases, phenobarbital, and 3-methyl-cholanthrene, did not change the kinetics of the reaction. Glucuronidation of the (+)-enantiomer, NNC 0756, was competitively inhibited in rat liver microsomes by the closely related structure, SCH 23388 [S-(-)-8-chloro-7-hydroxy-5-phenyl-3-methyl-2,3,4,5-tetrahydro-1H-3- benzazepine], with an apparent Ki value of 90 microM. Morphine and 4-hydroxybiphenyl, both known substrates of glucuronosyltransferase, did also inhibit the reaction with Ki values of 604 and 55 microM, respectively. In contrast to rats, glucuronidation of NNC 0756 in human liver microsomes followed Michaelis-Menten kinetics, suggesting the involvement of a single form of glucuronosyltransferase or possibly two forms, with similar affinity for NNC 0756.(ABSTRACT TRUNCATED AT 250 WORDS)


Stereoselective renal tubular secretion of a new uricosuric diuretic, 6,7-dichloro-5-(N,N-dimethylsulfamoyl)-2,3-dihydro-2-benzofurancarboxyl ic acid (S-8666), in cynomolgus monkeys. Plasma concentration-time curves and urinary excretion of individual enantiomers of unchanged S-8666 and its N-monodemethylated metabolite, M-1, in male cynomolgus monkeys were measured after oral administration of racemic S-8666 at doses of 5, 10, and 50 mg/kg and also after intravenous injection at doses of 1, 5, and 10 mg/kg. The Tmax values for individual enantiomers of S-8666 in fasted male monkeys were 30 min after oral administration. The AUC values for S(-)-S-8666 were greater than those for R(+)-S-8666 and oral dose-AUC relationships of both enantiomers showed a linearity over the dose range used. Most of the S-8666 and a trace of M-1 were excreted in the first 24-hr urine, with no evidence of stereoselectivity from the amounts excreted. Since large portions were recovered from the urine as unchanged S-8666 after intravenous injection, most excretion occurs via the kidney. The t1/2 beta values and the Vdss for S(-)-S-8666 after intravenous injection were smaller than those for R(+)-S-8666. The CLt and the CLr values decreased with increasing intravenous doses, indicating saturation at a renal excretion process at high plasma concentrations of S-8666. The CLt and CLr values for R(+)-S-8666 were greater than those for the S(-)-enantiomer. The unbound fraction of R(+)-S-8666 in plasma was significantly greater than that of S(-)-S-8666 [21.6% for R(+), 12.0% for S(-)]. Renal clearance for the unbound fraction of S(-)-S-8666 was greater than that of R(+)-S-8666, suggesting stereoselective renal tubular secretion.


Isolation of two immunosuppressive metabolites after in vitro metabolism of rapamycin. Rapamycin was incubated with human liver microsomes and an NADPH regenerating system, the metabolites were purified by semipreparative HPLC, and their structures were elucidated by direct chemical ionization and FAB-MS. At least six fractions were isolated containing rapamycin metabolites, indicating that rapamycin is metabolized by the human liver cytochrome P-450 system. One of these metabolites was identified as 41-O-demethyl-rapamycin. A second metabolite was hydroxylated in a yet unknown position. These two metabolites retained immunosuppressive activity in a phytohemagglutinin-stimulated human lymphocyte assay with IC50S of 1 and 1.5 nmol/liter, respectively. Rapamycin was metabolized by rat small intestinal microsomes to at least two metabolites, indicating extra-hepatic metabolism of rapamycin.


Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers. Urinary nicotine metabolic output was profiled for 11 smokers who smoked their regular cigarette brands ad libitum. Thermospray liquid chromatography/mass spectrometry was used to monitor nicotine and eight metabolites, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine that were determined indirectly using enzyme hydrolysis. These results were used to estimate an average, steady-state concentration in a 24-hr urine sample during ad libitum smoking and to assess interindividual variability in the excretion of these metabolites. The variability in absolute amount among the nine analytes ranged from 35 to 70% for these smokers. The glucuronide conjugates constituted an average of 29% of all urinary metabolites monitored in this study. trans-3'-Hydroxycotinine in the free form constitutes the largest single metabolite in smokers' urine, with an average of 35% of the total. The sums of nicotine metabolites determined here are very close to the Federal Trade Commission yields of nicotine for the total number of cigarettes smoked by these subjects during the urine collection interval. These results indicate that a large proportion of the nicotine absorbed while smoking can be accounted for as urinary metabolites of nicotine, including glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine.


Comparative metabolism and disposition of furfural and furfuryl alcohol in rats. The comparative metabolism and disposition of furfural (FAL) and furfuryl alcohol (FOL) were investigated following oral administration of approximately 0.001, 0.01, and 0.1 of the LD50, corresponding to approximately 0.127, 1.15, and 12.5 mg/kg for FAL and 0.275, 2.75, and 27.5 mg/kg for FOL. At all doses studied, at least 86-89% of the dose of FAL or FOL was absorbed from the gastrointestinal tract. FAL and FOL were extensively metabolized prior to excretion. The major route of excretion was in urine, where 83-88% of the dose was excreted, whereas 2-4% was excreted in the feces. Approximately 7% of the dose from rats treated with FAL at 12.5 mg/kg was exhaled as 14CO2. At 72 hr following administration, the pattern of tissue distribution of radioactivity was similar for both FAL and FOL. Liver and kidney contained the highest, and brain the lowest concentrations of radioactivity. Generally, the concentrations of radioactivity in tissues were proportional to the dose. Almost all of the urinary radioactivity was tentatively identified. No FAL or FOL was detected in urine. Furoylglycine was the major urinary metabolite (73-80% of dose), and furoic acid (1-6%) and furanacrylic acid (3-8%) were the minor metabolites following treatment with either FAL or FOL. Therefore, the initial step in the metabolism of FAL and FOL involves the oxidation to furoic acid, which is excreted unchanged and decarboxylated to form 14CO2, conjugated with glycine, or condensed with acetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolism of isoniazid by activated leukocytes. Possible role in drug-induced lupus. The tuberculostatic agent isoniazid has been implicated in inducing various idiosyncratic reactions including drug-induced lupus. The mechanism is unknown but may involve a reactive metabolite of the drug. Isoniazid was oxidized by activated leukocytes to isonicotinic acid. Myeloperoxidase is likely the enzyme in the leukocyte involved, since the oxidation was inhibited by azide, which inhibits myeloperoxidase, and by catalase, which catalyzes the breakdown of hydrogen peroxide. The same metabolic profile was observed when isoniazid was incubated with purified myeloperoxidase and hydrogen peroxide. The rate of the reaction was increased in the presence of chloride. Hypochlorous acid was also able to oxidize isoniazid to isonicotinic acid. Isoniazid, or an oxidative product, inhibited the reaction when high initial substrate concentrations were used. Isoniazid is oxidized by activated leukocytes, possibly to a reactive intermediate, which may have implications for isoniazid-induced lupus.


Systemic pharmacokinetics of acitretin, etretinate, isotretinoin, and acetylenic retinoids in guinea pigs and obese rats. Etretinate, a highly lipophilic retinoid, is known to accumulate in the human body with a slow systemic elimination (half-life approximately 100 days) after long-term treatment. Retinoids with high lipophilicity and slow body elimination have the propensity of eliciting teratogenic effects. Therefore, synthetic retinoids with reduced systemic retention are desired. In this study, we evaluated the systemic pharmacokinetics of acitretin, etretinate, isotretinoin, synthetic acetylenic retinoic acids (AGN 190121, AGN 190186, and AGN 190299), and acetylenic retinoates (AGN 190073, AGN 190089, and AGN 190168) in guinea pigs following iv doses. Their pharmacokinetics were also measured in obese rats to probe the effect of body fat on the drug disposition of retinoids. The acetylenic retinoates were hydrolyzed to their corresponding free acids at a much faster rate than etretinate in both animal species. All retinoates showed faster body clearance and larger volume of distribution than their free acids. In the obese rats, longer elimination half-lives and slower body clearance of the retinoids, except isotretinoin, were observed as compared to those in the normal rats. These results suggest that body fat has a significant effect on drug disposition and slows down the systemic clearance of retinoids. Since the synthetic acetylenic retinoates rapidly converted to their less lipophilic free acids after systemic absorption, the potential accumulation of these retinoids, as reported for lipophilic etretinate, were unlikely to occur in humans and animals.


