Cloning, expression, signaling mechanisms, and membrane targeting of P2Y(11) receptors in Madin Darby canine kidney cells.

The P2Y(11) receptor is hypothesized to link to both G(s) and G(q), although this proposition is based on expression and separate assays of G(s) and G(q) function in different cell types [J Biol Chem 1997;272:31969-31973]. We have cloned and characterized a canine P2Y(11)-like (cP2Y(11)) receptor from cultured Madin Darby canine kidney (MDCK-D1) cells. When cP2Y(11) receptors are expressed in canine thymocyte (CF2Th) cells that normally lack functional purinergic responses, ADP beta S stimulates phosphatidylinositol (PI) hydrolysis, Ca(2+) mobilization, and cAMP accumulation. Pharmacologic analysis indicates that the stimulation of cAMP production is direct and not a result of eicosanoid synthesis, activation of PKC, or elevation of cell Ca(2+). The rank order of potency for stimulation of PI hydrolysis by cP2Y(11) receptors (adenosine 5'-(2-O-thio) diphosphate = 2-methylthio-ADP >/= 2-methylthio-ATP >> ADP > ATP) differs from that of hP2Y(11) receptors. Microscopic examination of MDCK-D1 cells expressing carboxyl-terminal green fluorescent protein (GFP)-tagged cP2Y(11) (cP2Y(11)-GFP) receptors indicates primarily basolateral (BL) targeting. BL addition of 200 microM ADP beta S to confluent monolayers of MDCK-D1 cells produces an increase in short circuit current (I(sc)) (11.6 +/- 1.6 microA/cm(2)) whereas apical addition of agonist has no effect, confirming targeting of functional endogenous P2Y(11) receptors to the BL surface. In contrast, when either cP2Y(11) or cP2Y(11)-GFP is overexpressed in MDCK-D1 cells, the sensitivity of I(sc) to BL agonist increases by nearly 2 orders of magnitude, as if receptor density normally limited agonist potency; moreover, apical addition of ADP beta S now produces an increase in I(sc) but with low potency. The data support the BL localization of cP2Y(11) receptors and receptor coupling to changes in I(sc) in MDCK-D1 cells except in cases in which receptors are overexpressed; receptor overexpression leads to altered sensitivities and sites of coupling to physiologic responses.

Compartmentation of G protein-coupled signaling pathways in cardiac myocytes.

There is a large body of functional data that supports the existence of subcellular compartmentation of the components of cyclic AMP action in the heart. Data from isolated perfused hearts and from purified ventricular myocytes imply a fixed and hormone-specific spatial relationship amongst components of cyclic AMP synthesis, response, and degradation. Available data demonstrate that within a cardiac myocyte, not all cyclic AMP gains access to all cyclic AMP-dependent protein kinase (PKA), that not all PKA interacts with all possible cellular substrates of PKA, and that only a subset of the myocyte's phosphodiesterases (PDEs) may degrade cyclic AMP after a given synthetic stimulus. Molecular mechanisms contributing to compartmentation are being discovered: localization of receptors, G proteins, and adenylyl cyclases in caveolar versus noncaveolar regions of the sarcolemma; localization of PKA by A-kinase anchoring proteins; localization of PKA substrates, PDE isoforms, and phosphoprotein phosphatases in discrete subcellular regions; and differential regulation of multiple isoforms of adenylyl cyclase, phosphoprotein phosphatase, and PDE in distinct subcellular compartments.

P2Y(2) receptor of MDCK cells: cloning, expression, and cell-specific signaling.

Madin-Darby canine kidney (MDCK)-D1 cells, a canine renal epithelial cell line, co-express at least three different P2Y receptor subtypes: P2Y(1), P2Y(2), and P2Y(11) (24). Stimulation of P2Y receptors in these cells results in the release of arachidonic acid (AA) and metabolites and the elevation of intracellular cAMP. To define in more precise terms the signaling contributed by the MDCK-D1 P2Y(2) (cP2Y(2)) receptor, we have cloned and heterologously expressed it in CF2Th (canine thymocyte) cells, a P2Y(2)-null cell. Analysis by RT-PCR indicated that canine P2Y(2) receptors are expressed in skeletal muscle, spleen, kidney, lung, and liver. When expressed in CF2Th cells, cP2Y(2) receptors promoted phospholipase C-mediated phosphatidylinositol (PI) hydrolysis [uridine 5'-triphosphate > or = ATP > adenosine 5'-diphosphate > 2MT-ATP] and mobilization of intracellular Ca(2+). In contrast to their actions in MDCK-D1 cells, cP2Y(2) receptors did not stimulate formation of cAMP or AA release when expressed in CF2Th cells. The data indicate that cell setting plays an essential role in the ability of P2Y receptors to regulate AA release and cAMP formation. In particular, renal epithelial cells preferentially express components critical for cP2Y(2)-induced cAMP formation, including the expression of enzymes involved in the generation and metabolism of AA and receptors that respond to PGE(2).

beta-adrenergic stimulation of rat cardiac fibroblasts enhances induction of nitric-oxide synthase by interleukin-1beta via message stabilization.

