Coordinate, biphasic activation of p44 mitogen-activated protein kinase and S6 kinase by growth factors in hamster fibroblasts. Evidence for thrombin-induced signals different from phosphoinositide turnover and adenylylcyclase inhibition. In CCL39 cells transfected with m1-muscarinic receptors, carbachol stimulates phosphoinositide turnover and early events associated with mitogenesis as efficiently as thrombin but, in contrast to thrombin, fails to induce cell proliferation (Seuwen, K., Kahan, C., Hartmann, T., and Pouysségur, J. (1990) J. Biol. Chem. 265, 22292-22299). We show here that the action of the two agents can be dissociated at the level of S6 kinase and mitogen-activated protein kinase (MAP kinase) activation. Mitogenic concentrations of thrombin and basic FGF were found to stimulate S6 kinase activity, measured in whole cell lysates, with a biphasic time course; an early peak of activity is induced 10 min following stimulation and a sustained phase of activity can be measured over several hours. A very similar profile emerged for p44 MAP kinase (p44mapk), assayed in immunoprecipitates. In this case, the activity first peaks at 6-8 min, preceding S6 kinase. In contrast to thrombin and FGF, carbachol stimulates S6 kinase and MAP kinase only transiently, corresponding to the first peak of activity, but the sustained phase is not observed. Similarly, phorbol dibutyrate induces an early phase of activity only. Pertussis toxin (PTX), which is known to block thrombin mitogenicity efficiently, inhibited the first peak of thrombin-induced S6 kinase and MAP kinase activity only partially, but totally blocked the sustained phase. The toxin had no effect on FGF-induced kinase activities. The cAMP elevating hormone PGE1 did not inhibit p44mapk or S6 kinase activation by thrombin or FGF, demonstrating that the PTX-sensitive signal generated by thrombin does not depend on a Gi-mediated sustained inhibition of adenylylcyclase. Surprisingly, PGE1 was found to stimulate sustained phase S6 kinase activity both alone and in synergy with FGF or thrombin. This result, as well as the biphasic activation of S6 kinase by thrombin, could be qualitatively reproduced in immunocomplex kinase assays using an antiserum immunoprecipitating p70 S6 kinase (p70S6k). Our data show that activation of phosphoinositide turnover and PKC does not quantitatively explain thrombin action, in particular the sustained phase of kinase activities, which critically depends on a PTX-sensitive signal different from adenylylcyclase inhibition. We postulate that this signal does not exclusively originate from the recently identified G protein-coupled thrombin receptor.


The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors. The endothelial angiotensin I-converting enzyme (ACE; EC 3.4.15.1) has recently been shown to contain two large homologous domains (called here the N and C domains), each being a zinc-dependent dipeptidyl carboxypeptidase. To further characterize the two active sites of ACE, we have investigated their interaction with four competitive ACE inhibitors, which are all potent antihypertensive drugs. The binding of [3H] trandolaprilat to the two active sites was examined using the wild-type ACE and four ACE mutants each containing only one intact domain, the other domain being either deleted or inactivated by point mutation of the zinc-coordinating histidines. In contrast with all the previous studies, which suggested the presence of a single high affinity inhibitor binding site in ACE, the present study shows that both the N and C domains of ACE contain a high affinity inhibitor binding site (KD = 3 and 1 X 10(-10) M, respectively, at pH 7.5, 4 degrees C, and 100 mM NaCl). Chloride stabilizes the enzyme-inhibitor complex for each domain primarily by slowing its dissociation rate, as the k-1 values of the N and C domains are markedly decreased (about 30- and 1100-fold, respectively) by 300 mM NaCl. At high chloride concentrations, the chloride effect is much greater for the C domain than for the N domain resulting in a higher affinity of this inhibitor for the C domain. In addition, the inhibitory potency of captopril (C), enalaprilat (E), and lisinopril (L) for each domain was assayed by hydrolysis of Hip-His-Leu. Their Ki values for the two domains are all within the nanomolar range, indicating that they are all highly potent inhibitors for both domains. However, their relative potencies are different for the C domain (L greater than E greater than C) and the N domain (C greater than E greater than L). The different inhibitor binding properties of the two domains observed in the present study provide strong evidence for the presence of structural differences between the two active sites of ACE.


Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap. To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)


Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain I. Identification, purification, and structural comparisons. Two Ca(2+)-calmodulin (CaM)-dependent protein kinases were purified from rat brain using as substrate a synthetic peptide based on site 1 (site 1 peptide) of the synaptic vesicle-associated protein, synapsin I. One of the purified enzymes was an approximately 89% pure protein of M(r) = 43,000 which bound CaM in a Ca(2+)-dependent fashion. The other purified enzyme was an apparently homogenous protein of M(r) = 39,000 accompanied by a small amount of a M(r) = 37,000 form which may represent a proteolytic product of the 39-kDa enzyme. The 39-kDa protein bound CaM in a Ca(2+)-dependent fashion. Gel filtration analysis indicated that both enzymes are monomers. The 43- and 39-kDa enzymes are named Ca(2+)-CaM-dependent protein kinases Ia and Ib (CaM kinases Ia, Ib), respectively. The specific activities of CaM kinases Ia and Ib were similar (5-8 mumol/min/mg protein). CaM kinase Ia (but not CaM kinase Ib) activity was enhanced by addition of a CaM-Sepharose column wash (non-binding) fraction suggesting the existence of an "activator" of CaM kinase Ia. Both kinases phosphorylated exogenous substrates (site 1 peptide and synapsin I) in a Ca(2+)-CaM-dependent fashion and both kinases underwent autophosphorylation. CaM kinase Ia autophosphorylation was Ca(2+)-CaM-dependent and occurred exclusively on threonine while CaM kinase Ib autophosphorylation showed Ca(2+)-CaM independence and occurred on both serine and threonine. Proteolytic digestion of autophosphorylated CaM kinases Ia and Ib yielded phosphopeptides of differing M(r). These characteristics, as well as enzymatic and regulatory properties (DeRemer, M. F., Saeli, R. J. Brautigen, D. L., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13466-13471), indicate that CaM kinases Ia and Ib are distinct and possibly previously unrecognized enzymes.


Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain. II. Enzymatic characteristics and regulation of activities by phosphorylation and dephosphorylation. In addition to physical properties (DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13460-13465), enzymatic and regulatory characteristics indicate that calmodulin (CaM) kinase Ia and CaM kinase Ib are distinct entities. The Km values for ATP and site 1 peptide were similar between the two kinases, however, CaM kinase Ib is approximately 20-fold more sensitive to CaM than is CaM kinase Ia. The kinases also displayed differential sensitivities to divalent metal ions. For both kinases, site 1 peptide, synapsin I, and syntide-2 were highly preferred substrates relative to others tested. A 72-kDa protein from a heat-treated extract of rat pancreas was phosphorylated by CaM kinase Ib but not by CaM kinase Ia. CaM kinase Ia activity displayed a pronounced lag in its time course suggesting enzyme activation over time. Preincubation of CaM kinase Ia in the combined presence of Ca(2+)-CaM and MgATP led to a time-dependent increase in its site 1 peptide kinase activity of up to 15-fold. The extent of activation of CaM kinase Ia correlated with the extent of autophosphorylation. The enzyme retained full Ca(2+)-CaM dependence in the activated state which was rapidly reversible by treatment with protein phosphatase 2A catalytic subunit. Thus, the activation of CaM kinase Ia is a result of its Ca(2+)-CaM-dependent autophosphorylation. CaM kinase Ib was not activated by preincubation under autophosphorylating conditions yet lost approximately 90% of its activity toward either an exogenous substrate (site 1 peptide) or itself (autophosphorylation) after incubation with protein phosphatase 2A catalytic subunit. The deactivated state was not reversed by subsequent incubations under autophosphorylating conditions. Thus, CaM kinase Ib activity is dependent upon phosphorylation by a regulating kinase(s) which is resolved from CaM kinase Ib during purification of the latter.


Lymphocyte lineage-restricted tyrosine-phosphorylated proteins that bind PLC gamma 1 SH2 domains. Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.


A novel DNA helicase from calf thymus.

A novel DNA helicase from calf thymus. We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.