Correlations between conceptal concentrations of all-trans-retinoic acid and dysmorphogenesis after microinjections of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, or retinol in cultured whole rat embryos. Retinol (4,000 ng/ml), all-trans-retinoyl-beta-glucuronide (4,000 ng/ml), and 13-cis-retinoic acid (1,500 ng/ml) each produced dysmorphogenic effects qualitatively similar to those elicited by 250 ng/ml of all-trans-retinoic acid after microinjections of the respective individual retinoids into the amniotic cavities of cultured whole rat embryos. Subsequent HPLC analyses of the cultured whole conceptuses, embryos proper, yolk sacs, and culture media (24 hr after microinjections) indicated that conceptal biotransformation of each of the retinoids had occurred during the culture period. All-trans-retinoic acid was present in the embryos proper at quantitatively similar concentrations (20-100 nM) after microinjections of the selected quantities of each of the microinjected retinoids: retinol, all-trans-retinoyl-beta-glucuronide, 13-cis-retinoic acid, or all-trans-retinoic acid. The results suggested that all-trans-retinoic acid acted as an ultimate dysmorphogen for the retinoids tested with respect to the anomalies monitored in the embryo culture system.


Human hepatic microsomal metabolism of delta 1-tetrahydrocannabinol. Hepatic microsomal metabolism of delta 1-tetrahydrocannabinol (THC) has been extensively studied in many rodent species, but there have been few reports describing such metabolism in humans. Because several THC metabolites are known to be pharmacologically active, identifying the P-450 subfamilies responsible for their formation is of clinical importance. We have found that, in addition to catalyzing the formation of significant amounts of 7-hydroxy-THC, hepatic microsomes from nine human livers also formed 6 beta-hydroxy-THC at approximately the same rate. In addition, 1 alpha,2 alpha-epoxyhexahydrocannabinol (EHHC) was formed at approximately one-third the rate of 7-hydroxy- and 6 beta-hydroxy-THC, and small amounts of 6 alpha-hydroxy- and 6-keto-THC were also found. Immunoinhibition studies with antibodies raised against human hepatic P-450 2C9, or a mouse hepatic P-450 isozyme belonging to the P-450 3A subfamily, revealed that P-450 2C9 catalyzed the formation of 7-hydroxy-THC, whereas P-450 3A catalyzed the formation of 6 beta-hydroxy-THC, EHHC, and the relatively minor metabolites. In contrast, antibodies raised against human P-450 2C8 had no affect on human microsomal THC hydroxylation. Excellent correlations were found between hepatic microsomal P-450 2C9 and 3A content and 7-hydroxy- and 6 beta-hydroxy-THC formation, respectively. In addition, purified P-450 2C9 catalyzed the formation of 7-hydroxy-THC at a 7-fold higher rate than that observed with microsomes. Microsomal 7-hydroxy-THC formation varied less than 5-fold between the livers, suggesting that this activity is normally expressed and probably not subject to environmental influences.(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolism of sodium nifurstyrenate, a veterinary antimicrobial nitrofuran, in animals and fish. Sodium nifursyrenate [beta-(5-nitro-2-furyl)-p-carboxystyrene sodium salt, NSA-Na] is an antibacterial nitrofuran which has been widely used for prevention and treatment of bacterial infections in fish in Japan. When NSA-Na was anaerobically incubated with rabbit liver cytosol and 2-hydroxypyrimidine, 1-(p-carboxyphenyl)-5-cyano-3-oxo-1,4-pentadiene (cyano-pentadienone), 1-(p-carboxyphenyl)-5-cyano-3-oxo-1-pentanone (cyano-pentanone), and 1-(p-carboxyphenyl)-5-cyano-3-pentanone (cyano-pentanone) were isolated and identified as the metabolites of the nitrofuran. In addition, when cyano-pentenone and cyano-pentanone were aerobically incubated with the liver preparation and NADPH, 1-(p-carboxyphenyl)-5-cyano-3-hydroxy-1-pentene (cyano-pentenol) and 1-(p-carboxyphenyl)-5-cyano-3-pentanol (cyano-pentanol) were also isolated and identified as the metabolites of the nitrofuran in its further metabolism, respectively. The anaerobic incubation of NSA-Na with rat liver cytosol and 2-hydroxypyrimidine resulted in the formation of cyano-pentadienone and cyano-pentanone. In this case, however, cyano-pentenone was not detectable. On the other hand, when NSA-Na was anaerobically incubated with sea bream liver cytosol and NADPH, the formation of cyano-pentenone, cyano-pentanone, and cyanopentenol, but not cyano-pentadienone, was observed. Furthermore, cyano-pentanone was metabolized to cyano-pentanol by the fish liver preparation with NADPH under aerobic conditions. When NSA-Na was given orally to rabbits, cyano-pentanone, cyano-pentenol, cyano-pentanol, and beta-(acetamido-2-furyl)-p-carboxystyrene (acetamidofuran) were identified as the urinary metabolites of the nitrofuran.(ABSTRACT TRUNCATED AT 250 WORDS)


Urinary metabolic profiles in human and rat of 1,2-dimethyl- and 1,2-diethyl-substituted 3-hydroxypyridin-4-ones. The urinary metabolic profiles of two novel orally active iron chelators, 1,2-dimethyl-3-hydroxypyridin-4-one (CP20 or L1) and 1,2-diethyl-3-hydroxypyridin-4-one (CP94), have been studied in rats. The metabolism of CP20 was also studied in humans. Four novel metabolites of CP20, and a further three metabolites of CP94 were characterized. CP20 was found to undergo extensive phase II metabolism at the 3-hydroxy position, forming predominantly the O-glucuronide, which accounted for 44% of the dose administered in rat and greater than 85% of the dose administered in man. The 3-O-methylated CP20 metabolite (metabolite I) accounted for 1% of the administered dose in both species, whereas the unmetabolized CP20 amounted to 10.5% and 4% of the dose administered in the rats and man, respectively. In contrast, CP94 was extensively hydroxylated at the 2-ethyl position to give its 2-(1-hydroxyethyl) metabolite in the rat, which accounted for 40% of the administered dose. The O-glucuronide metabolite of CP94 accounted for 13.8% of the administered dose, whereas the unmetabolized CP94 amounted to 6.9% of the administered dose. At 72 hr, urinary levels of CP20 and CP94 and their metabolites in the rat accounted for about 55-60% of the administered dose. A large portion of the dose is therefore probably eliminated via the bile. The identity of the above metabolites was established using a combination of two or more of the following techniques: fast atom bombardment-mass spectroscopy, LC-MS, UV-VIS spectroscopy, NMR spectroscopy, specific enzyme hydrolysis assays, and chemical synthesis of compounds.