We have investigated factors modulating expression of inducible NO synthase (iNOS) in isolated adult rat cardiac fibroblasts. Treatment of cardiac fibroblasts with interleukin-1beta (IL-1beta) promotes induction of iNOS mRNA and protein and production of NO. Simultaneous incubation of cells with isoproterenol enhances the response to IL-1beta, even though isoproterenol alone is without effect. N(G)-nitro-L-arginine methyl ester inhibits the effect of isoproterenol + IL-1beta on NO production. beta(2)-Adrenergic receptors appear to mediate this effect of isoproterenol. Reverse transcriptase-polymerase chain reaction analyses show that beta(2)-receptor mRNA is the predominant beta-receptor message; in pharmacologic studies, ICI-118,551 significantly antagonizes isoproterenol-stimulated cyclic AMP production whereas CGP20712A does not. Dibutyryl-cyclic AMP and forskolin mimic the synergistic effect of isoproterenol on IL-1beta-induced NO production; H-89, a cyclic AMP-dependent protein kinase (PKA) inhibitor, antagonizes the enhancing effect of isoproterenol. Nuclear run-off experiments indicate that enhancement of iNOS by isoproterenol does not occur at the level of transcription. Message stability studies demonstrate that isoproterenol increases the half-life of iNOS mRNA from 1.0 to 1.9 h; this change is sufficient to account for the observed augmentation of iNOS mRNA and protein. Thus, cardiac fibroblasts produce significant amounts of NO in response to IL-1beta via induction of iNOS; beta-adrenergic stimulation enhances the IL-1beta effect by stabilizing the iNOS message. These data suggest that cardiac fibroblasts could participate in a paracrine mechanism whereby the direct positive inotropic effect of beta(1)-adrenergic stimulation of myocytes is opposed by beta(2)-adrenergic enhancement of NO production, a negative inotropic event, in neighboring fibroblasts.

Protein kinase C contributes to desensitization of ANG II signaling in adult rat cardiac fibroblasts.

We have studied G(q)-linked ANG II signaling [inositol phosphate (IP) accumulation, Ca(2+) mobilization] in primary cultures of rat cardiac fibroblasts (CFs) and have found that ANG II initiates a protein kinase C (PKC)-mediated negative feedback loop that rapidly terminates the ANG II response. Pharmacological inhibition of PKC by staurosporine and GF-109203X doubled IP production over that achieved in response to ANG II alone. Inhibition of PKC also led to larger Ca(2+) transients in response to ANG II, suggesting that Ca(2+) mobilization was proportional to G(q)-phospholipase C-IP(3) activity under the conditions studied. Depletion of cellular PKC by overnight treatment with phorbol 12-myristate 13-acetate (PMA) similarly augmented ANG II-induced IP production. Acute activation of PKC by PMA halved IP formation, with an EC(50) approximately 1 nM; 4alpha-PMA was inactive. Time course data demonstrated that ANG II-mediated IP production fully desensitized within 30 s; PKC inhibition reduced the rate and extent of this desensitization. In cells desensitized to ANG II, a purinergic agonist still mobilized intracellular Ca(2+), indicating that desensitization was homologous. The ANG II-induced Ca(2+) signal was fully resensitized within 30 min. The data demonstrate that a large portion of the IP-Ca(2+) responses of rat CFs to ANG II are short-lived because of rapid, PKC-mediated desensitization.

Characterization of G-protein signaling in ventricular myocytes from the adult mouse heart: differences from the rat.

We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3-6x10(6)cells/heart. The cells are rod-shaped, roughly 20 microM x 100 microM and Ca(++)tolerant, with viability of 65-80%. Binding studies with [(125)I]ICYP demonstrate the presence of beta -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the beta(1)-specific antagonist CGP 20712A on [(125)I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are beta(1)and 33% are beta(2), compared to 16-20%beta(2)in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC(50) approximately 110+/-20 n M. A functional G(i)pathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the G(q)-phospholipase C pathway, whereas carbachol, PGF(2 alpha)and alpha(1)-adrenergic receptor agonists show no significant effect. The inability of alpha(1)-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold alpha(1)-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.

Identification of G protein-coupled signaling pathways in cardiac fibroblasts: cross talk between G(q) and G(s).

