Cyclic nucleotide phosphodiesterase of rat pancreatic islets. Effects of Ca2+, calmodulin and trifluoperazine.
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ABSTRACT:
SUBMITTER: Sugden MC
PROVIDER: S-EPMC1163146 | biostudies-other | 1981 Aug
REPOSITORIES: biostudies-other
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Project description:Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown.Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo.Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro.These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.
| S-EPMC2803071 | biostudies-literature
Project description:We have attempted to identify islet Ca2+/calmodulin-dependent protein kinase (CaM kinase) by comparing its activity with purified brain CaM kinase II. Islet CaM kinase, in the presence of calmodulin and Ca2+, phosphorylated major endogenous substrates of 102, 57 and 53 kDa and also exogenous glycogen synthase; brain CaM kinase II phosphorylated glycogen synthase and peptides of 57 and 53 kDa. Alloxan (1 mM) inhibited the phosphorylation of glycogen synthase and the 102, 57 and 53 kDa islet peptides by islet CaM kinase; the phosphorylation of glycogen synthase and the 57 and 53 kDa substrates by brain CaM kinase II was also inhibited by alloxan. The Ca2+ and calmodulin-dependencies of phosphorylation of the endogenous islet substrates differed. In the presence of 400 nM calmodulin, half-maximal phosphorylation was attained at Ca2+ concentrations of 80 +/- 9, 401 +/- 61 and 459 +/- 59 nM for the 102, 57 and 53 kDa substrates respectively. In the presence of 10 microM Ca2+, half-maximal phosphorylation was attained at calmodulin concentrations of 9 +/- 2, 38 +/- 2.5 and 37 +/- 2 nM for the 102, 57 and 53 kDa substrates respectively. Differential centrifugation located the 102 kDa substrate in the post-100,000 g supernatant and the 57 and 53 kDa substrates in the particulate fraction. These data suggest that islet CaM kinase is similar to, if not identical with, brain CaM kinase II, but that phosphorylation of the endogenous 102 kDa substrate occurs by a distinct kinase which shows different sensitivities to Ca2+ and calmodulin. This kinase probably corresponds to CaM kinase III and the 102 kDa peptide to elongation factor 2 (EF-2), since the 102 kDa peptide was shown to undergo ADP-ribosylation in the presence of diphtheria toxin and NAD+.
| S-EPMC1132246 | biostudies-other
Project description:Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.
| S-EPMC1138026 | biostudies-other
Project description:The effects of Ca2+-calmodulin on adenylate cyclase activity in EGTA-washed, 27000 g particulate fractions of mouse and rat pancreatic islets were studied. Ca2+ (10 microM)-calmodulin (1 microM) stimulated adenylate cyclase activity 53.1 +/- 5.2 (N = 6)% in the particulate fraction of rat islets. Trifluoperazine (50 microM), a specific inhibitor of calmodulin, inhibited the Ca2+-calmodulin activation of the adenylate cyclase activity of this fraction of rat islets. These results confirm previous reports dealing with Ca2+-Calmodulin and rat islet adenylate cyclase [Valverde, Vandermeers. Anjaneyulu & Malaisse (1979) Science 206, 225-227; Sharp, Wiedenkeller, Kaelin, Siegel & Wollheim (1980) Diabetes 29, 74-77]. In contrast, however, Ca2+ (1-100 microM)-calmodulin (1-10 microM) did not stimulate the adenylate cyclase activity in the EGTA-washed particulate fraction of mouse islets, and trifluoperazine (50 microM) did not inhibit the adenylate cyclase activity of this fraction of mouse islets, although some remaining calmodulin [0.18 +/- 0.05 (n = 3) microgram/mg of protein] could be demonstrated. GTP (10 microM) enhanced islet adenylate cyclase activity considerably, but did not confer any sensitivity towards Ca2+-calmodulin on mouse islet adenylate cyclase. The results question the role of calmodulin in the Ca2+-dependent rise in cyclic AMP evoked by glucose in pancreatic islets.
| S-EPMC1158554 | biostudies-other
Project description:It has been suggested that the stimulatory effect of glucose on insulin release may be mediated by the adenylate cyclase-cyclic AMP phosphodiesterase system. In this study it was found that exposure of isolated pancreatic islets to an elevated extracellular glucose concentration for 1 week in vitro caused an increase of the cyclic AMP phosphodiesterase activity in the islet cells. These and previous data indicate that there is an increased turnover of cyclic AMP in B-cells exposed for a prolonged time to a high extracellular glucose concentration, which also causes an increased turnover rate of insulin.