A metabolic route of omeprazole involving conjugation with glutathione identified in the rat. A metabolic route of omeprazole involving glutathione has been established through identification of endproducts excreted in the urine of rats after oral administration of 400 mumol/kg of a mixture of [3H]- and [14C]omeprazole. The labeled positions enabled facile tracing of metabolites that were formed through fission of omeprazole, producing [3H]pyridine and [14C]benzimidazole metabolites. The structures of the metabolites were established by HPLC thermospray MS and MS/MS. Two of the metabolites were isolated and characterized by 1H NMR studies. The fact that the N-acetylcysteine derivative of the benzimidazole was one of the endproducts indicated that the initial reaction involved glutathione. Three metabolites reflecting the fate of the pyridine moiety were identified. Their proposed formation route is via initial reduction to the pyridylmethylthiol compound followed by S-methylation and S-oxidation to the corresponding sulfoxide or sulfone. The quantity of metabolites formed via the glutathione route identified in urine was about 10% of the dose given, both in male and female rats. The male and female rats excreted the same cleaved metabolites and approximately equal quantities thereof.


Stereoselective acetonyl side chain reduction of warfarin and analogs. Partial characterization of two cytosolic carbonyl reductases. The reductase activity mediating the ketone reduction of the acetonyl side-chain of warfarin and analogs has been partially purified from rabbit liver cytosol. The reductase activity is resolved in two different fractions (A and B) by DEAE-Sephacel chromatography. Both fractions reduce the acetonyl group of warfarin analogs with marked substrate (R-enantiomers), as well as product stereoselectivity (alcohols of the S-configuration). The reductases are NADPH-dependent, which is absolute for fraction B. The enzyme kinetics of the R-enantiomers of warfarin and three 4'-derivatives (4'-nitro-, 4'-chloro-, and 4'-methoxywarfarin) has been investigated. In contrast to fraction B, fraction A is sensitive in its KM for 4' substitution: the KM values of 4'-nitro- and chloro-analogs are approximately 6 times lower than the KM values of the 4'-methoxy analog or warfarin itself. On the other hand, the Vmax values of fraction A are all in the range of about 1 to 2 nmol/mg x min, whereas the Vmax values of fraction B vary from about 1(4'-methoxywarfarin) to 12 (the 4'-nitro analog). The intrinsic activities (Vmax/KM) of both enzymes show the same rank order: 4'-nitro greater than 4'-chloro greater than 4'-methoxy = warfarin. Warfarin reductase activity of both enzymes is not inhibited by pyrazole, sodium barbitone, or dicoumarol, but is strongly inhibited by quercetin, indomethacin, furosemide, and prostaglandin E2 (PGE2). In addition, fraction A is inhibited by menadione and androsterone; fraction B is inhibited by estrone. Various compounds were tested as substrates for these enzyme fractions.(ABSTRACT TRUNCATED AT 250 WORDS)


Availability of glycine and coenzyme A limits glycine conjugation in vivo. Using benzoic acid as substrate, this study tested the hypothesis that capacity limitation of glycine conjugation in vivo is due to substrate-induced depletion of hepatic cosubstrates (i.e., ATP, coenzyme A, and glycine) utilized in the conjugation reaction. Benzolyglycine formation was investigated by following the disappearance of benzoic acid from blood and appearance of benzoylglycine in blood and urine after administration of sodium benzoate (0.2-2 mmol/kg, iv) to anesthetized rats whose urine formation was stimulated by mannitol administration. Capacity limitation of glycine conjugation is indicated by (a) the gradual dose-dependent reduction of benzoate blood clearance from 39 ml/min/kg at 0.2 mmol/kg benzoate to 3.7 ml/min/kg at 2 mmol/kg, and (b) the tendency to attain maximal blood levels and urinary excretion rates of benzoylglycine after administration of 0.5-1 mmol/kg benzoate. The maximal urinary excretion rate of benzoylglycine after benzoylglycine administration exceeded the maximal excretion rate of endogenously formed benzoylglycine (approximately 5 mumol/kg/min) 5-fold. This suggests that the urinary excretion rate of endogenously formed benzoylglycine reflects the rate of its formation. Benzoate depleted hepatic glycine (to 40%) and coenzyme A (to 14%) in a dose-dependent fashion; however, it did not change ATP levels in liver. The pattern of this dose-dependent cosubstrate depletion suggests that benzoate primarily causes consumption of hepatic glycine which, at high substrate dosage, leads to marked depletion of coenzyme A in the liver. Thus, these observations indicate that capacity-limited glycine conjugation may be due to limited availability of glycine and coenzyme A for the conjugation process.


Human hepatic microsomal thiol methyltransferase. Assay conditions, biochemical properties, and correlation studies. Thiol methyltransferase (TMT) catalyzes the S-methylation of aliphatic sulfhydryl drugs and xenobiotic compounds. As a first step in the determination of whether genetic control of TMT activity in an easily accessible human cell, the red blood cell (RBC), might reflect the regulation of TMT in the human liver, we determined optimal conditions for the assay of human hepatic microsomal TMT activity. We then used those assay conditions to study the biochemical properties and regulation of TMT in the human liver for comparison with the properties of TMT in the human RBC. Substrate kinetic studies of hepatic microsomes performed with 2-mercaptoethanol (2-ME) revealed biphasic kinetics similar to those found in the human RBC, with apparent "high-" and "low"-affinity forms of TMT activity. The high-affinity form had an optimal pH of 7.2-7.6 and apparent KM values of 9.0 microM for 2-ME and 7.5 microM for S-adenosyl-L-methionine. The low-affinity form had an optimal pH of 8.8 and apparent KM values of 20 mM for 2-ME and 44 microM for S-adenosyl-L-methionine. Both forms were inhibited by compounds that also inhibited human RBC membrane TMT. The two kinetic forms of hepatic microsomal TMT activity were inactivated approximately 50% by heating for 15 min at 53 degrees C and, as was found with RBC TMT, had very similar thermal stability profiles.(ABSTRACT TRUNCATED AT 250 WORDS)


Guinea pig and rat hepatic microsomal metabolism of monocrotaline. The comparative metabolism of the pyrrolizidine alkaloid, [14C]monocrotaline, was studied using rat and guinea pig hepatic microsomes. Metabolites were quantified to the nanomole level using HPLC and radiometric detection. Triorthocresylphosphate and carbon monoxide were used to assess the involvement of carboxylesterases and cytochrome P-450 in the hepatic microsomal metabolism of monocrotaline, respectively. Esterase hydrolysis accounted for 92% of the metabolism in the guinea pig; the rat displayed no esterase activity. This result may explain the guinea pig's resistance to pyrrolizidine alkaloid toxicity. Dehydropyrrole was found to be the major pyrrolic metabolite in the guinea pig, although colorimetric analysis indicated multiple pyrrolic moieties in the rat microsomal incubations.


The metabolism of DuP 753, a nonpeptide angiotensin II receptor antagonist, by rat, monkey, and human liver slices. The in vitro metabolism of DuP 753, a novel nonpeptide angiotensin II receptor antagonist, has been investigated in incubations with liver slice preparations from rats, monkeys and humans. Metabolites were identified by HPLC/MS, FAB/MS, Cl/MS, and/or 1H NMR. In the rat, the primary route of metabolism was oxidative, leading to either monohydroxylated or oxidized (carboxylic acid) metabolites, whereas in monkeys, glucuronidation of the tetrazole moiety predominated. An equal mixture of both oxidized and glucuronic acid-conjugated metabolites was isolated from incubations with human liver slices. All metabolites were tested in an in vitro assay to determine their activity as angiotensin II receptor antagonists. The monohydroxylated products and glucuronic acid conjugates were determined to be much less active than DuP 753. Biotransformation to the carboxylic acid, however, was shown to dramatically increase the activity of this agent. The in vivo duration of action of DuP 753 has been observed to be much longer in the rat than in the monkey. This may be explained, at least in part, by these in vitro metabolism studies. The predominance of glucuronidation observed in incubations with monkey liver slices would yield metabolites with diminished activity and might be expected to shorten the in vivo duration of DuP 753 in that species. The oxidative conversion to the carboxylic acid metabolite, along with the low level of glucuronidation observed in incubations with rat liver slices, may be responsible for the prolonged duration observed in vivo in the rat.


