Adenylyl cyclase type VI corrects cardiac sarcoplasmic reticulum calcium uptake defects in cardiomyopathy

Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart.     

Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes

Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.     

Adenylyl cyclase activity and function are decreased in rat cardiac fibroblasts after myocardial infarction

Myocardial infarction (MI) results in left ventricular remodeling (e.g., ventricular hypertrophy, dilatation, and fibrosis). Fibrosis contributes to increased myocardial stiffening, impaired ventricular filling and function, and reduced cardiac output. Adenylyl cyclase (AC) expression and activity are reduced in animal models of heart failure. Stimulation of AC can inhibit extracellular matrix production in isolated cardiac fibroblasts; however, a role for reduced AC expression and activity in fibrosis associated with cardiac remodeling after chronic MI has never been determined. We tested the hypothesis that AC expression and activity are reduced in cardiac fibroblasts after chronic (18 wk) MI. Rats underwent coronary artery ligation or sham surgery (control), and echocardiography was used to assess left ventricular remodeling 1, 3, 5, 7, 10, 12, and 18 wk after surgery. Cardiac fibroblasts were isolated from the noninfarcted myocardium and compared for differences in AC activity and collagen synthesis. End-diastolic dimension was increased [control: 0.76 +/- 0.02 cm and MI: 1.0 +/- 0.02 cm (means +/- SE), P < 0.001] and fractional shortening was decreased (control: 44 +/- 2% and MI: 17 +/- 2%, P < 0.001) in MI compared with control rats. Basal and forskolin-stimulated cAMP production were decreased by 90% and 93%, respectively, and AC5/6 expression was decreased 39% in fibroblasts isolated from MI rats compared with sham controls. Serum-stimulated collagen production was increased twofold and forskolin-mediated inhibition of collagen synthesis was reduced in fibroblasts from MI rats compared with controls. Our data demonstrate that AC expression and activity are reduced and collagen production is increased in cardiac fibroblasts of rats after MI.     

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca(2+) channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current (I_Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca²⁺ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5-E11.5) than late developmental stage (LDS, E16.5-E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I_Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca²⁺ influx via LTCCs on AC activity, leading to the inability to inhibit AC.     

Adenylyl cyclase type II domains involved in Gbetagamma stimulation

Mammalian particulate adenylyl cyclases contain two transmembrane regions (M(1) and M(2)) and two cytosolic domains (C(1) and C(2)) forming the catalytic core. The cytosolic domains are subdivided into a highly conserved region (part a) and a region with lower similarity (part b). Hypothetical models exist that account for the mechanism by which Galpha(s) and forskolin stimulate mammalian adenylyl cyclase. In contrast, little is known about how Gbetagamma dimers regulate catalysis. The so-called QEHA region located in the C(2a) domain of type II adenylyl cyclase has been proposed to represent a site of interaction. Here we show (i) that the QEHA region directly interacts with Gbetagamma but (ii) that it is of minor importance for the stimulation of type II adenylyl cyclase because it can be replaced by corresponding, nonidentical regions of other adenylyl cyclase isoforms without altering the stimulatory effect of Gbetagamma and (iii) that the C(1b) region is necessary for Gbetagamma to exert a stimulatory effect on adenylyl cyclase type II as in a C(1b) deletion mutant the Gbetagamma regulation was specifically impeded whereas the Galpha(s)- and forskolin-mediated stimulation was maintained.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

Gbetagamma activation site in adenylyl cyclase type II. Adenylyl cyclase type III is inhibited by Gbetagamma

The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.     

Calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of phospholamban

Summary Active calcium transport by cardiac sarcoplasmic reticulum assumes a central role in the excitation-concentration coupling of the myocardium, in that Ca²⁺-dependent ATPase (mol.wt. 100 000) of cardiac sarcoplasmic reticulum serves as an energy transducer and a translocator of Ca²⁺ across the membrane. During the translocation of Ca²⁺, the ATPase undergoes a complex series of reactions during which the phosphorylated intermediate EP is formed. We documented how the elementary steps of the ATPase reaction are coupled with calcium translocation, and provided evidences to indicate that two key steps of ATPase correspond to the conformational change of the enzyme, and appear to alter the affinity of the enzyme for Ca²⁺.A line of evidence also indicated that Ca²⁺-dependent ATPase of cardiac sarcoplasmic reticulum is regulated by a specific protein named phospholamban (mol.wt. 22 000), which serves as a substrate for cyclic AMP-dependent protein kinase. Cyclic AMP-dependent phosphorylation of phospholamban resulted in a marked increase in the rate of turnover of the ATPase, by enhancing the rates of the key elementary steps, i.e. the steps at which the intermediate EP is formed and decomposed. Thus phospholamban is putatively thought to serve as a modulator of Cat²⁺-dependent ATPase of cardiac sarcoplasmic reticulum. A working model was proposed to interpret the mechanism. Also documented is a possibility that another protein kinase activatable by Ca²⁺ and calmodulin is functional in regulating the phospholamban-ATPase system, thus suggesting the existence of a dual control system, in which both cyclic AMP- and calmodulin-dependent phosphorylation are in control of the Cat²⁺-dependent ATPase.Such a control mechanism may provide the interpretation, at the cellular level, that catecholamines exert actions on myocardial contractility. Thus, catecholamine-mediated increases in intracellular cyclic AMP could enhance calcium fluxes across the membrane of sarcoplasmic reticulum, thus resulting in the increased rates of relaxation and, at the same time, the increased rate and extent of contraction. Such a mechanism could also be operational in the tissues, other than the myocardium, in which catecholamines and other hormones serve as the first messenger, producing intracellular cyclic AMP as the second messenger.     