Identification of determinants that confer ligand specificity on the insulin receptor. We have previously shown, using truncated soluble recombinant receptors, that substituting the 62 N-terminal amino acids of the alpha subunit from the insulin-like growth factor I receptor (IGFIR) with the corresponding 68 amino acids from the insulin receptor (IR) results in a chimeric receptor with an approximately 200-fold increase in affinity for insulin and only a 5-fold decrease in insulin-like growth factor I (IGFI) affinity (Kjeldsen, T., Andersen, A. S., Wiberg, F. C., Rasmussen, J. S., Schäffer, L., Balschmidt, P., Møller, K. B., and Møller, N. P. H. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4404-4408). We demonstrate that these 68 N-terminal amino acids of the IR also confer insulin affinity on the intact IGFI holoreceptor both in the membrane-bound state and when solubilized by Triton X-100. Furthermore, this domain can be subdivided into two regions (amino acids 1-27 and 28-68 of the IR alpha subunit) that, when replacing the corresponding IGFIR sequences, increases the insulin affinity of truncated soluble receptor chimeras 8- and 20-fold, respectively, with only minor effects on the IGFI affinity. Within the latter of these two regions, we found that amino acids 38-68 of the IR, representing 13 amino acid differences from IGFIR, confer the same 20-fold increase in insulin affinity on the IGFIR. Finally, the amino acids from position 42 to 50 are not responsible for this increase in insulin affinity. We thus propose that at least two determinants within the 68 N-terminal amino acids of the insulin receptor are involved in defining the ligand specificity of the insulin receptor, and that one or a combination of the remaining seven amino acid differences between position 38 and 68 are involved in conferring insulin affinity on the insulin receptor.


MyoD binds to the guanine tetrad nucleic acid structure. A high affinity interaction between a protein and the guanine tetrad nucleic acid structure is described. Recombinant MyoD, a transcription factor that can initiate myogenesis, specifically bound to helical structures formed by stacks of guanine residues in square planar arrays. The N-7 methylation of a set of consecutive dG residues in a single-stranded probe of the creatine kinase enhancer interfered with the formation of this nucleic acid structure and prevented protein binding. Recombinant MyoD also bound to a guanine tetrad formed with a telomeric DNA probe, and it had a higher affinity for the four-stranded structure than for the double-stranded E-box-binding site. These data are the first report of a direct interaction between a protein and this nucleic acid conformation. The potential biological significance of this finding is discussed.


The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies. The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.


Dual roles of the 90-kDa heat shock protein hsp90 in modulating functional activities of the dioxin receptor. Evidence that the dioxin receptor functionally belongs to a subclass of nuclear receptors which require hsp90 both for ligand binding activity and repression of intrinsic DNA binding activity. Signal transduction by dioxin (2,3,7,8-tetrachloro-dibenzo-p-dioxin) is mediated by the intracellular dioxin receptor which, in its dioxin-activated state, regulates transcription of target genes encoding drug metabolizing enzymes such as cytochrome P-450IA1 and glutathione S-transferase Ya. Upon binding of dioxin the receptor translocates from the cytoplasm to the nucleus in vivo and is converted from a latent non-DNA binding form to a species which binds to dioxin-responsive positive control elements in vitro. The latent receptor form is associated with an inhibitory protein (the 90-kDa heat shock protein, hsp90), the release of which is necessary to unmask the DNA binding activity of the receptor. Here we have established a protocol to disrupt the hsp90-receptor complex in the absence of ligand. We show that it was possible to covalently cross-link with dioxin only the hsp90-associated form of dioxin receptor. In contrast, the disrupted hsp90-free form of receptor did not form a stable complex with dioxin but bound DNA constitutively. Moreover, we could partially reconstitute the ligand binding activity of the salt-disrupted hsp90-free dioxin receptor by incubation with hsp90-containing reticulocyte lysate but not by incubation with wheat germ lysate which lacks immuno-detectable levels of hsp90. Thus, we demonstrate that the dioxin receptor loses its high affinity ligand binding activity following release of hsp90 and that it is possible to reverse this process. In conclusion, hsp90 appears to play dual roles in the modulation of functional activities of the dioxin receptor: (i) it represses the intrinsic DNA binding activity of the receptor and (ii) it appears to determine the ability of the receptor to assume and/or maintain a ligand binding conformation.


Isolation and characterization of a cDNA encoding the putative distal colon H+,K(+)-ATPase. Similarity of deduced amino acid sequence to gastric H+,K(+)-ATPase and Na+,K(+)-ATPase and mRNA expression in distal colon, kidney, and uterus. A series of Northern blot hybridization experiments using probes derived from the rat gastric H+,K(+)-ATPase cDNA and the human ATP1AL1 gene revealed the presence of a 4.3-kilobase mRNA in colon that seemed likely to encode the distal colon H+,K(+)-ATPase, the enzyme responsible for K+ absorption in mammalian colon. A rat colon library was then screened using a probe from the ATP1AL1 gene, and cDNAs containing the entire coding sequence of a new P-type ATPase were isolated and characterized. The deduced polypeptide is 1036 amino acids in length and has an Mr of 114,842. The protein exhibits 63% amino acid identity to the gastric H+,K(+)-ATPase alpha-subunit and 63% identity to the three Na+,K(+)-ATPase alpha-subunit isoforms, consistent with the possibility that it is a K(+)-transporting ATPase. Northern blot analyses show that the 4.3-kilobase mRNA is expressed at high levels in distal colon; at much lower levels in proximal colon, kidney, and uterus; and at trace levels in heart and forestomach. The high mRNA levels in distal colon and the similarity of the colon pump to both gastric H+,K(+)- and Na+,K(+)-ATPases suggest that it is the distal colon H+,K(+)-ATPase. Furthermore, expression of its mRNA in kidney raises the possibility that the enzyme also corresponds to the H+,K(+)-ATPase that seems to play a role in K+ absorption and H+ secretion in the distal nephron.


The platelet-derived growth factor beta-receptor kinase insert confers specific signaling properties to a chimeric fibroblast growth factor receptor. Signal transduction by tyrosine kinase growth factor receptors involves ligand-induced phosphorylation of substrates for the kinase, resulting in mediation of common or receptor-specific biological signals. We have compared signal transduction pathways for the fibroblast growth factor receptor-1 (FGFR-1), the platelet-derived growth factor beta-receptor (PDGFR-beta), and a chimeric FGFR-1 molecule, FGFRchim, in which the FGFR-1 kinase insert was replaced with that of the PDGFR-beta. The different receptors were characterized and found to be functional as ligand-stimulatable kinases, after expression of the respective human cDNAs in porcine aortic endothelial cells. Substrates for the receptors were analyzed by ligand stimulation of [32P]orthophosphate-labeled cells and immunoprecipitation with phosphotyrosine antiserum. A number of phosphoproteins were induced in all the different types of cells, but components specifically induced after stimulation of FGFR-1 and PDGFR-beta expressing cells could also be detected. Examination of receptor-associated substrates by in vitro kinase assays revealed phosphoproteins of 65 and 85 kDa, which were associated with PDGFR-beta and FGFRchim, but not with FGFR-1. The 85-kDa phosphoprotein could correspond to the regulatory subunit of phosphatidylinositol 3' kinase (PI3-K), since phosphatidylinositol 3' kinase activity was detected after ligand stimulation of FGFRchim- and PDGFR-beta- but not FGFR-1-expressing cells. In addition, ligand stimulation of FGFRchim- and PDGFR-beta-expressing cells, but not FGFR-1-expressing cells, led to induction of actin reorganization in the form of circular membrane ruffling. Thus, replacement of a discrete segment of the intracellular domain of the FGFR-1 with the corresponding stretch from the PDGFR-beta resulted in transfer of PDGFR-beta-specific signaling properties to the chimeric molecule.


Collagen binding site in collagenase can be determined using the concept of sense-antisense peptide interactions. Tissue degradation and invasion are hallmarks of the metastatic phenotype. While several extracellular matrix components can be digested by proteases, degradation of interstitial collagen is selectively initiated by collagenase. It is obvious that inhibitors of collagenase activity would be extremely useful in preventing tissue destruction and tumor cell invasion and thus prove invaluable therapeutic agents. We describe here the possible development of such inhibitors through the use of the principle of complementary hydropathy. A peptide was deduced from the nucleotide sequence complementary to that coding for the region in interstitial collagen surrounding the bond between Gly775 and Ile776 which is cleaved by the enzyme. Labeled collagen binds specifically and quantitatively to this peptide. A polyclonal mouse serum raised against this peptide recognized purified human collagenase, was able to immunoprecipitate collagenase from cultured human keratinocyte supernatants and was effective in inhibiting collagenolytic activity with a K(iapp) = 0.3 microM.