Metabolic fate of menthofuran in rats. Novel oxidative pathways. Metabolic fate of menthofuran (II) in rats was investigated. Menthofuran (II) was administered orally (200 mg/kg of the body weight/day) to rats for 3 days. The following metabolites were isolated from the urine of these animals: p-cresol (VI), 5-methyl-2-cyclohexen-1-one (VII), 3-methylcyclohexanone (VIII), 3-methylcyclohexanol (IX), 4-hydroxy-4-methyl-2-cyclohexen-1-one (V), geranic acid (XI), neronic acid (XII), benzoic acid (XIII), and 2-[2'-keto-4'-methylcyclohexyl]propionic acid (X). Incubation of menthofuran (II) with phenobarbital-induced rat liver microsomes in the presence of NADPH and oxygen resulted in the formation of a metabolite tentatively identified as 2-Z-(2'-keto-4'-methylcyclohexylidene)propanal (III; alpha,beta-unsaturated-gamma-keto-aldehyde). The structure assigned was further supported by trapping this metabolite (III) as a cinnoline derivative. Phenobarbital-induced rat liver microsomes also converted 4-methyl-2-cyclohexenone (IV) to 4-hydroxy-4-methyl-2-cyclohexenone (V) and p-cresol (VI) in the presence of NADPH and oxygen. On the basis of both in vivo and in vitro studies, a possible mechanism for the formation of p-cresol from menthofuran has been proposed.


The influence of vitamin K3 treatment on the pharmacokinetics and metabolism of (+)-propranolol in the rat. The effects of vitamin K3 treatment on the pharmacokinetics and metabolism of (+)-propranolol and the consequences of hepatic injury associated with vitamin K3 treatment were examined in groups of male Sprague-Dawley rats. When vitamin K3 (20 mg/kg) in polyethylene glycol 300 (PEG 300) was coinfused with (+)-propranolol (2 mg/kg) into the pyloric vein (a tributary flowing directly into the hepatic portal vein), a significant decrease in the intrinsic clearance of total drug (CLint) from 94.1 +/- 50.1 to 32.9 +/- 11.5 ml/min/kg was observed (p less than 0.01 vs. vehicle control). However, a lower dose of vitamin K3 (2 mg/kg in PEG 300) had little effect on this parameter. Interestingly, the PEG 300 vehicle control group exhibited a significantly (p less than 0.05) higher CLint than that observed in a saline control group (94.1 +/- 50.1 vs. 45.9 +/- 13.7 ml/min/kg). This difference appeared to be due to an increase in the free fraction of propranolol caused by PEG 300, because in vitro addition of this solvent to serum (at estimated in vivo concentrations) with or without added vitamin K3 doubled propranolol free fraction. Furthermore, rats that received the high dose vitamin K3 (20 mg/kg) treatment exhibited a pronounced increase in the serum concentration of enzymes of hepatic origin (alanine aminotransferase and sorbitol dehydrogenase) and in the incidence of hepatic necrosis. It was also observed that high-dose vitamin K3 treatment caused only minor changes in the urinary recovery of propranolol metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)


Pharmacokinetics of caffeine and its demethylated metabolites in lactating adult rabbits and neonatal offspring. Predictions of breast milk to serum concentration ratios. The purpose of this study was to assess the pharmacokinetics of caffeine and its metabolites in the lactating rabbit and suckling pup. The ability of a diffusional model to predict milk-to-serum drug concentration ratios (M/S) observed in vivo from in vitro experiments was established. The distribution into milk of caffeine, paraxanthine, theobromine, and theophylline was measured in lactating New Zealand White rabbits following an iv bolus dose of caffeine (5 mg/kg). M/S ratios were determined in vivo (M/Sobs; caffeine = 0.875 +/- 0.052; paraxanthine = 0.358 +/- 0.019; theobromine = 0.829 +/- 0.038; and theophylline = 0.412 +/- 0.054) under single dose conditions using area under the milk and serum concentration-time profiles. Predicted M/S values (M/Spred; caffeine = 0.797 +/- 0.040; paraxanthine = 0.316 +/- 0.029; theobromine = 0.692 +/- 0.062; and theophylline = 0.385 +/- 0.039) were calculated from in vitro measurements of the unbound fractions of drug in skim milk and serum (fm and fs, respectively), the skim-to-whole milk drug concentration ratio (S/W), milk and serum pH, and the pKa of the model compound. The pharmacokinetic profile of caffeine in the suckling pup following iv bolus administration (5 mg/kg) was more prolonged compared with adult rabbits. The mean systemic clearance of total caffeine (CIs) in the adults and the pups was 3.83 +/- 1.94 and 1.14 +/- 0.80 ml/min/kg, respectively. The mean unbound systemic clearance (CIs,u) for caffeine was 5.09 +/- 2.60 ml/min/kg in the adults and 1.41 +/- 0.71 ml/min/kg in the pups.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha-glucoside formation of xenobiotics by rat liver alpha-glucosidases. We investigated enzymes participating in alpha-glucoside formation, a novel metabolic pathway of xenobiotics in a metabolic study of indeloxazine hydrochloride in rats. When rat tissue homogenates and the indeloxazine metabolite trans-4-(2-morpholinylmethoxy)-1,2-indandiol (M-2) were incubated, M-2-alpha-glucoside formation was observed in liver. This reaction was almost completely inhibited by the alpha-glucosidase inhibitor acarbose. The liver homogenate was then separated into subcellular fractions and an acid alpha-glucosidase in lysosomes and two neutral alpha-glucosidases in microsomes and cytosol were partially purified. The chromatographic behavior and optimum pH of the glucosyltransferase activity of each of the enzyme preparations were almost identical with those of alpha-glucosidase (hydrolase) activity of the same specimen, suggesting the former activity to be also due to alpha-glucosidase. Agreeing with their hydrolytic substrate specificities, the acid enzyme transferred glucose to M-2 from a series of glucose derivatives, ranging from low molecular maltosaccharides to high molecular glycogen, whereas the neutral enzymes took only low molecular maltosaccharides as glucosyl donors. These results led to the conclusion that the formation of alpha-glucoside conjugates is catalyzed by more than one alpha-glucosidase in the liver and uses maltosaccharides or glycogen as glucosyl donors. Several other diol structure-bearing compounds were found in vitro to give rise to alpha-glucoside conjugates, and the mechanism of alpha-glucoside formation is discussed.