| S-EPMC1163768 | biostudies-other
Project description:The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.
| S-EPMC1565993 | biostudies-literature
Project description:The occurrence of endogenous substrate proteins for Ca2+-dependent protein kinase, augmented by either phospholipid or calmodulin, and for cyclic AMP-dependent protein kinase was examined in homogenates and subcellular fractions of mouse pancreatic islets. Islet protein phosphorylation was enhanced by Ca2+-calmodulin; the major endogenous substrates in the homogenate were two proteins of Mr 53000 and 100000. The Mr-100000 phosphoprotein was localized to a 27000g-supernatant fraction, whereas the Mr-53000 phosphoprotein was present in a 27000g particulate fraction of mouse islets. In the presence of Ca2+, phosphatidylserine stimulated phosphorylation of 15 proteins, of Mr 17000-190000, in a 27000g-supernatant fraction. No effects of Ca2+ plus phosphatidylserine were observed in a 27000g particulate fraction of mouse islets. Examination of cyclic AMP-dependent protein phosphorylation revealed five substrate proteins, of Mr 23000-72000, present in the 27000g supernatant of mouse islets. No common substrates for either the two Ca2+-dependent phosphorylation systems or for the cyclic AMP-dependent and the Ca2+-calmodulin-dependent phosphorylation systems were noted. On the other hand, the actions of the cyclic AMP-sensitive and the Ca2+-phospholipid-sensitive systems may be overlapping, since two common substrates for them were noted in the 27000g-supernatant fraction. The results are consistent with the hypothesis that protein phosphorylation may play a role in the regulation of insulin secretion by Ca2+ and cyclic AMP. The extensive stimulatory effect of phosphatidylserine furthermore suggests that the Ca2+-phospholipid-sensitive protein kinase may prove to be a prominent phosphorylation system in pancreatic islets.
| S-EPMC1144026 | biostudies-other
Project description:This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.
| S-EPMC1147887 | biostudies-other
Project description:Oleic acid, phosphatidylserine and pyrenedecanoic acid were found to activate calmodulin-deficient cyclic nucleotide phosphodiesterase at concentrations above their critical micellar concentration. In contrast with calmodulin these activators do not require the presence of Ca2+ for their action. It is shown that the size of phosphatidylserine vesicles is of crucial importance with respect to the activating potency of phosphatidylserine. Fluorescence measurements with the probe pyrenedecanoic acid revealed that micelles rather than monomers are the active species for stimulation of phosphodiesterase. There are indications that this result also may be applied to the other activators.
| S-EPMC1153913 | biostudies-other
Project description:The effects of KS-505a, a novel microbial metabolite, on the activity of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaM-PDE) were investigated. (1) KS-505a potently inhibited the purified 61 kDa isoenzyme of CaM-PDE from bovine brain and required much higher doses to inhibit the purified 59 kDa isoenzyme of CaM-PDE from bovine heart. The inhibition of both isoenzymes was observed only in the presence of calcium-activated calmodulin (Ca2+/CaM). The IC50 values for the 61 and 59 kDa isoenzymes were 0.17 and 13 microM respectively with 20 microM cAMP as a substrate. (2) Kinetic analysis indicated that the inhibitory mode of KS-505a for the 61 kDa isoenzyme was competitive with respect to Ca2+/CaM; the K1 for KS-505a was 0.089 microM. The inhibition was not competitive with respect to the substrates cAMP or cGMP. (3) KS-505a did not interfere with the interaction between Ca2+/CaM and n-phenyll-naphthylamine, a hydrophobic fluorescent probe, nor was it adsorbed to CaM-conjugated gels in the presence of Ca2+, thereby indicating that KS-505a does not bind to Ca2+/CaM. (4) Trypsin-activated 61 kDa isoenzyme, which lacked the Ca2+/CaM-binding domain, was not inhibited by KS-505a at less than micromolar concentrations. Taken together, these results suggest that KS-505a apparently bound to a site in the Ca2+/CaM-binding domain of the 61 kDa isoenzyme and selectively inhibited Ca2+/CaM-activated 61 kDa isoenzyme activity. (5) In rat hippocampal slices, KS-505a at 10 micronM increased the intracellular cAMP concentration to approximately three times the basal level, whereas in rat striatal slices it had no effect on the cAMP concentration at concentrations of 1.0-10 microM, suggesting that each CaM-PDE isoenzyme functions differentially in these regions. These results demonstrate that KS-505a is a highly potent selective inhibitor both in vitro and in vivo and distinguishes between subfamily members within the CaM-PDE family.
| S-EPMC1217340 | biostudies-other