Treatment results of the thioether lipid ilmofosine in patients with malignant tumours. In a multicentre study patients with liver metastases stratified to the histology of the primary tumour were investigated. A total of 102 patients with colorectal adenocarcinoma, non-small-cell lung cancer, pancreatic cancer, primary liver carcinoma and malignant melanoma were treated with the thioether lipid ilmofosine. The drug was administered orally as a tablet at a dosage of 150-300 mg/day (75 mg/tablet). The tolerability of ilmofosine was poor. There was a dose-limiting gastrointestinal toxicity with nausea, vomiting and loss of appetite (WHO grade II-IV) in 67% of patients. During the period of therapy (1-29 weeks, 8.5 weeks mean) no complete remission and no partial response were observed. We thus conclude that treatment with oral ilmofosine is not effective in patients with liver metastases due to various malignancies.


Intercellular calcium signaling via gap junctions in glioma cells. Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.


The effect of dopamine on pulmonary hemodynamics and tissue damage after inhalation injury in an ovine model. Hypoxic pulmonary vasoconstriction and reduced blood flow occur as a result of smoke inhalation. The aim of this study was to investigate how the amelioration of blood flow reduction by the vasodilator dopamine affects histopathologic outcome. We exposed the left lungs of chronically instrumented sheep (n = 12) to smoke, awakened them, and studied them for 24 hours. Six hours after inhalation injury, the sheep received randomized infusions of dopamine (9 micrograms/kg/min) or equal volumes of 0.9% saline solution. Pulmonary resistance in the left lungs of animals in the group that received saline solution rose continuously throughout the study period (624 +/- 48 dyne.sec.cm-5/m2 to 1747 +/- 140 dyne.sec.cm-5/m2, baseline to 24 hours after injury). Dopamine treatment caused a significantly lower vascular resistance in the injured lung than did saline solution between 8 and 24 hours after injury. The histologic evaluation of the injured lungs showed epithelial necrosis and cast formation in both groups in addition to an increased wet/dry ratio. No difference in lung injury between the groups could be distinguished. We conclude that the amelioration of blood flow reduction by treatment with dopamine in the lungs that were exposed to smoke did not affect pulmonary damage after inhalation injury.


Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex. We performed an immunocytochemical analysis to study the transfer of a marker protein (G glycoprotein coded by vesicular stomatitis virus ts 045 strain) from the intermediate compartment to the Golgi stacks in infected Vero cells. The intermediate compartment seemed to consist of about 30-40 separate units of clustered small vesicles and short tubules. The units contained Rab2 protein and were spread throughout the cytoplasm, with a ratio of about 6:4 in the peripheral versus perinuclear site. Time-course experiments revealed a progressive transfer of G glycoprotein from the intermediate compartment to the Golgi stacks, while the tubulo-vesicular units did not appear to change their intracellular distribution. Moreover, the labeling density of peripheral and perinuclear units decreased in parallel during the transfer. These results support the notion that the intermediate compartment is a station in the secretory pathway, and that a vesicular transport connects this station to the Golgi complex.


Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation. Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.


Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells. Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.


Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange. The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.


Protein kinase C has both stimulatory and suppressive effects on macrophage superoxide production. Unlike resident peritoneal macrophages (RPM) or tumor necrosis factor alpha (TNF alpha)-primed bone marrow-derived macrophages (BMM), unprimed BMM do not generate superoxide in response to the protein kinase C (PKC) activator, phorbol myristate acetate (PMA). However, these cells do contain significant levels of PKC activity. In contrast to PMA, zymosan induces the generation of superoxide in unprimed BMM, as well as in TNF alpha-primed BMM and RPM. Staurosporine, a potent PKC inhibitor, failed to affect the zymosan-induced production of superoxide by unprimed and TNF alpha-primed BMM and RPM, in spite of substantial inhibition of PMA-induced superoxide production by the primed BMM and RPM. However, when PKC was depleted from unprimed BMM by prolonged (24 h) treatment with phorbol dibutyrate (PdBt) (10(-7) M) the ability of zymosan to induce the production of superoxide was greatly diminished. Such a result could be interpreted as suggesting a role for PKC in the zymosan-induced response, a conclusion which contrasts with the inhibitor data. However, PKC depletion, in this case, is achieved via the PdBt-induced activation of PKC. It is thus possible that it is the initial activation of PKC, rather than its depletion, that suppresses superoxide production. Consistent with this interpretation, the co-stimulation of unprimed BMM with both zymosan and PMA resulted in a reduced superoxide release compared to zymosan alone. The activation of PKC therefore appears to have a suppressive effect on the generation of superoxide by unprimed cells. We thus conclude that PKC is not required for zymosan-induced superoxide production by either primed or unprimed macrophages and suggest that PKC may be involved in regulatory mechanisms restricting superoxide production by macrophages. However, since PMA alone can initiate the release of superoxide from primed BMM and RPM, it would appear that PKC can mediate both stimulatory and suppressive signals for macrophage superoxide production.


Rapid inverse changes in alpha 1B- and beta 2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells. In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.


Insulin/IGF-I receptor hybrids: a mechanism for increasing receptor diversity. Insulin and IGF-I receptors are homologous disulfide linked alpha 2 beta 2 tetramers. These tetramers are formed biosynthetically when proreceptors containing alpha and beta subunits in a single uninterrupted linear peptide form disulfide linked homodimers and are subsequently proteolytically cleaved at the alpha-beta junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor alpha and beta subunit, and one IGF-I receptor alpha and beta subunit. These presumably form by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF-I receptor. Regulatory mechanisms that involve either insulin or IGF-I receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.


Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits. Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.


Enzymic preparation of dioxygen-18 labelled leukotriene E4 and its use in quantitative gas chromatography-mass spectrometry. A simple and rapid method is described for the preparation of a stable isotope oxygen-18 labelled leukotriene E4 (LTE4). Oxygen-18 labelling of LTE4 methyl ester in oxygen-18 water catalysed by a pig liver esterase resulted in the incorporation of two oxygen-18 atoms in the carboxylic group of LTE4 to the extent of 89.8% ([18O2]LTE4) and one oxygen-18 atom to the extent of 9.4% ([16O18O]LTE4), with only 0.7% remaining unchanged ([16O2]LTE4). [18O2]LTE4 was found not to back-exchange following incubation in acidified urine (pH 4.0) at 4 degrees C for up to 20 h. [18O2]LTE4 was demonstrated to be a useful internal standard in a method for the quantitative determination of LTE4 in human urine involving high-performance liquid chromatography and gas chromatography with negative-ion chemical ionization tandem mass spectrometry: the concentration of LTE4 in a 24-h urine sample of a healthy subject was determined to be 68.1 pg/ml.


Quantitation of 6-(pyridin-3-yl)quinolin-2(1H)-one, a cardiac stimulant, and its N-oxide metabolite in dog plasma by high-performance liquid chromatography. A method is described for the determination of UK-57,400 (I), a 6-substituted quinoline cardiotonic, and its pyridyl-N-oxide in dog plasma. The analytes are selectively retained from plasma (1 ml) on a solid-phase extraction column and eluted with 1 ml of methanol. After evaporation to dryness, the residue is reconstituted in 100 microliters of the mobile phase. Chromatography is carried out on a Spherisorb 5 microns phenyl high-performance liquid chromatography column, with ultraviolet detection. Calibration curves are linear for concentrations from 10 to 100 ng ml-1. For I, the coefficients of variation at highest and lowest concentrations are 1 and 14%, respectively, while the corresponding figures for the pyridyl-N-oxide metabolite are 4 and 10%, respectively. Sample recovery from extraction is greater than 90%. The limit of detection is 4 ng ml-1 for both analytes.


Comparison of detergents for extraction and ion-exchange high-performance liquid chromatography of Sendai virus membrane proteins. The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e., pentaethylene glycol monolauryl ether (C12E5), lauryldimethylamine oxide (LDAO) and dodecyldimethylammoniopropane-1-sulphonate (SB12), respectively. The extracts were subjected to ion-exchange high-performance liquid chromatography (HPIEC) using 0.1% of the detergent in the eluent on four different columns (MA7Q, Zorbax BioSeries SAX, Mono Q and PL-SAX) with a quaternary amine as interacting ligand and with different pore sizes: non-porous and 30, 80 nm and 400 nm, respectively. The relative recoveries of protein were similar for all the columns. The highest recovery of HN and F protein and the best separation were obtained with C12E5. Analysis of HPIEC fractions with monoclonal antibodies directed against conformational epitopes showed that C12E5 had less effect on the conformation than the other two detergents.


Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells. Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.


