Journal of cyclic nucleotide and protein phosphorylation research最新文献

{"title":"Desensitization in rat parotid to beta-adrenergic agonists and counteracting effects of forskolin are conserved in membrane and detergent-solubilized adenylate cyclase catalyst activity.","authors":"J F Harper","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Cyclic AMP accumulation in rat parotid slices is only transiently stimulated by isoproterenol (Harper, J.F. and Brooker, G. Molec. Pharmacol. 13:1048-1059, 1977); the progressive loss of isoproterenol effect is termed desensitization. In this report we show that desensitized cyclic AMP accumulation is associated with desensitization of adenylate cyclase in subsequently prepared membranes and in adenylate cyclase that has been detergent-solubilized from desensitized membranes. Adenylate cyclase in membranes made from isoproterenol-desensitized tissue is desensitized to both the stimulating effects of isoproterenol with 6 mM MgCl2 and of forskolin with 30 mM MnCl2. We have previously determined (Harper, J.F. J. Cyclic Nucleo. Prot. Phosphoryl. Res. 9:401-414, 1984) that cyclic AMP accumulation desensitized to isoproterenol is rapidly counteracted by 1 microM forskolin but not 0.1 microM forskolin. Similarly, if 1 microM forskolin was included in the desensitizing incubation with isoproterenol then adenylate cyclase subsequently prepared was not desensitized. Development of desensitized adenylate cyclase was only partially affected by 0.1 microM forskolin. Desensitization is counteracted by forskolin only on intact cells. Once tissue is homogenized, desensitized adenylate cyclase does not respond as well to forskolin as does control adenylate cyclase. The site of desensitization appears to be at or near the adenylate cyclase catalytic unit. Desensitization of adenylate cyclase catalytic activity remains demonstrable after membranes are solubilized with CHAPS. The adenylate cyclase activity remaining in the supernatant following solubilization of desensitized membranes is depressed to nearly the same extent as found in the membranes. Further, desensitized adenylate cyclase in membrane preparations and after solubilization is desensitized to stimulatory effects of forskolin with 30 mM MnCl2, a condition under which forskolin is probably acting directly on the adenylate cyclase catalytic unit. Desensitization appears not to be dependent on activity of the inhibitory guanine nucleotide regulatory protein (Gi), since pertussis toxin is without effect on desensitization of cyclic AMP accumulation to isoproterenol.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"11 3","pages":"167-76"},"PeriodicalIF":0.0,"publicationDate":"1986-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14013847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calmodulin is required for a full activation of the calcium slow channels in heart cells.","authors":"G Bkaily,&nbsp;N Sperelakis","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The role of calmodulin in regulating the functioning of the calcium slow channels in cultured heart cell reaggregates was determined by using the liposome method for intracellular delivery of calmodulin, calmidazolium (calmodulin inhibitor), inhibitor of adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent protein kinase (PrK), and the catalytic subunit of cAMP-dependent protein kinase. The cells exhibited a naturally-occurring slowly-rising action potential (APs) having a maximum rate of rise (+Vmax) of less than 25 V/s. Injection of calmodulin inhibitor (calmidazolium) blocked the spontaneously occurring slow APs and depolarized the membrane. Simultaneous injections of calmidazolium and the inhibitor of cAMP-dependent protein kinase further depolarized the membrane. Injection of calmodulin did not restore the slow APs, but a subsequent injection of the catalytic subunit of cAMP-dependent protein kinase did. If the catalytic subunit of cAMP-PrK was injected before the injection of calmodulin, the slow APs recovered only partially; full recovery of the slow APs required a subsequent injection of calmodulin. These findings suggest a potentiating effect of calmodulin in the regulation of myocardial slow calcium APs and thus suggest that the myocardial slow channels protein or an associated regulatory protein(s) must be phosphorylated by the Ca2+-calmodulin-dependent protein kinase and the catalytic subunit of cyclic AMP-dependent protein kinase in order to make the channel fully available for voltage activation.