Involvement of the cytochrome P-450IID subfamily in minaprine 4-hydroxylation by human hepatic microsomes. 4-Hydroxylation of minaprine was measured on microsomal fractions prepared from 25 different human liver samples. In vitro formation of 4-hydroxyminaprine exhibited a large interindividual variability. Indeed, minaprine 4-hydroxylase activity ranged between 0.033 and 0.421 nmol/min/mg microsomal protein. Two samples presented a particularly low enzyme activity. Minaprine 4-hydroxylation followed Michaelis-Menten kinetics with KM and Vmax values of 5.26 microM and 0.478 nmol/min/mg microsomal protein, respectively, for one particular representative sample. The effects of various compounds (substrates or inhibitors of cytochrome P-450 isoforms) on 4-hydroxyminaprine formation were investigated. Selective substrates for P-450IA [benzo(a)pyrene, theophylline, and phenacetin], IIC (hexobarbital), IIE (aniline), and IIIA (erythromycin, nifedipine, and troleandomycin) cytochrome subfamilies did not inhibit 4-hydroxyminaprine formation. The nonspecific cytochrome P-450 inhibitor, cimetidine, slightly inhibited minaprine 4-hydroxylation. The classical substrates of the P-450IID cytochrome subfamily (debrisoquine, propranolol, and sparteine) inhibited minaprine 4-hydroxylation, as did the known P-450IID specific inhibitor, quinidine. These compounds inhibited minaprine 4-hydroxylase with Ki values of 16.5 (debrisoquine), 14.4 (propranolol), 61.9 (sparteine), and 0.146 microM (quinidine). 4-Hydroxyminaprine formation rate was shown not to be correlated with the activity of both erythromycin N-demethylase (r = 0.29, non-significant) and aniline hydroxylase (r = -0.15, NS). In contrast, minaprine 4-hydroxylase was well correlated with both debrisoquine 4-hydroxylase activity (r = 0.501, p less than 0.05) and immunoquantified cytochrome P-450IID6 (r = 0.579, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Metabolic inversion of (R)-ibuprofen. Epimerization and hydrolysis of ibuprofenyl-coenzyme A. Ibuprofen [(racemic)2-(4-isobutylphenyl)propionic acid] has been proposed but not directly demonstrated to undergo unidirectional inversion from the (R)- to the (S)-configuration via a coenzyme A (CoA) thioester intermediate. Chemically synthesized (R)- and (S)-ibuprofenyl-CoA, and rat and human liver homogenates were used to investigate the relative rates of ibuprofenyl-CoA epimerization and hydrolysis. Rat whole liver homogenate completely epimerized (R)- or (S)-ibuprofenyl-CoA, whereas hydrolysis of this intermediate occurred at a much slower rate. Rat liver mitochondria was the most efficient at both epimerizing and hydrolyzing ibuprofenyl-CoA, whereas rat liver microsomes hydrolyzed ibuprofenyl-CoA at a rate similar to whole liver homogenate but had very little epimerization activity. Rat liver cytosol was the poorest at hydrolyzing ibuprofenyl-CoA but had substantial epimerization capability. Whole liver homogenate from human tissue was less efficient at epimerizing but as efficient at hydrolyzing ibuprofenyl-CoA as rat whole liver homogenate. No stereoselectivity of either epimerization or hydrolysis was noted for any of the enzyme preparations studied. This study demonstrates that the inversion of (R)-ibuprofen occurs, at least in part, via the epimerization of the metabolic intermediate, ibuprofenyl-CoA, in both rat and human liver tissues.


Molecular analysis of the deletion mutants in the E homeotic complex of the silkworm Bombyx mori. The E loci in Bombyx mori are expected to contain a homeotic gene complex specifying the identities of the larval abdominal segments. However, the molecular structure of this complex remains to be determined. We have started to analyze the structural changes in the E complex mutations. We used three newly isolated Bombyx homeobox genes as probes. These genes are probably homologues of the Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) in the Drosophila bithorax complex, because the amino-acid sequences of the homeobox regions in these Bombyx genes are almost identical to those of Drosophila genes. We found that the Bombyx Ubx and abd-A genes are deleted in the EN chromosome, and the Bombyx abd-A gene is deleted in the ECa chromosome. From these results, we conclude that the Bombyx E complex consists of the Ubx, abd-A and possibly Abd-B genes, which may play similar roles to their homologues in the Drosophila bithorax complex.


SUPERMAN, a regulator of floral homeotic genes in Arabidopsis. We describe a locus, SUPERMAN, mutations in which result in extra stamens developing at the expense of the central carpels in the Arabidopsis thaliana flower. The development of superman flowers, from initial primordium to mature flower, is described by scanning electron microscopy. The development of doubly and triply mutant strains, constructed with superman alleles and previously identified homeotic mutations that cause alterations in floral organ identity, is also described. Essentially additive phenotypes are observed in superman agamous and superman apetala2 double mutants. The epistatic relationships observed between either apetala3 or pistillata and superman alleles suggest that the SUPERMAN gene product could be a regulator of these floral homeotic genes. To test this, the expression patterns of AGAMOUS and APETALA3 were examined in superman flowers. In wild-type flowers, APETALA3 expression is restricted to the second and third whorls where it is required for the specification of petals and stamens. In contrast, in superman flowers, APETALA3 expression expands to include most of the cells that would normally constitute the fourth whorl. This ectopic APETALA3 expression is proposed to be one of the causes of the development of the extra stamens in superman flowers. The spatial pattern of AGAMOUS expression remains unaltered in superman flowers as compared to wild-type flowers. Taken together these data indicate that one of the functions of the wild-type SUPERMAN gene product is to negatively regulate APETALA3 in the fourth whorl of the flower. In addition, superman mutants exhibit a loss of determinacy of the floral meristem, an effect that appears to be mediated by the APETALA3 and PISTILLATA gene products.


Sequence and expression pattern of pax-6 are highly conserved between zebrafish and mice. Despite obvious differences in the patterns of early embryonic development, vertebrates share a number of developmental mechanisms and control genes, suggesting that they use similar genetic programs at some stages of development. To examine this idea, we isolated and characterized one such gene, pax-6, a member of the pax gene family, from the zebrafish Brachydanio rerio and determined the evolutionary conservation in the structure and expression of this gene by comparison to its homolog in mice. We found two alternatively spliced forms of the zebrafish pax-6 message. Sequence and expression pattern of the zebrafish pax-6 gene are remarkably similar to its murine homolog. pax-6 expression begins during early neurulation. A stripe of cells in the neuroectoderm, including the prospective diencephalon and a part of the telencephalon, expresses pax-6 as well as the hindbrain and the ventral spinal cord extending from the level of the first rhombomere to the posterior end of the CNS. During later development more limited regions of the brain including the eye, the olfactory bulb and the pituitary gland express pax-6. Cells at the midbrain-hindbrain junction express eng genes and are separated from the neighboring pax-6 regions by several cells that express neither gene, indicating a complex subdivision of this region. pax-6 expression appears during processes when cell-to-cell signalling is thought to be important, for example during induction of the eye and regionalization of the spinal cord and brain, suggesting that it may be one component mediating the response to inductive interactions.


An early marker of axial pattern in the chick embryo and its respecification by retinoic acid. Chick Ghox 2.9 protein, a homeodomain-containing polypeptide, is first detected in the mid-gastrula stage embryo and its levels increase rapidly in the late gastrula. At this time, the initially narrow band of expression along the primitive streak expands laterally to form a shield-like domain that encompasses almost the entire posterior region of the embryo and extends anteriorly as far as Hensen's node. We have found that this expression domain co-localizes with a morphological feature that consists of a stratum of refractile, thickened mesoderm. Antibody-staining indicates that Ghox 2.9 protein is present in all cells of this mesodermal region. In contrast, expression within the ectoderm overlying the region of refractile mesoderm varies considerably. The highest levels of expression are found in ectoderm near the streak and surrounding Hensen's node, regions that recent fate mapping studies suggest that primarily destined to give rise to neurectoderm. At the definitive streak stage (Hamburger and Hamilton stage 4) the chick embryo is especially sensitive to the induction of axial malformations by retinoic acid. Four hours after the treatment of definitive streak embryos with a pulse of retinoic acid the expression of Ghox 2.9 protein is greatly elevated. This ectopic expression occurs in tissues anterior to Hensen's node, including floor plate, notochord, presumptive neural plate and lateral plate mesoderm, but does not occur in the anteriormost region of the embryo. The ectopic induction of Ghox 2.9 is strongest in ectoderm, and weaker in the underlying mesoderm. Endoderm throughout the embryo is unresponsive. At stage 11, Ghox 2.9 is normally expressed at high levels within rhombomere 4 of the developing hindbrain. In retinoic-acid-treated embryos which have developed to this stage, typical rhombomere boundaries are largely absent. Nevertheless, Ghox 2.9 is still expressed as a discrete band, but one that is widened and displaced to a more anterior position.