Mechanisms for differences in lipolysis between human subcutaneous and omental fat cells. Catecholamine-regulation of lipolysis and beta-adrenoceptor binding isoterms were studied in human sc and omental isolated fat cells from 24 subjects undergoing elective cholecystectomy. The lipolytic sensitivity of the nonselective beta-agonists epinephrine and isoprenaline as well as the selective agonists norepinephrine (beta 1) and terbutaline (beta 2) was significantly increased 5-10 times in omental fat cells. On the other hand, no regional difference in antilipolytic sensitivity was seen for the alpha 2 agonist clonidine. No regional difference in lipolytic action was seen when measuring the effect of forskolin, (Bu)2cAMP or enprofylline, which act at different postadrenoceptor steps in the lipolytic cascade. Lipolysis data showed no sex differences. A beta 1-pattern was seen in both regions when lipolysis dose-response curves were arranged in order of potency. Radioligand saturation experiments with the nonselective beta-antagonist 125I-cyanopindolol and competition experiments between this radioligand and the selective antagonists CGP-20,712-A (beta 1) and ICI-118551 (beta 2) showed a 2-fold increase in the amount of beta 1- and beta 2-adrenergic receptors in omental as compared to sc fat cells (P less than 0.02). Competition studies with the same radioligand and the nonselective beta-agonist isoprenaline showed no regional differences in terms of receptor affinity (Kd high 10 nM and Kd low 1 microM) or in relative fraction of receptors in the high affinity state (35%). It is concluded that an increased lipolytic sensitivity for beta 1- and beta 2-agonists can be due to an increase in the amount of the two adrenoceptor subtypes in omental fat cells and thereby explain why catecholamines are more lipolytic in omental cells than in sc fat cells.


Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal vitamin D receptor concentrations in normal women. Although aged rats reportedly have reduced intestinal vitamin D receptor (VDR) concentrations, it is unclear whether an analogous age-related defect occurs in man. Thus, we assessed the interrelationship among serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium absorption and intestinal VDR in 44 healthy, ambulatory women, ages 20-87 yr. Fractional calcium absorption was measured after oral administration of 45Ca (20 mg CaCl2 as carrier); serum 1,25-(OH)2D3, by the calf thymus binding assay; and serum intact PTH, by a two-site immunochemiluminometric assay. Vitamin D receptor concentration was measured, by a new immunoradiometric assay, in biopsy specimens taken from the second part of the duodenum during gastroduodenoscopy in 35 of the women. Despite an age-related increase in serum PTH (r = 0.48; P less than 0.001) and in serum 1,25-(OH)2D3 concentration (r = 0.32; P less than 0.05), intestinal VDR concentration decreased with age (r = -0.38; P = 0.03) and fractional calcium absorption did not change with age. Although a contribution of decreased 25-hydroxyvitamin D 1 alpha-hydroxylase activity to the blunting of the increase in serum 1,25-(OH)2D3 concentration late in life is not excluded, the data are far more consistent with impaired intestinal responsiveness to 1,25-(OH)2D3 action. This defect could lead to compensatory increases in PTH secretion and 1,25-(OH)2D3 production which maintain calcium absorption and serum ionic calcium, but at the expense of increased bone loss.


Antiproliferative effects of luteinizing hormone-releasing hormone agonists on the human prostatic cancer cell line LNCaP. Highly potent LH-releasing hormone (LHRH) agonists have been recently introduced in therapy for the treatment of the carcinoma of the prostate, an androgen-dependent pathology. These peptides are believed to act mainly by inhibiting the pituitary-testicular axis and, consequently, by reducing testosterone levels. The recent observation that binding sites for LHRH analogs are present on prostatic tumor tissue suggests that these drugs could also act directly on the tumor. To verify this hypothesis, the effects of two potent LHRH agonists [Zoladex (Z) and Buserelin (B)] have been studied on the proliferation of the human prostatic cancer cell line LNCaP (lymph node carcinoma of the prostate). LNCaP cells were treated for 9 days with different doses of either Z or B (concentrations from 10(-12)-10(-6) M). Both analogs significantly inhibited cell proliferation at doses between 10(-9)-10(-6) M. The antiproliferative action of the two LHRH agonists was shown to be dose dependent, with IC50 values of 0.82 and 1.79 nM for Z and B, respectively. A similar treatment with B was without any significant effect on the proliferation of a mouse embryo fibroblast cell line (Swiss 3T3), which was used as a nontumoral control. The inhibitory action of both LHRH agonists (10(-8) M) on LNCaP cell proliferation was completely antagonized by the simultaneous treatment of the cells with a potent LHRH antagonist (Nal-Arg-LHRH; 10(-8) M); when given alone at the dose selected, the antagonist did not affect cell growth. These results clearly suggest that the antiproliferative effect of LHRH agonists on LNCaP cells may be mediated by specific receptors. The presence of binding sites for [125I]B was consequently demonstrated on the membranes of LNCaP cells cultured in a medium containing charcoal-stripped fetal calf serum, i.e. in the absence of steroids. The affinity of these binding sites for the ligand was lower than that observed for the same receptors on rat pituitary membranes. To clarify the mechanism of the antiproliferative action of the LHRH agonists, the effects of both Z and B on the incorporation of [3H]thymidine and [14C]methionine into LNCaP cells were investigated. During a short incubation period (3 h), the two LHRH agonists rapidly inhibited [3H]thymidine incorporation into the cells. The same treatment did not affect the incorporation of [14C]methionine into proteins.(ABSTRACT TRUNCATED AT 400 WORDS)


Sleep shift dissociates the nocturnal peaks of parathyroid hormone (1-84), nephrogenous cyclic adenosine monophosphate, and prolactin in normal men. The secretion of PTH(1-84) and PRL over a 24-h period in normal subjects has been shown to be highly correlated, with changes in PRL occurring approximately 2 h after those in PTH(1-84). It has been postulated that there may be neuroendocrine control common to PTH(1-84) and PRL. As the secretion of PRL is known to be strongly influenced by sleep we investigated the effect of a 7-h acute sleep shift on the nocturnal secretion of PTH(1-84), PRL, and nephrogenous cAMP, a marker of PTH(1-84) bioactivity. Six normal male subjects were studied on two occasions (study A sleep, 0100-0800 h; study B, 0800-1400 h) with samples withdrawn at 30-min intervals. Sleep shift produced the expected shift in PRL secretion to new time of sleep. The overall timing of the PTH(1-84) nocturnal peak (0200-0600 h) was not altered by sleep shift. However, the start of the rise in PTH(1-84) (0200-0300 h) was blunted (P less than 0.05), and the peak of nephrogenous cAMP, coincident with the nocturnal rise in PTH(1-84) in study A, was markedly attenuated (P less than 0.01). Thus whereas the results of this study argue against a direct neuroendocrine link between PTH(1-84) and PRL, it is postulated that sleep shift disrupts a high degree of temporal organization which, under normal conditions, may allow concerted metabolic effects between PTH(1-84) and other hormones over a 24-h period.


Diagnosis and therapy surveillance in Addison's disease: rapid adrenocorticotropin (ACTH) test and measurement of plasma ACTH, renin activity, and aldosterone. The rapid ACTH injection test is an indirect screening test for adrenocortical insufficiency. As a supplement to this test, we evaluated the practicability of single measurements of plasma cortisol, ACTH, aldosterone, and PRA as a definitive diagnostic test of primary adrenocortical insufficiency (PAI). We also tested the value of PRA measurements during treatment with hydro- and fludrocortisone (HC and FC) as a guide for correct mineralocorticoid substitution. In 45 patients with PAI, results of the rapid ACTH test and single measurements of the four hormones (all tests between 0800-0900 h) were compared. Single hormone measurements were also made in 55 normal subjects and 46 patients with pituitary disease (cortisol and ACTH only), most of them with mild to severe secondary adrenocortical insufficiency (SAI). The rapid ACTH test was abnormal in 100% of 41 patients with PAI tested. Plasma ACTH, PRA, and the ratios of ACTH/cortisol and PRA/plasma or urinary aldosterone were clearly elevated in 100% of the patients with PAI. The ACTH/cortisol ratio also distinguished 100% of patients with PAI from those with SAI, but not always control subjects from those with SAI. Thus, dynamic tests (CRH or insulin tests) are indicated if SAI is suspected. PAI and involvement of zona fasciculata and glomerulosa function can be diagnosed with high reliability by measuring cortisol, ACTH, aldosterone, and PRA either together with the rapid ACTH test or later, after a short interval of steroid substitution. PRA measurements during treatment with HC and FC correlated better with the mineralocorticoid dose than plasma potassium and sodium levels. PRA measurement is a valuable guide for FC replacement therapy. It should be titrated into the upper normal range to avoid under- and overtreatment.