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"11 1","pages":"25-34"},"PeriodicalIF":0.0,"publicationDate":"1986-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13570938","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiple molecular forms of phosphodiesterase and the regulation of cardiac muscle contractility.","authors":"R E Weishaar,&nbsp;D C Kobylarz-Singer,&nbsp;M M Quade,&nbsp;R P Steffen,&nbsp;H R Kaplan","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Two approaches were taken to examine the role which the different forms of phosphodiesterase present in cardiac muscle play in regulating contractility. In an initial study, the effect of selective inhibitors of i) the calmodulin-stimulated phosphodiesterase (M & B 22, 948), ii) the cyclic GMP-stimulated phosphodiesterase (dipyridamole), and iii) the low Km, cyclic AMP-specific phosphodiesterase (imazodan) on the contractility of isolated guinea pig left atria was examined. Of the three selective phosphodiesterase inhibitors, only imazodan increased atrial contractility. In a subsequent study, the effect of imazodan on in vivo contractility was evaluated. Imazodan was found to potently increase contractility in the dog and the Rhesus monkey, while exerting only modest-to-minimal effects of contractility in the guinea pig and the hamster. Imazodan produced no positive inotropic effect in the rat. These species differences can apparently be attributed to i) the presence of subclasses of the low Km, cyclic AMP-specific phosphodiesterase (PDE III) in cardiac muscle, one of which is potently inhibited by the selective PDE III inhibitors imazodan, cyclic GMP and cilostamide, and the other which is selectively inhibited by rolipram and Ro 20-1724, and ii) variations in the intracellular localization of imazodan-sensitive subclass of PDE III. Thus, the maximum inotropic response to imazodan was observed only in those species in which the imazodan-sensitive subclass of PDE III was present and was membrane-bound, e.g., Rhesus monkey and dog. In the dog, the imazodan-insensitive subclass PDE III does not appear to play an important role in regulating cardiac contractility. These observations provide further support for the hypothesis that the inotropic response to imazodan, amrinone and related cardiotonics is due to their inhibitory effects on the cyclic AMP-specific form of phosphodiesterase, and also provides new insight into the relationship between cyclic AMP, phosphodiesterase and myocardial contractility.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"11 7","pages":"513-27"},"PeriodicalIF":0.0,"publicationDate":"1986-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13969643","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On the role of protein kinase subunits in the control of eukaryotic gene expression.","authors":"D Schlichter,&nbsp;H Miller,&nbsp;W D Wicks","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Transcriptional regulation by cAMP has been demonstrated for several eukaryotic genes; however, the identity of the protein kinase subunit involved has been a source of debate. Based on homologies with the procaryotic cAMP-binding catabolite activator protein, a recent hypothesis has invoked the regulatory protein RII as the mediator. The evidence currently available on the effects of microinjected kinase subunits suggests, however, that the catalytic subunit is the active factor. Moreover, the proposed homologies between the catabolite activator protein and RII are difficult to reconcile with its proposed mediatory role. We suggest as an alternative hypothesis that a phosphoprotein other than RII may mediate the effects of cAMP on eukaryotic gene expression.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"11 3","pages":"149-54"},"PeriodicalIF":0.0,"publicationDate":"1986-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14152275","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Phorbol ester-induced augmentation and inhibition of epinephrine-stimulated adenylate cyclase in S49 lymphoma cells.","authors":"J A Johnson,&nbsp;T J Goka,&nbsp;R B Clark","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The effects of 4-beta phorbol 12-myristate 13-acetate (PMA) on hormone and forskolin-stimulated adenylate cyclase were evaluated in S49 lymphoma cells. Treatment of wild type (WT) S49 cells with PMA caused stimulation, inhibition or had no effect on epinephrine stimulation of cAMP accumulation. The effect observed was dependent on the length of PMA treatment, the concentration of PMA and the concentration of hormone (or forskolin) used to stimulate cAMP accumulation. Longer treatment times with PMA and higher PMA concentrations favored the inhibitory effects. Pretreating WT with 0.5 microM PMA for 18 min caused an increase in the EC50 and maximal levels for epinephrine stimulation of cAMP accumulation. Thus inhibition was seen at relatively low epinephrine concentrations and augmentation with high concentrations. The inhibitory effects of PMA on epinephrine-stimulated adenylate cyclase activity were observed only at low free Mg++ concentrations (0.75 mM). The effects of PMA on PGE1-stimulated cAMP accumulation were similar to those observed for epinephrine. In S49 WT cells 100 nM PMA augmented 5 microM forskolin-stimulated cAMP accumulation; however with 100 microM forskolin, PMA effects were minimal. PMA also attenuated Gi-mediated Gpp(NH)p inhibition of forskolin-stimulated adenylate cyclase in both WT and cyc- membranes, resembling the effects of pertussis toxin. The effects of various phorbol analogues on epinephrine-stimulated cAMP accumulation were as follows: 4 beta-phorbol 12,13-didecanoate had similar effects to PMA, 4 alpha-phorbol 12,13-didecanoate had no effects and 1-oleoyl, 2-acetylglycerol augmented epinephrine-stimulated cAMP accumulation at concentrations greater than or equal to 5 microM. Our results are consistent with a dual mechanism of PMA action on adenylate cyclase involving protein kinase C-mediated phosphorylation of Gi and of the beta-adrenergic receptor, the former leading to augmentation and the latter to inhibition of hormone-stimulated adenylate cyclase.