Function of an Ultrabithorax minigene in imaginal cells. An Ultrabithorax (Ubx) minigene constructed from three key Ubx control regions is capable of supporting development of Ubx null mutants throughout larval life and beyond to pharate flies, thereby rescuing the larval lethality due to the homeotic mutation. The cuticle of these flies shows that the minigene provides at least partial Ubx function in each of the four compartments whose morphogenetic pathways are determined by Ubx. We analyse beta-galactosidase patterns in imaginal discs conferred by each individual Ubx control region. From the comparison of these patterns with Ubx expression in Cbx mutants, we infer that long-range repressor elements in the chromosomal Ubx gene play an important role in the generation of Ubx expression patterns in imaginal discs. Expression and function of our Ubx minigenes indicate that Ubx control regions are capable of functioning properly out of context and detached from their normal chromosomal location within the homeotic gene complex.


The mouse has a Polycomb-like chromobox gene. The Drosophila gene Polycomb (Pc) has been implicated in the clonal inheritance of determined states and is a trans-regulator of the Antennapedia-like homeobox genes. Pc shares a region of homology (the chromobox) with the Drosophila gene Heterochromatin Protein 1 (HP1), a component of heterochromatin. The Pc chromobox has been used to isolate a mouse chromobox gene, M33, which encodes a predicted 519 amino acid protein. The M33 chromodomain is more similar to that in the Pc protein, than that in the HP1 protein. In addition to the chromodomain, the M33 and Pc proteins also share a region of homology at their C termini. The temporal and spatial expression patterns of M33 have been studied by in situ hybridization and northern analysis. During the final 10 days of embryonic development, M33 expression mirrors that of the cell-cycle-specific cyclin B gene. It is therefore suggested that the rate of cellular proliferation controls M33 expression. From comparisons of the characteristics of M33 with those of Pc it is proposed that M33 is a Pc-like chromobox gene. The roles of M33 and Pc in models of cellular memory are examined and implications of the memory models addressed.


The mis-expression of posterior Hox-4 genes in talpid (ta3) mutant wings correlates with the absence of anteroposterior polarity. Developing chicken wings homozygous for the talpid (ta3/ta3) mutation are polydactylous and have defects in the establishment of their anteroposterior polarity. We analysed the expression domains of the posteriorly restricted homeobox Hox-4 genes in such mutant wings. The Hox-4 genes are now expressed right across the anteroposterior axis instead of being expressed just posteriorly. This correlates well with the absence of clear morphological differences between the talpid3 digits and reinforces the idea that vertebrate Hox-4 genes are involved in setting up the limb anteroposterior asymmetry.


Dopamine agonists and pituitary tumor shrinkage. The primary aim of this review has been to clarify the tumor shrinking effects of dopamine agonists on pituitary macroadenomas of different cell types. Shrinkage is most dramatic for macroprolactinomas and is due to cell size reduction. Seventy-nine percent of 271 definite macroprolactinomas were reduced in size by at least 25%, and 89% shrank to some degree. Most shrinkage occurs during the first 3 months of treatment, although in a minority shrinkage is delayed. Dopamine agonist resistance during long-term therapy is exceptional. Drug withdrawal nearly always leads to a return of hyperprolactinemia, even after several years treatment, although early tumor reexpansion is unusual. About 10% of true macroprolactinomas do not shrink with dopamine agonists; the molecular mechanisms of such resistance have yet to be determined. Alternative formulations of BC and new dopamine agonists (CV 205-502 and cabergoline) are useful for the minority of patients unable to tolerate oral BC, but do not seem to further improve overall shrinkage rates. The risks of pregnancy have probably been overstated, and BC is suitable primary treatment for women with prolactinomas of all sizes; the drug can be used safely during pregnancy in the event of clinically relevant tumor expansion. The interpretation of different degrees of hyperprolactinemia is discussed and management strategies suggested. Most patients with macroprolactinomas now avoid surgery, but drug-induced, time-dependent tumor fibrosis should be remembered if surgery is contemplated. Nonfunctioning pituitary tumors are mostly of gonadotroph cell origin and may be associated with significant disconnection hyperprolactinaemia. Seventy-six of 84 well-characterized tumors showed no tumor shrinkage during dopamine agonist therapy. Possible explanations include abnormalities of dopamine receptor number and function. Preliminary evidence suggests that dopamine agonists may restrain the growth of some functionless tumors; most of these tumors, however, can be satisfactorily debulked using transsphenoidal surgery. In contrast to macroprolactinomas, other functioning pituitary tumors (GH-, TSH-, and ACTH-secreting) rarely shrink during dopamine agonist therapy, although the number of tumors studied is small.


Basic counterpoint: mechanisms and pathways of gonadal steroid modulation of growth hormone secretion. In spite of the different patterns of GH secretion observed in male and female rats, it can be argued that there are limited differences between the mechanism of action of androgens and estrogens as reported in the literature. However, we feel that it is possible to organize the available data into a unique physiological model explaining the sex-based differences in GH secretion in the rat. Thus, it can be proposed that the greater spontaneous GH peaks observed in the male with respect to the female rat may be due to an androgen-mediated enhancement of both GHRH secretion at the hypothalamic level and GH secretion at the pituitary level. The lower GH troughs observed in the male as compared to the female rat may be due to increased interpeak somatostatin secretion induced by the androgens with respect to the estrogens. It is likely that these high GH peaks and low GH troughs establish a recycling mechanism through established feedback mechanisms. That is, the high GH peak, induced by GHRH, stimulates somatostatin secretion such that a very low GH trough follows. In turn, this low GH trough, in the high somatostatin environment, establishes the correct neuroendocrine milieu for the next high GH peak, and so on. Additional studies will help clarify this model and hopefully provide a better understanding of the mechanisms regulating the interaction between gonadal steroids and GH.


Is xamoterol safe in chronic airflow obstruction? We have demonstrated in a group of 10 patients with CAO that treatment with xamoterol (200 mg b.i.d. for 7 days) did not alter lung function or respiratory symptoms. These results suggest that xamoterol can be used to treat mild heart failure in patients with CAO.


Neuroendocrine effects of ipsapirone on the hypothalamic-pituitary adrenal axis: CRF, ACTH and cortisol in healthy volunteers. The neuroendocrine effects (changes in plasma CRF, ACTH and cortisol) of single and multiple (t.d.s. for 2 days) doses of ipsapirone (BAY Q 7821) 5 and 10 mg have been investigated in 6 healthy male volunteers. The study followed a balanced complete block, placebo-controlled and double blind design with two baseline phases (pre and post-treatment). Volunteers were investigated on identical days during 5 successive weeks. The results do not show a specific effect of ipsapirone on the hypothalamic-pituitary-adrenal axis when doses in the range of 5-30 mg per day were given.


Azelastine reduces allergen-induced nasal response: a clinical and rhinomanometric assessment. The effect of azelastine 2 mg b.d. p.o. for 10 days on grass pollen-induced nasal responses in 16 patients with grass pollen allergic rhinitis has been assessed. The study was a double blind, randomized, placebo controlled, crossover trial, with a 10-14 day wash-out period. Patients were challenged with grass pollen before and after placebo and azelastine. The response was assessed by measurement of nasal resistance using active posterior rhinomanometry, by weighing nasal secretions, and by counting sneezes. The sensation of nasal obstruction was assessed with a visual analogue scale. After measurement of baseline total nasal resistance, doubling doses of allergen were sprayed into both nostrils at 15 min intervals until the nasal resistance was doubled. Cumulative doses of allergen that doubled prechallenge nasal resistance, numbers of sneezes and the amounts of nasal secretions were similar before azelastine as well as before and after placebo (cumulative dose, mean, (microgram): 2.3, 4.2 and 2.1 respectively, N.S.). After azelastine, the cumulative dose of allergen was increased (7.3 micrograms), and nasal secretions and the number of sneezes were decreased. The visual analogue scores were similar before and after azelastine as well as before and after the placebo. It is concluded that azelastine reduced the allergen-induced nasal responses.