Hereditary adrenocortical unresponsiveness to adrenocorticotropin with a postreceptor defect. We report two cases in one pedigree with hereditary adrenocortical unresponsiveness to ACTH (HACUA) where it is suggested that the pathogenic defect occurs after cAMP generation. Although the patients showed increased plasma ACTH, decreased plasma cortisol and dehydroepiandrosterone, and no steroidogenic response to exogenous ACTH, they responded normally to both furosemide administration and to a low sodium diet by showing increases in plasma aldosterone. The peripheral blood mononuclear leukocytes (MNLs) from these patients possessed ACTH receptors similar to adrenocortical ones, which was in contrast to a previously reported case with a deficiency of ACTH receptors in the MNLs. Furthermore, ACTH receptors in the patients' MNLs were functionally coupled to adenylate cyclase. Dibutyryl cAMP infusion did not, however, increase plasma cortisol nor aldosterone in these patients in a sharp contrast to its remarkable increase in a normal control subject. These results suggest that these patients represent a new subtype of HACUA with a failure of intracellular reception of the cAMP message in adrenocortical cells. We propose to classify our patients with a postreceptor defect as HACUA type II using an analogy to pseudohypoparathyroidism type II.


Inhibition of progestin accumulation by activin-A in human granulosa cells. The effects of activin and inhibin on steroidogenesis in the human ovary were investigated. Granulosa cells harvested from follicles of women undergoing oocyte recovery for in vitro fertilization were maintained in culture for 4 days before treatment in serum-free medium. Human recombinant inhibin-A and activin-A at concentrations of 100 ng/mL did not affect basal progesterone secretion (P greater than 0.05). Progesterone concentrations were increased 2- to 6-fold by hCG or FSH. Activin-A inhibited the progesterone response to hCG compared with that of cells treated with hCG alone (P less than 0.01). The effect of activin-A was dose dependent and significant at 16-18 h of treatment (P less than 0.01). Inhibin-A at the same concentrations as activin-A had no effect on the progesterone responses to hCG and FSH. The hCG-induced accumulation of 20 alpha-hydroxyprogesterone was also attenuated by simultaneous activin-A treatment compared to that in cells treated with hCG alone (P less than 0.01). To investigate the mechanism of action of activin-A, cells were treated with a cAMP analog (8-bromo-cAMP) or an activator of adenylate cyclase (forskolin), with or without activin-A. Activin-A had no effect on 8-bromo-cAMP-stimulated progesterone accumulation. Likewise, forskolin-stimulated progesterone accumulation was not affected by activin-A. The hCG-induced increase in intracellular cAMP was decreased by activin-A in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine (P less than 0.01). Thus, activin-A may inhibit progesterone production by suppression of gonadotropin-induced cAMP production. These results support an autocrine role of activin-A in the steroidogenic capacity of human ovarian cells.


Fetal and maternal endocrine responses to reduced uteroplacental blood flow. In clinical practice, falling or reduced maternal estrogen levels are commonly regarded as indicators of fetal distress. However, experimental studies in primate animal models demonstrate that changes in maternal estradiol concentrations vary in response to reduced uteroplacental blood flow and are elevated during fetal hypoxemic stress, suggesting an increase in fetal adrenal precursor steroids. We studied the effects of graded reductions in maternal distal aortic blood flow (Qda) on the fetal MCR of dehydroepiandrosterone (D; MCR-D), the fetal production rate of D (PR-D), and changes in maternal and fetal plasma concentrations of D, D sulfate, cortisol, androstenedione, estrone (E1), and estradiol (E2) and in fetal plasma ACTH, PRL, and LH. A continuous i.v. infusion of [7-3H]D was administered to fetuses in five pregnant baboons (Papio anubis) at 155-165 days gestation (term, 184 days) for 270 min. A 50% reduction in mean distal aortic blood flow was imposed after 60 min by means of partial occlusion of the aorta with a snare device, which was released at 180 min. Maternal and fetal blood samples were collected at 10-min intervals from 30-60, 120-180, and 240-270 min. Equilibrium concentrations of [3H]D in fetal plasma were determined, and the MCR-D and PR-D were calculated for each of the three levels of Qda corresponding to the control, occlusion, and release intervals. Concentrations of steroid and peptide hormones in maternal and fetal plasma were determined by RIA, and arterial blood pH, pO2, and pCO2 were measured. Control fetal PR-D (mean +/- SE, 4.4 +/- 2.0 mg/day) rose significantly during aortic occlusion accompanied by fetal hypoxemia (11.8 +/- 3.1 mg/day; P less than 0.05) and remained elevated with release of the aortic constriction (13.8 +/- 2.9 mg/day). Changes in fetal MCR-D were variable and not statistically significant. Among the maternal plasma steroids, only E1 and E2 increased significantly, doubling from control values during aortic occlusion and increasing by another 50% after release (P less than 0.05). There was a significant correlation between fetal PR-D and maternal plasma E2 and E1 concentrations (r2 = 0.76 and 0.71, respectively; P less than 0.01). Fetal hypoxemia was associated with dramatic increases in fetal plasma D, D sulfate, androstenedione, E1, and E2. No significant change occurred in fetal plasma cortisol, which tended to decline throughout the study. We observed a dramatic and sustained increase in fetal plasma ACTH during the period of reduced Qda and for 90 min thereafter, but no change in PRL or LH.(ABSTRACT TRUNCATED AT 400 WORDS)


Murine mucosal T cells have VIP receptors functionally distinct from those on intestinal epithelial cells. Reports suggest that vasoactive intestinal peptide (VIP) binds to lymphocytes and modulates immune responses. The intestines are richly innervated with VIP-producing nerves. Thus, VIP from nerves or other sources may participate in mucosal immunoregulation. To explore this hypothesis further, murine intestinal mucosal inflammatory cells were scrutinized for functional VIP receptors. An [125I]VIP competitive binding assay characterized VIP receptors. Unfractionated lamina propria inflammatory cells bound [125I]VIP specifically. This binding was abrogated by T cell depletion. The VIP receptor on lamina propria T cells was of a single class with a Kd of 9.08 x 10(-9) M. It bound PHI and other peptide analogs poorly. The intestinal epithelial cell had a high-affinity VIP receptor (Kd 4.17 x 10(-10) M) that bound one VIP analog with moderate affinity. Both VIP and ConA stimulated mucosal inflammatory cells to release interleukin-5 (IL-5). Mucosal inflammatory cells depleted of T cells did not release IL-5 in response to VIP or ConA. It is concluded that: (1) some murine mucosal T lymphocytes have VIP receptors that may be distinct from those displayed on mucosal epithelial cells; (2) VIP affects mucosal T lymphocyte function.


Characterization of ACTH mediated suppression of MHC class II expression by murine peritoneal macrophages. The effect of ACTH on the expression of major histocompatibility complex (MHC) class II (I-A) glycoprotein by murine peritoneal macrophages was evaluated. ACTH suppressed the expression of I-A by macrophages in a time- and dose-dependent manner. ACTH mediated its effect by decreasing the level of I-A mRNA. ACTH suppressed the expression of I-A by macrophages from mice that are susceptible to the in vivo growth of mycobacteria but did not affect the expression of I-A by macrophages from Mycobacterium bovis strain (BCG)-resistant mice. The concentrations of ACTH required to suppress I-A expression were greater than that required for an effect on adrenal steroid production and may be related to the localized production of ACTH by lymphocytes and macrophages.


Autocrine and paracrine regulation of astrocyte function by transforming growth factor-beta. Recent evidence indicates that astrocytes have a wide range of functions, usually attributed to cells of the immune system, which are critical for maintaining a balanced homeostatic environment in the central nervous system (CNS). Moreover, these cells are known to participate in inflammatory events within the CNS by secreting cytokines such as transforming growth factor-beta (TGF-beta). In this study we have investigated the ability of TGF-beta to influence astrocyte functions. TGF-beta 1 mRNA is constitutively expressed by astrocytes in vitro, and when cultures are stimulated with exogenous TGF-beta 1 an increase in the expression of this mRNA can be shown, suggesting both autocrine and paracrine regulation. In in vitro assays, TGF-beta 1 is chemotactic for astrocytes in a dose-dependent fashion and inhibits astrocyte proliferation. These results indicating signal transduction by TGF-beta 1-prompted studies to explore receptor-ligand interactions on isolated astrocyte populations. In a receptor binding assay, we demonstrate that astrocytes appear to express three distinct TGF-beta receptor subtypes with nearly 10,000 receptors per cell. Thus, TGF-beta may play an important role in regulating astrocyte functions pivotal to the evolution of intracerebral immune responses including recruitment and activation of glial cells at local inflammatory sites within the CNS.