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"11 3","pages":"199-215"},"PeriodicalIF":0.0,"publicationDate":"1986-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14154046","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Relationship between inhibition of cardiac muscle phosphodiesterases, changes in cyclic nucleotide levels, and contractile response for CI-914 and other novel cardiotonics.","authors":"R E Weishaar,&nbsp;M M Quade,&nbsp;J A Schenden,&nbsp;D B Evans","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>In the present study, the inhibitory effects of CI-914, a novel cardiotonic agent, as well as other recently identified positive inotropic agents and reference phosphodiesterase (PDE) inhibitors on the different molecular forms of cardiac PDE were examined, and correlated with changes in tissue levels of cyclic AMP and cyclic GMP, and the contractility of isolated guinea pig left atria produced by these agents. CI-914 was found to be a potent, selective inhibitor of peak III PDE, which is a low Km, cyclic AMP-specific form of the enzyme, with little effect on peak I or peak II PDE, both of which hydrolyze cyclic AMP as well as cyclic GMP. CI-914 also exerts a selective effect on cyclic nucleotides; increasing cyclic AMP levels in a concentration-dependent manner, while having no significant effect on cyclic GMP levels. Increases in contractility produced by CI-914 correlated with changes in the tissue level of cyclic AMP. Non-selective phosphodiesterase inhibitors such as theophylline, and less selective inhibitors such as papaverine, carbazeran, and milrinone increased tissue levels of both cyclic AMP and cyclic GMP. Increases in cyclic GMP, or the cyclic AMP/cyclic GMP ratio, did not appear to have an effect on contractility. These results provide support for the hypothesis that CI-914 increases contractility by selectively inhibiting the activity of the peak III phosphodiesterase. These results also indicate that cyclic AMP and cyclic GMP do not act in an opposing \"yin-and-yang\" fashion to regulate atrial contractility.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 6","pages":"551-64"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14136662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of a homogeneous class of beta 2-adrenoceptors in human platelets by (-)-125I-iodopindolol binding.","authors":"X L Wang,&nbsp;O E Brodde","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The highly specific beta-adrenoceptor radioligand (-)-125I-iodopindolol (IPIN) was used to label in membranes from human platelets beta-adrenoceptors. Binding of IPIN was at 25 degrees C saturable (Bmax = 7.4 +/- 2.5 fmoles bound/mg protein, N = 10) of high affinity (KD-value = 34.8 +/- 3.3 pM, N = 10), rapid and reversible. beta-Adrenoceptor antagonists inhibited binding of IPIN with monophasic displacement curves and pseudo Hill coefficients (nH) not significantly different from 1.0. The beta 2-selective antagonist ICI 118,551 was much more potent in inhibiting IPIN binding than the beta 1-selective antagonist bisoprolol. IPIN binding was stereospecific as indicated by the 100-times greater potencies of (-)-alprenolol and (-)-isoprenaline in inhibiting binding than their (+)-isomers. beta-Adrenoceptor agonists inhibited binding with slightly shallow displacement curves and nH-values of 0.85-0.88; the order of potency was: isoprenaline greater than adrenaline greater than noradrenaline. These results demonstrate that IPIN labels (in human platelet membranes with high affinity) a homogeneous population of beta 2-adrenoceptors with low capacity.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 5","pages":"439-50"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14002858","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Regulation of cyclic 3',5'-adenosine monophosphate phosphodiesterase in Friend erythroleukemia cells.","authors":"M Mason,&nbsp;S Narindrasorasak,&nbsp;B D Sanwal","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Friend erythroleukemia cells contain only a single form of cAMP phosphodiesterase with high affinity for substrate. It is activated by treatment with various proteases including those present in snake venom. The activity of this enzyme is increased about 2-fold when the cells are either cultivated in the presence of cAMP or in the presence of compounds which are capable of generating cAMP in vivo, such as isoproterenol, epinephrine and prostaglandins. The enzyme produced in the presence of cAMP is modified in such a way that it is no longer susceptible to activation by treatment with proteases. The activation and modification obtained in vivo can be duplicated in cell-free extracts of Friend cells, if they are incubated in the presence of cAMP and ATP. A possibility thus exists that the phosphodiesterase is activated by its substrate by phosphorylation.