The effect of ethanol on responses of the isolated rabbit ileocolic artery. Antagonist drugs were used to separate the purinergic and adrenergic contributions involved in the sympathetic vasopressor responses produced by electrical field stimulation in the ileocolic artery of the rabbit. Blocking drugs were applied either alone or in various combinations and sequences. The effect of ethanol was studied in conditions of alpha-adrenoceptor blockade, P2x purinoceptor desensitisation, or in the absence of antagonists. From these studies it is concluded that ethanol has a selective potentiating effect on alpha-adrenoceptor-mediated responses.


Different effects of peptidase inhibitors on dermorphin- and on [D-Arg2]dermorphin-induced antinociceptive activity. The antinociceptive effects produced by the intracerebroventricular (i.c.v.) injection of dermorphin and [D-Arg2]dermorphin were compared in conscious mice, using the combined administration of peptidase inhibitors. Nociception was assessed using a tail pressure assay. Dermorphin-induced antinociception was not potentiated by simultaneous administration of amastatin or captopril as judged from the ED50 values. Co-administration of dermorphin and amastatin gave a longer duration than with dermorphin alone, whereas there was no significant effect on duration with captopril. The antinociceptive activity of dermorphin was significantly enhanced when the heptapeptide was injected simultaneously with both peptidase inhibitors. This result indicates that the heptapeptide sequence is required for the full expression of intrinsic opioid activity of dermorphin. In contrast, co-administration of amastatin brought about a significant enhancement of the antinociceptive activity induced by i.c.v. administration of [D-Arg2]dermorphin, whereas the effect of [D-Arg2]dermorphin was markedly decreased by the concurrent administration of captopril or thiorphan. The potency of captopril was much greater than that of thiorphan. The present results suggest that [D-Arg2]dermorphin may be transformed metabolically to a peptide which has potent antinociceptive activity.


The effect of some alpha-adrenoceptor antagonists on spontaneous myogenic activity in the rat portal vein and the putative involvement of ATP-sensitive K+ channels. In the present study we showed that the alpha-adrenoceptor antagonists phentolamine, yohimbine, prazosin, corynanthine and idazoxan, when cumulatively applied in high concentrations (1-100 mumol/l), can increase spontaneous myogenic activity in the rat portal vein. 5-Methyl-urapidil and rauwolscine were ineffective in this respect. Pretreatment with phenoxybenzamine in a concentration of 1 mumol/l (20 min), which results in alkylation of all functional alpha-adrenoceptors in the rat portal vein, was unable to antagonize the increase in spontaneous myogenic activity elicited by phentolamine. Antazoline (1-100 mumol/l), a H1 antagonist and 2-substituted imidazoline which is devoid of alpha-adrenoceptor blocking properties, exhibited similar effects on spontaneous myogenic activity as its structurally closely related analogue phentolamine. Since phentolamine is reported to interact with ATP-sensitive K+ channels we investigated the role of K+ channels in more detail. The K+ channel openers cromakalim and diazoxide elicited a decrease in spontaneous myogenic activity. Glibenclamide (0.3-3 mumol/l), a selective blocker of ATP-sensitive K+ channels in cardiac and pancreatic tissues, and phentolamine (1-10 mumol/l) shifted the concentration-response curves of cromakalim and diazoxide concentration dependently to the right. Yohimbine showed only a modest effect in the highest concentration (100 mumol/l) applied. E-4031 (0.01-0.3 mumol/l), a sotalol derivative and one of the most selective blockers of the delayed rectifier current (Ik) in cardiac tissue, was a potent contractile agent when added to the rat portal vein in the same way as the alpha-adrenoceptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects on body temperature in mice differentiate between dopamine D2 receptor agonists with high and low efficacies. The effects of different dopamine (DA) D2 receptor agonists and the DA D2 receptor antagonist, emonapride, on body temperature were studied in male mice. The aim of the study was to test whether DA D2 receptor agonists ranging from full agonists to agonists with low efficacy could be differentiated by means of their effect on body temperature. Talipexole induced a marked hypothermia (maximum decrease of 6.5 degrees C). Apomorphine, quinelorane, (+)-4-propyl-9-hydroxy-naphtoxazine((+)-PHNO), and (-)-N-n-propylnorapomorphine ((-)-NPA) induced a maximum hypothermia of 3.5-4.1 degrees C. Quinpirole and (+)-3-(3-hydroxyphenyl)-N-n-propylpiperidine ((+)-3-PPP) induced a less pronounced hypothermia (1.7 and 1.5 degrees C), and preclamol ((-)-3-PPP), terguride and 3-(4-(4-phenyl-1,2,3,6-tetrahydropyridyl)-(1))-butyl)-indole (EMD 23448) had no or only a slight effect. Emonapride induced significant hyperthermia at high doses. Apomorphine-, quinelorane- and talipexole-induced hypothermia was reversed by terguride and preclamol, whereas EMD 23448 partially reversed the apomorphine-induced hypothermia. The alpha 2-adrenoceptor antagonist, idazoxan, partly reversed the effect of talipexole. Quinpirole had no effect on the hypothermic effect of the above-mentioned agonists. Pretreatment with the catecholamine synthesis inhibitor, alpha-methyl-m-tyrosine, increased significantly the hypothermic response to quinpirole, whereas the effect of quinelorane was unchanged. It is suggested that the effect of DA D2 agonists on body temperature in mice can be used to differentiate between agonists with different efficacies.(ABSTRACT TRUNCATED AT 250 WORDS)


Persistent supersensitivity of sigma receptors develops during repeated methamphetamine treatment. Functional changes in sigma receptors were examined after behavioral sensitization induced by repeated methamphetamine treatment. Rats received either saline or 4 mg/kg methamphetamine for 14 days. (+)3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ((+)-3-PPP), a sigma receptor agonist, was given as challenge after various periods of abstinence. (+)-3-PPP at doses greater than 6 mg/kg stimulated several forms of behavior in naive rats. (+)-3-PPP at 12 and 24 mg/kg produced more frequent rearing and more intense stereotyped sniffing and repetitive head movements in rats previously sensitized with methamphetamine than in saline-pretreated rats. The augmented response to (+)-3-PPP in methamphetamine-treated rats was maintained for at least one month. The augmented response to (+)-3-PPP was reversed by the combined administration of 100 mg/kg (+/-)-sulpiride, a D2 dopamine receptor antagonist, and 30 mg/kg BMY 14802, a sigma receptor antagonist. These results suggest that repeated methamphetamine treatment induces persistent supersensitivity in sigma receptors and that it may subsequently activate the dopamine system.


Haemodynamic and humoral effects of omega-conotoxin GVIA in normotensive and spontaneously hypertensive rats. Omega-conotoxin GVIA, a 27-amino acid peptide, has been shown to be a potent and selective inhibitor of N-type voltage-operated calcium channels (VOCCs). A single intravenous dose of 10 micrograms/kg conotoxin slowly lowered blood pressure by 41.3 +/- 4.4 and 73.3 +/- 4.6 mm Hg in conscious Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) respectively without changing the heart rate. Plasma renin activity was significantly increased after conotoxin. In anaesthetized rats, conotoxin (3 and 10 micrograms/kg) lowered blood pressure and heart rate and produced a marked increase in renal vascular conductances. Baroreceptor heart rate reflex experiments using methoxamine and sodium nitroprusside before and after treatment with conotoxin showed that conotoxin almost totally abolished the sympathetic component of the reflex without affecting the vagal tone to the heart in both rat strains. Because conotoxin does not affect directly the vasculature and heart contractile properties, we suggest that the control of presynaptic calcium influx and of neurotransmitter release mostly depends on conotoxin-sensitive N-type VOCCs in the peripheral sympathetic system of the rat.