Role of sulfhydryl groups in the binding of vasoactive intestinal peptide to its receptor on murine lymphocytes. The effect of sulfhydryl-containing compounds on the specific binding of the neuropeptide vasoactive intestinal peptide (VIP) to murine lymphocytes was studied. Both 2-mercaptoethanol (2ME) and dithiothreitol (DTT) inhibited VIP-binding to lymphocytes at millimolar concentrations. A sulfhydryl-containing analogue of VIP, [Cys2]VIP, was synthesized. This compound competed for the binding of [125I]VIP about 150,000 x more effectively than 2ME, but was approximately 100 x less effective than VIP itself. Both VIP and [Cys2]VIP increased intracellular cyclic AMP and inhibited the proliferative response of lymphocyte cultures to concanavalin A (ConA), but the molar potency of [Cys2]VIP on these lymphocyte activities was approximately 100 x less than that of VIP. The effects of VIP and [Cys2]VIP on intracellular cyclic AMP and ConA-stimulated proliferation were competed for by the VIP receptor antagonist [4Cl-D-Phe6,Leu17]VIP. Replacement of serine2 with L-cysteine disrupts the ability of VIP to occupy and activate lymphocyte VIP receptors. This may reflect a role of serine2 in hydrogen-bond formation during ligand-receptor interactions, or a functional role of sulfhydryl-containing residues of the VIP receptor in maintaining the integrity of the binding site of the VIP receptor on lymphocytes.


Effects of stimulus duration on the response properties of Pacinian corpuscles: implications for the neural code. Based on physiological and psychophysical data, it has been suggested that the neural code for threshold detection in the P channel, mediated by Pacinian corpuscles (PCs), may be 2-4 neural impulses/stimulus (Bolanowski et al., 1988). To further test the efficacy of this code, responses from PCs were measured at different vibratory burst durations. Since it is known that the P channel has the capacity to summate vibratory stimuli temporally within the central nervous system, the effect should also be present in the physiological results. Temporal summation predicts a decrease in threshold at a rate of -3 dB/doubling of stimulus duration. Responses from single PCs were integrated by counting neural spikes over the entire burst duration. Furthermore, real-time responses were integrated with a low-pass filter, more accurately modeling the central process. For the spike-counting scheme, a criterion of 4 impulses/stimulus showed a decrease in stimulus amplitude for increases in duration similar to that obtained psychophysically. The amplitude-duration function obtained with the low-pass filter, however, resulted in a function which did not follow that obtained psychophysically, regardless of the number of impulses/stimulus. Since the P channel is known to have a central integrator, it has been concluded that activity of a single PC afferent probably is not sufficient to signal threshold in the P channel.


Clinical and epidemiological features of acute gastroenteritis caused by human rotavirus subgroups. Rotavirus was detected in 44 (15.28%) of 288 hospitalised children suffering from gastroenteritis in Delhi over a period of one year. Of these 44 children, subgroup I and II rotaviruses were detected in 13 (29.55%) and 25 (56.82%) children respectively. Samples from 5 (11.36%) children did not react with either subgroup I or II monoclonal antibodies, indicating the presence of a third subgroup. Both subgroups I and II were detected in one (2.27%) case. The median ages of the children infected by subgroup I and II rotavirus were 7 and 12 months respectively. Rotavirus was more prevalent in boys than in girls. The peak of rotavirus infection was seen in the early winter months. The difference of maximum and minimum monthly temperatures correlated significantly (p less than 0.05) with the incidence of rotavirus infection. Subgroup II was the predominant group throughout the period of study and caused a significantly higher incidence of severe diarrhoea than did subgroup I.


Azithromycin and clarithromycin: overview and comparison with erythromycin. Azithromycin and clarithromycin are erythromycin analogues that have recently been approved by the FDA. These drugs inhibit protein synthesis in susceptible organisms by binding to the 50S ribosomal subunit. Alteration in this binding site confers simultaneous resistance to all macrolide antibiotics. Clarithromycin is several-fold more active in vitro than erythromycin against gram-positive organisms, while azithromycin is 2- to 4-fold less potent. Azithromycin has excellent in vitro activity against H influenzae (MIC90 0.5 microgram/ml), whereas clarithromycin, although less active against H influenzae (MIC90 4.0 micrograms/ml) by standard in vitro testing, is metabolized into an active compound with twice the in vitro activity of the parent drug. Both azithromycin and clarithromycin are equivalent to standard oral therapies against respiratory tract and soft tissue infections caused by susceptible organisms, including S aureus, S pneumoniae, S pyogenes, H influenzae, and M catarrhalis. Clarithromycin is more active in vitro against the atypical respiratory pathogens (e.g., Legionella), although insufficient in vivo data are available to demonstrate a clinical difference between azithromycin and clarithromycin. Superior pharmacodynamic properties separate the new macrolides from the prototype, erythromycin. Azithromycin has a large volume of distribution, and, although serum concentrations remain low, it concentrates readily within tissues, demonstrating a tissue half-life of approximately three days. These properties allow novel dosing schemes for azithromycin, because a five-day course will provide therapeutic tissue concentrations for at least ten days. Clarithromycin has a longer serum half-life and better tissue penetration than erythromycin, allowing twice-a-day dosing for most common infections. Azithromycin pharmacokinetics permit a five-day, single daily dose regimen for respiratory tract and soft tissue infections, and a single 1 g dose of azithromycin effectively treats C trachomatis genital infections; these more convenient dosing schedules improve patient compliance. Azithromycin and clarithromycin also are active against some unexpected pathogens (e.g., B burgdorferi, T gondii, M avium complex, and M leprae). Clarithromycin, thus far, appears the most active against atypical mycobacteria, giving new hope to what has become a difficult group of infections to treat. Gastrointestinal distress, a well known and major obstacle to patient compliance with erythromycin, is relatively uncommon with the new macrolides. Further clinical data and experiences may better define and expand the role of these new macrolides in the treatment of infectious diseases.


Effect of nicardipine treatment upon cardiac hypertrophy in spontaneously hypertensive rats: a morphometric and ultrastructural study. The present study was designed to investigate the effect of nicardipine administration upon systolic blood pressure (SBP) and cardiac hypertrophy in spontaneously hypertensive rats (SHR). SBP, heart: and left ventricle: body weight ratios, the cross-sectional area of cardiocytes, and the ultrastructure of the left ventricle were evaluated. Ten-week old male SHR and age-matched normotensive Wistar-Kyoto rats were studied for 12 weeks. One group of SHR was treated for 12 weeks with a daily oral dose of 1 mg/kg nicardipine and another group with 1 mg/kg hydralazine; Wistar-Kyoto rats were used as a normotensive control group. Light and electron microscope techniques associated with image analysis and morphometry were used. Nicardipine administration normalized SBP values and significantly reduced the heart: and left ventricle: body weight ratios. Moreover, administration reduced the cross-sectional area of cardiocytes by approximately 38% in subendocardium and by 24% in subepicardium. Hydralazine administration significantly reduced SBP values but had no effect upon heart: or left ventricle: body weight ratios or the cross-sectional area of cardiocytes. Electron microscopy showed that nicardipine treatment was able to reduce the hypertension-dependent changes in cardiac ultrastructure consisting of alternations to intercalated discs and line Z morphology as well as in the decrease of the mitochondria: myofibrils ratio. The above data indicate that nicardipine administration is able to reduce SBP and to counter the development of structural and ultrastructural changes in cardiac morphology which represent a common complication of arterial hypertension.


Enhanced spontaneous calcium efflux and decrease of calcium-dependent calcium release from the isolated perfused heart of spontaneously hypertensive rats. The aim of this study was to clarify the further details of calcium handling in hypertension. By preserving the physiological environment of cell membrane, whole hearts were used for comparison of calcium flux between spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Hearts from SHR and WKY rats were perfused with Krebs-Henseleit solution under constant flow and the effluent collected. After labelling of the heart with 45Ca2+ (100 mumol/l), 45Ca2+ binding was found to be saturated, and washing with calcium-free perfusion solution showed two exponential curves for calcium dissociation, indicating a fast (alpha-) and slow (beta-) phase. The half-lives of the beta-phase for both 4- and 8-week-old SHR were significantly shorter than those for age-matched WKY. Also in this phase, infusion of non-radioactive Ca2+ caused a transient dose-dependent release of 45Ca2+. A significant reduction in the amount of 45Ca2+ release induced by 2 mmol/l Ca2+ was observed in both 4- and 8-week-old SHR compared with age-matched WKY rats. Infusion of lanthanum, caffeine, ionomycin (calcium ionophore) and treatment of the hearts with ethyleneglycol-bis-(beta-aminoethylether)-N,N,N,',N'-tetraac etic acid did not alter 45Ca2+ release by non-radioactive Ca2+. From these observations, 45Ca2+ is presumably released from the intracellular calcium pool, and not from extracellular binding sites or sarcoplasmic reticulum. These findings suggest that an abnormal calcium-handling defect (enhanced calcium efflux and reduction of membrane-bound Ca2+) exists under physiological conditions before and after the onset of hypertension, and that this may be a primary characteristic of SHR.