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 2","pages":"129-42"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"13724617","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Intracellular localization of the catalytic subunit from cyclic AMP-dependent protein kinase in mutant Chinese hamster ovary cells deficient in this enzyme.","authors":"C V Byus,&nbsp;W H Fletcher","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A direct cytochemical procedure that specifically locates free catalytic subunits (C) from cAMP-dependent protein kinase has been used to follow the kinetics of kinase dissociation in parental Chinese hamster ovary cells (CHO 10001) and 4 mutant CHO cell lines variously deficient in this enzyme (CHO 10215, 10248, 10260, 10265) (1-5). When cultures of wild-type cells were stimulated with 8BrcAMP a time- and dose-dependent dissociation of kinase was observed. The catalytic unit appeared first in the cytoplasm and nucleolus and with time in the nucleoplasm as well. At peak protein kinase activation (30 min) more than 80% of the cells possessed abundant C in all of these subcellular compartments. The data indicate that both the type I and type II isozymes of the cAMP-dependent protein kinase are localized in similar areas of the cell. Stimulation of the mutant cell lines with 8-BrcAMP revealed that they each contained activatable kinase, determined cytochemically, that paralleled in amount the total assayable protein kinase determined biochemically (1-6). There were differences in the basal (unstimulated) levels of free C in the mutants relative to each other and to wild-type cells. For example, wild-type and mutants 10248, and 10260 had barely detectable cytoplasmic catalytic-subunit in unstimulated cultures whereas mutant 10215 possessed a significant amount of free C. Upon stimulation with 8-BrcAMP, the subcellular distribution of C was in all cases similar; although there were significant quantitative disparities between the wild type and various mutant cell lines. All cell lines examined had roughly equivalent amounts of nucleolar C but differed predominantly in the quantity of cytoplasmic kinase. Specifically, two of the mutant lines (10260 and 10248) contained barely detectable amounts of nucleoplasmic C whereas the other mutants had moderate (10265) to near normal (10215) amounts of nucleoplasmic enzyme. Mutants having only type I isozyme (10248) and those with only type II isozyme (10265) gave similar patterns of free C localization after 8BrcAMP stimulation.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 1","pages":"9-22"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"15151446","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evidence for two specific affinity states of 3H-antagonist binding to cardiac beta-adrenergic receptors and influence of Gpp(NH)p.","authors":"P H Lang,&nbsp;B Lemmer","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Binding of the lipophilic antagonist ligand 3H-DHA and the hydrophilic antagonist ligand 3H-CGP 12177 to beta-adrenergic receptors of rat heart ventricular membranes was studied. Quantitative analysis of the binding data indicated the existence of two specific affinity states of the beta-adrenergic receptor population aside from a third non-specific binding site for 3H-DHA. In order to exclude that the biphasic saturation isotherm may be due to retained endogenous agonist, crude membranes as well as modified membranes were used. In the latter a 99% reduction of noradrenaline concentration was obtained by washing and preincubation or by catecholamine depletion. Two affinity states of antagonist binding could be demonstrated independently from the kind of membrane suspension. A biphasic dissociation of 3H-DHA by unlabelled (-)-alprenolol was also found in kinetic studies. In crude or washed membranes of untreated rats the guanine nucleotide Gpp(NH)p affected saturation and antagonist competition curves. However, this was not observed in catecholamine-depleted membranes of reserpine-treated rats. Stereoselectivity of the high and the low affinity state was demonstrated in competition experiments with (-) - and (+) -alprenolol in catecholamine-depleted membranes. The data are best explained by assuming a ternary complex model (1) in which antagonists, instead of passively occupying binding sites, play an active role in receptor mechanisms. Based on this model, it is assumed that beta-adrenergic antagonists bind with high affinity to the free form of the receptor and with low affinity to the precoupled form. Furthermore, an interaction of Gpp(NH)p with the regulatory component is proposed.</p>","PeriodicalId":15406,"journal":{"name":"Journal of cyclic nucleotide and protein phosphorylation research","volume":"10 4","pages":"341-60"},"PeriodicalIF":0.0,"publicationDate":"1985-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"14126841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}