Role of phospholamban in cyclic GMP mediated signaling in cardiac myocytes.

We tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes were mediated through phospholamban (PLB) and activation of sarcoplasmic reticulum Ca(2+)-ATPase. Using ventricular myocytes from wild type (WT, n=10) and PLB knockout (PLB-KO, n=10) mouse hearts, functional changes were measured using a video edge detector at baseline and after 10(-6), 10(-5)M 8-bromo-cyclic GMP (cGMP), 10(-8), 10(-7)M C-type natriuretic peptide (CNP), or 10(-6), 10(-5)M S-nitroso-N-acetyl-penicillamine (SNAP, nitric oxide donor). Changes in cytosolic Ca(2+) concentration were assessed in fura 2-loaded WT and PLB-KO myocytes. Cyclic GMP dependent phosphorylation analysis was also performed in WT and PLB-KO myocytes. 8-bromo-cGMP 10(-5)M caused a significant decrease in %shortening (3.6+/-0.2% to 2.3+/-0.1%) in WT, but little change in PLB-KO myocytes (3.4+/-0.1% to 3.2+/-0.2%). Similarly, CNP and SNAP reduced %shortening of WT, but not PLB-KO myocyte. Changes in other contractile parameters such as maximum rate of shortening and relaxation were consistent with the changes in % shortening. Intracellular Ca(2+) transients changed similarly to cell contractility in WT and PLB-KO myocytes treated with cGMP and CNP; i.e. Ca(2+) transients decreased with cGMP or CNP in WT myocytes, but were unchanged in PLB-KO myocytes. cGMP dependent phosphorylation analysis showed that some proteins were phosphorylated by cGMP to a lesser extent in PLB-KO compared with WT myocytes, suggesting impaired cGMP-kinase function in PLB-KO cardiac myocytes. These results indicated that cGMP-induced reductions in cardiac myocyte function were at least partially mediated through the action of phospholamban.     

Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.

The application of electrophoretic resolution of the different phosphorylation species of pentameric phospholamban as a measure of phosphorylation stoichiometry was examined and verified. This enabled a critical evaluation of a number of issues central to current models of calcium pump regulation in cardiac sarcoplasmic reticulum. The phospholamban content of numerous preparations was calculated from 32P incorporation at a given stoichiometry, and compared with the respective calcium pump concentration (derived by comparison with a Coomassie-stained calibration curve of the fast-twitch skeletal muscle isozyme). A relationship of 2 mol of phospholamban:1 mol of ATPase resulted (phospholamban monomer:ATPase monomer), which was maintained throughout all vesicle subpopulations. The precise mechanism of coupling of phospholamban phosphorylation to calcium pump stimulation was probed, with particular emphasis on the individual contributions of each phosphorylated species (P1 to P5). This relationship could be adequately explained in three ways: (i) each phosphorylation event contributed equally to calcium pump stimulation; (ii) P1 and P2 were incapable of stimulating calcium pump activity, but full stimulation occurred upon generation of species P3; or (iii) the phosphospecies P1 was without effect on basal calcium pump activity, but successive phosphorylations contributed equally to stimulation. Finally, the functional implication of dual site phosphorylation of phospholamban (cAMP- and the endogenous calmodulin-dependent kinases) was examined. No change in calcium pump activity accompanied the second tier of phosphorylation over that achieved by the first.     

Monoacylglycerol lipase activity in cardiac myocytes

Monoacylglycerol lipase activity in homogenates of isolated myocardial cells (myocytes) from rat hearts was recovered in both particulate and soluble subcellular fractions. The activity present in the microsomal (100,000 X g pellet) fraction was solubilized by treatment with Triton X-100 and combined with the 100,000 X g supernatant fraction; the properties of monoacylglycerol lipase were investigated with this soluble enzyme preparation. The Km for the hydrolysis of a 2-monoolein substrate was 16 microM. The rates of hydrolysis of 1-monoolein and 2-monoolein were identical, and 1-monoolein was a competitive inhibitor (Ki = 20 microM) of the hydrolysis of 2-monoolein. Monoacylglycerol lipase activity was regulated by product inhibition according to the following order of potency: fatty acyl CoA greater than free fatty acids greater than fatty acyl carnitine.     

Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes.

We have assessed the effects of endothelin-1 (ET-1) on transmembrane signaling in adult rat ventricular myocytes. ET-1 stimulates phosphoinositide hydrolysis with an EC50 of 0.3-0.8 nM. This stimulation is linear for up to 30 min in the presence of a protease inhibitor, is additive with the effects of other stimulators of phosphoinositide hydrolysis, is not inhibited by the Ca2+ entry blocker, nifedipine, and is insensitive to pertussis toxin. ET-1 also reduces cyclic AMP production in myocytes in response to isoproterenol and forskolin (EC50, 1 nM). This cyclic AMP-lowering effect of ET-1 is sensitive to pertussis toxin, can be demonstrated directly in assays of adenylate cyclase activity of myocyte membranes, and seems to be mediated by Gi. These data indicate that the effects of endothelin on adult cardiac myocytes involve multiple signaling pathways, including enhanced activity of the inositol phosphate pathway and a decrease in cyclic AMP-mediated responses, neither of which seems likely to account for the positive contractile effects of endothelin.     

Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle

Skeletal muscle insulin sensitivity improves with short-term reduction in calorie intake. The goal of this study was to evaluate changes in the abundance and phosphorylation of Akt1 and Akt2 as potential mechanisms for enhanced insulin action after 20 days of moderate calorie restriction [CR; 60% of ad libitum (AL) intake] in rat skeletal muscle. We also assessed changes in the abundance of SH2 domain-containing inositol phosphatase (SHIP2), a negative regulator of insulin signaling. Fisher 344 x Brown Norway rats were assigned to an AL control group or a CR treatment group for 20 days. Epitrochlearis muscles were dissected and incubated with or without insulin (500 microU/ml). Total Akt serine and threonine phosphorylation was significantly increased by 32 (P < 0.01) and 30% (P < 0.005) in insulin-stimulated muscles from CR vs. AL. Despite an increase in total Akt phosphorylation, there was no difference in Akt1 serine or Akt1 threonine phosphorylation between CR and AL insulin-treated muscles. However, there was a 30% decrease (P < 0.05) in Akt1 abundance for CR vs. AL. In contrast, there was no change in Akt2 protein abundance, and there was a 94% increase (P < 0.05) in Akt2 serine phosphorylation and an increase of 75% (P < 0.05) in Akt2 threonine phosphorylation of insulin-stimulated CR muscles compared with AL. There was no diet effect on SHIP2 abundance in skeletal muscle. These results suggest that, with brief CR, enhanced Akt2 phosphorylation may play a role in increasing insulin sensitivity in rat skeletal muscles.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

A phospholamban protein phosphatase activity associated with cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum vesicles contain intrinsic phospholamban protein phosphatase activity, which is also effective in dephosphorylating phosphorylase a. The phosphatase associated with sarcoplasmic reticulum membranes was solubilized with Triton X-100 and subjected to chromatography on Mono Q HR 5/5 and polylysine-agarose. A single peak of phosphatase activity was eluted from each column and it was coincident for both phospholamban and phosphorylase a, used as substrates. Thermal denaturation of the enzyme resulted in progressive and coincident loss of both phospholamban and phosphorylase a phosphatase activities. Enzymic activity was partially inhibited by protein phosphatase inhibitor 1. Migration of the enzyme during sucrose density gradient ultracentrifugation corresponded to a globular protein with an apparent Mr of 46,000. This enzyme preparation could dephosphorylate both the calcium-calmodulin-dependent as well as the cAMP-dependent sites on phospholamban. Thus, dephosphorylation of phospholamban by this sarcoplasmic reticulum-associated phosphatase may participate in modulating sarcoplasmic reticulum function in cardiac muscle.     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

Chromosomal mapping of human adenylyl cyclase genes type III,type V and type VI

Adenylyl cyclase activity plays a central role in the regulation of most cellular processes. At least eight different adenylyl cyclases have been identified, which are endowed with various and sometimes opposing regulatory properties. Recently we have localized the human genes encoding two of these adenylyl cyclases: the gene for type 11 adenylyl cyclase is located on chromosome 2 (sub-band 2p15.3), the gene for type VIII is located on chromosome 8 (sub-band 8824.2). More recently the type I gene has been located on chromosome 7 (sub-band 7pl2–7p13). Using in situ hybridization, we have now localized the genes for three other adenylyl cyclases: the type III gene has been localized on chromosome 2 in the sub-band 2p22–2p24, the type V gene on chromosome 3 at position 3q13.2–3q21, and the type VI gene on chromosome 12 at position 12q12–12q13. It therefore appears that all adenylyl cyclase genes, known at present are located on different chromosomes and thus are likely to be independently regulated.