Contractile properties of ventricular myocytes isolated from spontaneously hypertensive rat. To determine whether there are any differences in contractile properties of individual cardiac myocytes isolated from the spontaneously hypertensive rat (SHR) in comparison with its normotensive control--the Wistar-Kyoto (WKY) rat. The effects of cardiac hypertrophy upon individual myocytes from SHR have not been studied previously. Isolated cardiac myocytes do not suffer from a number of problems inherent in experiments on multicellular preparations. Seven SHR and eight WKY animals were studied. Age-matched animals were compared at 60 and 100 days old. Ventricular myocytes were isolated enzymatically. Myocyte length and width was measured. The cells were stimulated with extracellular electrodes and contraction was measured optically. The effects of altering stimulus rate and extracellular calcium concentration upon contraction were studied. SHR myocytes were found to be significantly wider than WKY myocytes. The contraction (i.e. unloaded cell shortening) of SHR myocytes at stimulation rate of 0.3, 1, 2 and 3 Hz was significantly increased. The time-course of contraction was altered, with SHR myocytes having an increased maximal velocity of shortening and relaxation. The response to changes in bathing calcium was similar in both strains. Individual cardiac myocytes isolated from SHR have an increased contraction. This indicates that cardiac hypertrophy, at least in the early stages, is a protective adaptation allowing the heart to overcome the increased afterload resulting from hypertension.


Chronic alcohol consumption lowers blood pressure but enhances vascular contractility in Wistar rats. To determine the effect of chronic alcohol consumption upon blood pressure, blood pressure reactivity and vascular contractility in Wistar rats. Wistar rats were fed a liquid diet containing 36% ethanol; control rats were pair fed. Rats were maintained on diets for 18 weeks. Indirect systolic blood pressure was measured weekly. Catheters were implanted for assessment of direct arterial pressure and blood pressure reactivity to norepinephrine, angiotensin II and ethanol injections. In a subgroup of rats, contractility of isolated mesenteric resistance vessels was measured. In comparison with simultaneously pair-fed controls, ethanol-treated rats developed significantly lower blood pressure within 3 weeks of exposure to alcohol; this continued throughout the study. Despite the reduction in blood pressure, in vitro assessment of vascular contractility in mesenteric resistance vessels indicated that ethanol consumption significantly enhanced vascular contractility to norepinephrine and attenuated the vasodepressive effects of ethanol. Measurement of blood pressure reactivity to infused pressor agents showed no difference between controls and ethanol-treated rats in response to norepinephrine but a significantly attenuated pressor response to angiotensin II was observed in ethanol-treated rats. The blood pressure results contrast with reports of elevated blood pressure in Wistar rats given ethanol in drinking water. This disparity may be due to nutritional factors. Increased vascular contractility combined with hypotension suggests that cardiovascular regulatory systems offset the direct effects of ethanol upon the vasculature. This view is reinforced by the lack of difference between groups in blood pressure reactivity to norepinephrine. The attenuated angiotensin II responses in the ethanol-treated rats suggests altered levels of circulating angiotensin II in this group.


Adenosine dephosphorylates myosin light chain in primary cultures of vascular smooth muscle cells from normotensive and spontaneously hypertensive rats. The aim was to determine whether vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) respond differently to adenosine than those from normotensive Wistar-Kyoto (WKY) rats. Confluent primary cultures of VSMC derived from SHR and WKY aorta and mesenteric arteries and cerebral arteries were used. The effect of adenosine upon cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) formation and the phosphorylation of myosin light chain (MLC) was studied. MLC phosphorylation was estimated by subjecting VSMC extracts incubated with 32P to gel electrophoresis, followed by autoradiography and laser densitometry. cAMP and cGMP levels were measured by radioimmunoassay. Baseline MLC phosphorylation levels were not significantly different in SHR and WKY VSMC. Adenosine caused dephosphorylation of MLC in a time- and dose-dependent manner. A maximal response of approximately 40% below control values was observed 5 min after addition of 10(-5) mol/l adenosine in SHR and WKY VSMC with no significant difference between the two strains. The maximally effective concentration of 10(-5) mol/l adenosine evoked increases in both cAMP and cGMP in VSMC from SHR and WKY rats to the same degree. We conclude that the overall ability of VSMC to relax, as evidenced by a marked decrease in MLC phosphorylation in response to adenosine, is unaltered in SHR.


Effect of angiotensin converting enzyme inhibitors upon brain angiotensin II binding. The major objective of the current study was to determine whether administration of converting enzyme (CEI) inhibitors produces an alteration in angiotensin II receptor binding in the spontaneously hypertensive rat (SHR). These studies were designed to test the hypothesis that administration of CEI to SHR is associated with a downregulation of the brain angiotensin II receptor. Ten to fifteen rats per group were used as mating pairs to produce the pups used for cell culture. The specific binding of 125I-angiotensin II to cell membranes was determined in primary neuronal enriched cultures (PNC) developed from whole brains of 1-day-old SHR pups treated in utero with captopril compared with control SHR pups. We then determined the effect of short-term incubation of PNC with captopril or lisinopril upon angiotensin II binding kinetics. 125I-angiotensin II binding in PNC from captopril-treated SHR was decreased compared with cells from control rats. Scatchard analysis revealed no differences in receptor affinity but maximum density of binding sites was less in captopril-treated rats than in controls. Short-term incubation of cells with captopril or lisinopril did not affect angiotensin II binding in neuronal cultures from normotensive rats; however, angiotensin II binding was decreased in SHR cells treated with captopril. Angiotensin II binding was also decreased in SHR treated in utero with captopril. These data suggest that CEI decrease 125I-angiotensin II binding in brains from SHR and, furthermore, that the angiotensin II receptor in SHR may be regulated differently from that of normotensive rats.


High cardiac zinc levels in spontaneously hypertensive rats. Higher cardiac zinc levels have been observed previously in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto (WKY) rats. However, this difference was established in adult males only and needed to be confirmed on a larger number of animals of both sexes. We also explored the respective roles of genetic and environmental factors in the genesis of this zinc anomaly as well as the causal relations with hypertension. Cardiac zinc levels were determined in adult male and female SHR and WKY rats originating from various colonies and submitted to various experimental conditions (anaesthesia, stress). These determinations were also performed in 3-week prehypertensive SHR and in adult Wistar rats submitted or not to deoxycorticosterone acetate-salt-induced hypertension. Zinc levels were measured by flame atomic absorption spectrophotometry. In adults, cardiac zinc content was significantly higher in SHR than in WKY rats irrespective of sex and experimental conditions. In young prehypertensive rats, the difference between SHR and WKY cardiac zinc levels was also very significant. Experimental hypertension induced in Wistar rats did not entail any significant rise in cardiac zinc levels. These findings indicated that the higher cardiac zinc of SHR is not secondary to blood pressure elevation. High erythrocyte zinc, previously described in SHR, together with the present data suggest the occurrence of a primary genetic defect leading to high intracellular zinc in SHR. The possible role of this zinc anomaly in the development of hypertension and/or cardiac hyperplasia is discussed.


Depressor systems contribute to hypertension induced by glucocorticoid excess in dogs. The aim of this study was to investigate the role of depressor systems in glucocorticoid-induced hypertension. The serial changes in cardiorenal hemodynamics, urinary excretions of kallikrein and prostaglandins (PGE2 and the prostacyclin derivative 6-keto-PGF1 alpha) before, and during the administration of both low and high doses of dexamethasone (9 alpha-fluoro-16 alpha-methylprednisolone) and after the cessation of dexamethasone were examined in conscious trained dogs. In addition, pressor responses to prostaglandin, bradykinin, bradykinin antagonist and indomethacin were studied during the administration of dexamethasone. High-dose dexamethasone induced a significant elevation in mean arterial pressure (MAP) that was accompanied by a significant reduction in the urinary excretion of kallikrein, PGE2 and 6-keto-PGF1 alpha. In contrast, low-dose dexamethasone treatment had no significant effect upon MAP but induced a transient elevation in the urinary excretion of kallikrein, PGE2 and 6-keto-PGF1 alpha. Furthermore, additional oral administration of indomethacin produced a significant elevation in MAP in dogs treated with low-dose dexamethasone; but did not affect the hemodynamics of animals with high-dose dexamethasone. Whilst i.v. administration of either bradykinin or prostacyclin induced a significant reduction in MAP in high-dose but not low-dose dexamethasone-treated dogs, administration of a competitive bradykinin antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin induced a significant elevation in MAP in low-dose but not high-dose dexamethasone-treated dogs. Depressor systems play an important role in regulation of blood pressure in glucocorticoid-treated dogs.