Membrane stabilizing activity and beta-adrenoceptor antagonist-induced bradycardia in conscious dogs. The atrial effective refractory period (AERP) and atrial and ventricular chronotropic effects of the stereoisomers of propranolol, pindolol, metoprolol and penbutolol were studied in conscious atrio-ventricular blocked dogs. Atrial beta-adrenoceptor blocking activity was assessed for all the drugs against isoprenaline. All the drugs except dextro-pindolol lengthened AERP and decreased ventricular rate dose relatedly. At comparable levels of atrial beta-adrenoceptor blockade, dextro-propranolol, dextro-metoprolol and dextro-penbutolol were more potent to induce AERP lengthening than their respective levo-isomers, whereas dextro-pindolol was less potent than levo-pindolol. In addition, levo-pindolol and levo-metoprolol were more potent to produce ventricular bradycardia than the corresponding dextro-isomers, whereas the levo- and dextro-isomers of propranolol and penbutolol were equipotent. These results confirm that the ventricular bradycardia induced by the different beta-adrenoceptor antagonists is partly due to ventricular beta-adrenoceptor blockade and to the membrane stabilizing activity of these drugs, and partly to another as yet unknown factor seen especially with the levo-isomers and particularly marked with metoprolol.


Effect of atrial natriuretic peptide on apomorphine-induced stereotyped cage-climbing behavior in mice. In previous experiments, it was observed that rat atrial natriuretic peptide-(1-28) (ANP-(1-28)) participated in fear-induced learning and memory processes via dopaminergic and cholinergic mediation. Since cage-climbing behavior is described as a simple test for studying dopaminergic activity in the central nervous system, a systemic study was carried out with ANP-(1-28) in order to confirm or to exclude the possible involvement of dopamine in the ANP-induced action in the brain. The present study demonstrate that ANP-(1-28) facilitated cage-climbing behavior in mice in a dose-dependent manner. When combined with apomorphine, the peptide potentiated the effect of the dopamine agonist. The effect of ANP-(1-28) in combination with apomorphine could be antagonized by a selected dose of haloperidol. These data suggest that ANP might be regarded as a dopamine agonist-modulating agent and that a dopaminergic mechanism is a possible mode of action of ANP in the fear-induced learning studied earlier.


Haloperidol-induced vacuous chewing in rats: suppression by alpha-methyl-tyrosine. Chronic treatment of rats with haloperidol (1 mg/kg twice daily for 4 weeks) induced repetitive vacuous chewing movements (VC), that persisted for over 72 h after haloperidol withdrawal. Haloperidol-induced VC were inhibited by the s.c. administration of the specific dopamine D1, receptor antagonist, SCH 23390 (0.025-0.100 mg/kg), in a dose-dependent manner, and were totally suppressed by an acute challenge with haloperidol (2 mg/kg i.p.) and by the dopamine synthesis inhibitor, alpha-methyl-tyrosine (AMT) (200 mg/kg i.p.). In AMT-treated rats, VC were reinstated by the administration of the selective D1 agonist, SKF 38393. The results support the hypothesis that chronic haloperidol-induced VC are mediated by dopamine acting selectively upon D1 receptors.


Molecular cloning of the cDNAs coding for the two subunits of soluble guanylyl cyclase from human brain. Complementary DNA clones corresponding to the 70 and 82 kDa subunits of soluble guanylyl cyclase from human adult brain have been isolated and sequenced. Their respective open reading frames correspond to 619 amino acids (M(r) 70,469) and 717 amino acids (M(r) 81,324). Southern blots of human genomic DNA using these clones as probes give patterns which might be compatible with the presence of more than one copy per gene, or pseudogenes, for each subunit in the human genome. Comparison of the protein sequence of the large subunit from adult brain with the subunit cloned from human fetal brain (Harteneck, C., Wedel, B., Koesling, D., Malekewitz, J., Böhme, E., and Schultz, G. (1991) FEBS Lett. 292, 217-222) revealed only 34% homology. This result demonstrates the existence of a novel large subunit isoform for soluble guanylyl cyclase in human tissues.


Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells. Evidence for a differential induction of two distinct isoenzymes. Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.


Volume-activated Cl- secretion and transepithelial vinblastine secretion mediated by P-glycoprotein are not correlated in cultured human T84 intestinal epithelial layers. The relationship between the P-glycoprotein-mediated vinblastine secretion and cell-swelling activated Cl- secretion (conductance) in intact epithelial layers of human colonic adenocarcinoma T84 cells has been investigated. Whereas vinblastine secretion is effectively inhibited by 100 microM 1,9-dideoxy-forskolin, volume-stimulated Cl- secretion is unaffected. In contrast, 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) inhibited the volume-stimulated Cl- secretion, but was without effect upon transepithelial vinblastine secretion. In addition, it was noted that some epithelial layers failed to express a volume-stimulated Cl- secretion but maintained a normal level of secretory vinblastine flux.


ATP and GTP as alternative energy sources for vinblastine transport by P-170 in KB-V1 plasma membrane vesicles. Purified plasma membrane vesicles isolated from multidrug-resistant human KB-V1 cells accumulate [3H]vinblastine in an energy-dependent manner. The accumulation of [3H]vinblastine in the presence of ATP is a saturable process. ATP can be replaced by other purine nucleotide triphosphates, of which GTP is the most efficient.


Heat shock proteins of barley mitochondria and chloroplasts. Identification of organellar hsp 10 and 12: putative chaperonin 10 homologues. Tissue slices from barley seedlings were subjected to heat shock and metabolically labelled with [35S]methionine and [35S]cysteine. Mitochondria and chloroplasts were isolated and shown to contain two novel heat shock proteins of 10 and 12 kDa, respectively. The possibility that these proteins, like a mitochondrial 10 kDa stress protein recently isolated from rat hepatoma cells [(1992) Proc. Natl. Acad. Sci. 89, in press] represent eukaryotic chaperonin 10 homologues is discussed.


Identification of the atrial natriuretic factor-R1C receptor subtype (B-clone) in cultured rat aortic smooth muscle cells. The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF-R1C (B-clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP-(82-103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP-(82-103) binds to a specific receptor subtype which is insensitive to the ANF-R2 specific ligand, C-ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP greater than pBNP greater than rANF. Furthermore it was possible to covalently photolabel this receptor subtype with underivatized]125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.


Restriction endonuclease analysis of chloroplast DNA from streptomycin-resistant mutants of Nicotiana plumbaginifolia. Chloroplast DNA isolated from wild-type Nicotiana plumbaginifolia and 12 maternally inherited streptomycin-resistant mutants was digested with various restriction enzymes and the resultant patterns were compared. No gross structural alterations of the chloroplast genome were detected in any mutants; however, variant patterns owing to the gain or loss of a restriction site were found in three mutants, SR1007, SR1019, and SR1022. The variant patterns in SR1019 and SR1022 are identical and are the results of mutation in the psbG gene coding for a chloroplast membrane protein G, and that in SR1007 is due to mutation in the 16S rRNA gene. Inheritance of the variant patterns in mutants SR1007 and SR1019 was studied. The results showed that the variant patterns and streptomycin resistance were co-transmitted in reciprocal crosses.