Platelet and erythrocyte Mg2+, Ca2+, Na+, K+ and cell membrane adenosine triphosphatase activity in essential hypertension in blacks. To assess the relationship between intracellular Mg2+, Ca2+, Na+ and K+ and cell membrane adenosine triphosphatase (ATPase) activity in normotensive and hypertensive blacks. Intracellular cations and cell membrane ATPase activity were studied in black patients with untreated essential hypertension and age-, weight- and height-matched normotensive controls. Platelet, erythrocyte and serum Mg2+, Ca2+, Na+ and K+ levels as well as platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities were measured in all subjects. Intracellular Na+ and K+ were measured by flame photometry and Mg+ and Ca+ by atomic absorption spectrophotometry. Cell membrane ATPase activity was determined by a colorimetric method. The hypertensive group consistently demonstrated depressed activity of each ATPase studied, with significantly lower serum Mg2+, serum K+, erythrocyte Mg2+ and platelet Mg2+ levels compared with the normotensive group. Platelet Na+ and Ca2+ and erythrocyte Ca2+ were significantly elevated in the hypertensive group. In the hypertensive group, mean arterial pressure (MAP) was inversely correlated with platelet and erythrocyte membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase. Serum Mg2+, serum Ca2+ and platelet Mg2+ were negatively correlated with MAP in the hypertensive group whilst erythrocyte and platelet Ca2+ were positively correlated. In the normotensive group, platelet Mg2+ and MAP were negatively, and erythrocyte Ca2+ and MAP, positively correlated. Black patients with essential hypertension have widespread depression of cell membrane Na+,K(+)-ATPase, Ca(2+)-ATPase and Mg(2+)-ATPase activities with serum and intracellular Mg2+ depletion and cytosolic Na+ and Ca2+ overload, which may reflect an underlying membrane abnormality in essential hypertension. These cellular abnormalities may be related to the defective transport mechanisms that in turn may be aggravated by Mg2+ depletion.


Increased activity of the Na(+)-dependent Cl(-)-HCO3- anion exchanger in erythrocytes of patients with essential hypertension. To study the activity of the Na(+)-dependent Cl(-)-HCO3- anion exchanger in erythrocytes of patients with essential hypertension. The study was performed in cells from 48 untreated essential hypertensive patients and 30 normotensive controls with similar age- and sex- distribution. The activity of the Na(+)-dependent anion exchanger was determined by measuring the 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) sensitive Li+ influx in fresh cells incubated into a medium containing Li2CO3. The DIDS-sensitive Li+ influx was higher in hypertensives than controls. With the 100% confidence (upper) limit of the normotensive population as a cut-off point, a subgroup of 11 hypertensives had an abnormally high activity of the Na(+)-dependent Cl(-)-HCO3- anion exchanger. Compared with patients with normal exchanger activity, patients with increased exchanger activity were characterized by the following: higher frequency of family history of hypertension; lower serum high-density lipoprotein cholesterol levels and higher plasma aldosterone concentrations. After multiple regression analysis, the DIDS-sensitive Li+ influx was inversely correlated with high-density lipoprotein cholesterol levels. These results show the presence of a new abnormality of erythrocyte Na+ transport in essential hypertension--increased activity of the Na(+)-dependent Cl(-)-HCO3- anion exchanger. In addition, our findings suggest that from the clinical point of view, patients with this transport abnormality represent a particular subset of essential hypertensives.


Withdrawal of antihypertensive drug treatment: time-course for redevelopment of hypertension and effects upon left ventricular mass. To examine: (1) in how many treated hypertensive patients it was possible to discontinue drug treatment; (2) the time-course for redevelopment of hypertension after discontinuation of therapy; and (3) whether drug withdrawal was associated with an increase in left ventricular mass (LVM). Fifty-four men with primary hypertension treated for a mean period of 6 years (primarily beta 1-selective beta-blockade) were evaluated for withdrawal of treatment. Exclusion criteria were signs of organ damage, severe hypertension, other serious disease and unwillingness. Treatment was reinstituted if blood pressure increased above a safety level or if symptoms occurred. Echocardiographic estimations of LVM were obtained before withdrawal and 1, 4 and 8 weeks after withdrawal or before return to treatment. Outpatient clinic in a city hospital. A random sample of 56-year-old hypertensive men. Gradual discontinuation of treatment with close follow-up of blood pressure. Number of patients who could withdraw from treatment and who had to return to pharmacological therapy; time-courses for development of hypertension; absolute changes in LVM. Thirty-two patients withdrew from treatment for 1-1000 days. Therapy was reinstituted in all owing to hypertension or symptoms. Serial echocardiograms were obtained in 22 patients. During the drug-free period, relative wall thickness increased, but LVM did not. Patients with rapid redevelopment of hypertension had larger prewithdrawal LVM than patients whose blood pressure increased more slowly. It was possible to withdraw treatment and obtain readable echocardiograms in a minority of the patients. After drug-withdrawal, relative wall thickness increased, but not LVM. We suggest that previously treated patients should be avoided in studies examining reversal of left ventricular hypertrophy.


Short-term representatives of daytime and night-time ambulatory blood pressures. To see whether measurements of ambulatory blood pressure during short-term daytime and night-time periods can represent complete daytime and night-time pressures accurately. Short-term measurements would be less uncomfortable for patients, easier to perform and could lead to fewer missing values, outliers or artefacts than full-day measurements, especially when repeated monitorings are required. Ambulatory blood pressure was measured every 15 min for 24 h in 254 subjects with normal or borderline office blood pressure. Each pressure profile included at least 80 valid readings. Mean blood pressures for different 1-, 2-, 3-, 4-, 5- and 6-h spans were calculated and compared with mean daytime and night-time values using paired Student's t-test. One or two-hour spans of daytime blood pressure poorly represented mean daytime pressure. In contrast, 4-h readings, selected between 1000-2200 h represented daytime blood pressure with good accuracy. Over the total sample, 4-h mean blood pressure readings from 1000-2200 h differed from daytime readings by less than 2 mmHg and 2-h mean readings from 0300-0700 h differed from mean night-time readings by less than 1 mmHg. We suggest that 4-h measurements of ambulatory blood pressure during the daytime and 2-h measurements at night (with time spans selected as indicated as above) may be of value.


[Activated macrophages in the peritoneal fluid of women with endometriosis: examination of the intracytoplasmic localization of endogenous peroxidase and interleukin-1]. In order to study the effect of peritoneal macrophages (M phi) on conception in patients with endometriosis, the total numbers of peritoneal M phi and the proportion of the exudate type, defined on the basis of ultracytochemical localization of endogenous peroxidase (PO) activity, were investigated in 21 patients with endometriosis, five with uterine leiomyoma, three with tubal obstruction and three with carcinomatous peritonitis. An immunocytochemical observation of interleukin-1 (IL-1) was also performed in three patients with endometriosis, one with tubal obstruction, and one male patient with cholelithiasis. The total numbers of peritoneal M phi in patients with endometriosis were significantly higher than in uterine leiomyoma (2.11 x 10(7) v.s. 0.68 x 10(7), p less than 0.025). The total numbers of peritoneal M phi in tubal obstruction (0.96 x 10(7)) were not statistically different from those in uterine leiomyoma. The peritoneal M phi were remarkably increased in number in patients with carcinomatous peritonitis. On ultracytochemical observation of endogenous PO activity, the proportion of exudate M phi to whole M phi was significantly larger in endometriosis than that in uterine leiomyoma (13.3% v.s. 3.5%, p less than 0.025). This type of M phi increased even in stage I endometriosis (p less than 0.005). These data suggest that the abdominal cavity in women with endometriosis is in the stimulated conditions which may lead to infertility. A positive reaction to anti-IL-1 antibody on the cell membrane of all M phi examined in each patient suggests that an immunocytochemical study of IL-1 in M phi is not suitable for evaluating the degree of activation of M phi.