Cardiac fibroblasts (CFs) are an important cellular component of myocardial responses to injury and to hypertrophic stimuli. We studied G protein-coupled receptors to understand how CFs integrate signals that activate G(q), G(s), and G(i). We predicted that the second messenger pathways present in CFs were distinct from those in cardiac myocytes and that unique signaling interactions existed in the CFs. ANG II, bradykinin, ATP, and UTP stimulated inositol phosphate (IP) production 2.2- to 7-fold. Each of these agonists elevated intracellular Ca(2+) concentration ([Ca(2+)](i)) via release from the intracellular Ca(2+) storage compartment. Endothelin-1 (ET-1), carbachol, and norepinephrine failed to increase either IP production or [Ca(2+)](i). Although agonists that activated IP and Ca(2+) transients had no effect on cAMP production when administered alone, these agents potentiated the beta(2)-adrenergic response two- to fourfold. Hormones known to inhibit adenylyl cyclase activity in cardiac myocytes, such as ET-1 and carbachol, failed to lower the beta-adrenergic response in fibroblasts. Order of potency and inhibitor data indicate that the functional receptor subtypes in these cells are beta(2), P2Y(2), and AT(1) for isoproterenol, ATP, and ANG II, respectively. We conclude that CFs express functional G protein-linked receptors that couple to G(q) and G(s), with little or no coupling to G(i). The expression of receptors and their coupling to G(q)- but not to G(i)-linked responses distinguishes the signaling in CFs from that in myocytes. Furthermore, agonists that activate G(q) in CFs potentiate stimulation of G(s), an example of signaling cross talk not observed in adult myocytes. These data suggest that G protein-mediated signaling in CFs is unique and may contribute to the specificity of hormone and drug action on individual cell types within the heart.

Inhibition of protein kinases by balanol: specificity within the serine/threonine protein kinase subfamily.

Balanol is a potent inhibitor of cyclic AMP-dependent protein kinase and protein kinase C, acting competitively with ATP with an affinity 3000 times that of ATP. We tested the capacity of balanol to inhibit representative serine- and threonine-specific protein kinases from the protein kinase subfamily that shares a common conserved catalytic core with cyclic AMP-dependent protein kinase. Balanol's pattern of interactions indicates considerable diversity of the ATP/balanol-binding sites of protein kinases within familial groups and even among isoforms of the same kinase. We propose that balanol is a protean structure that may be modified to produce selective, high-affinity inhibitors and probes of the ATP-binding sites of serine/threonine protein kinases.

Differential and selective inhibition of protein kinase A and protein kinase C in intact cells by balanol congeners.

The fungal metabolite balanol is a potent inhibitor of protein kinase A (PKA) and protein kinase C (PKC) in vitro that acts by competing with ATP for binding (K(i) approximately 4 nM); congeners of balanol show specificity for PKA over PKC. We have characterized the effects of balanol and 10"-deoxybalanol in intact cells to determine whether these compounds cross the cell membrane and whether the potency and specificity noted in vitro are preserved in vivo. In neonatal rat myocytes and cultured A431 cells transiently transfected with a cyclic AMP response element-luciferase reporter construct, balanol inhibits the induction of luciferase activity by isoproterenol, indicating inhibition of PKA. Western analysis shows that both balanol and 10"-deoxybalanol reduce phosphorylation of cAMP response element-binding protein in isoproterenol-stimulated A431 cells; inhibition is concentration dependent with an IC(50) value of approximately 3 microM. Balanol, but not 10"-deoxybalanol, inhibits phosphorylation of the myristoylated alanine-rich C kinase substrate protein, a PKC substrate, in phorbol ester-stimulated A431 cells (IC(50) approximately 7 microM). Our data demonstrate that balanol is a potent inhibitor of PKA and PKC in several whole-cell systems and causes no obvious toxicity. In addition, balanol congeners inhibit PKA and PKC with the specificity and potency predicted by in vitro experiments.

Crystal structure of the potent natural product inhibitor balanol in complex with the catalytic subunit of cAMP-dependent protein kinase.

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanol's specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

The quasi-irreversible nature of endothelin binding and G protein-linked signaling in cardiac myocytes.

In experiments on neonatal and adult rat ventricular myocytes, endothelin (ET) binding and the effects of ET on transmembrane signaling are quasi-irreversible. The ET(A) receptor antagonist BQ123 competes for binding and biochemical effects if added simultaneously with ET; when added after ET, the antagonist prevents neither binding nor activation of the Gi and Gq pathways. At 4 degrees C, at which internalization of the ligand should be minimized, the interaction of [125I]ET is still irreversible. After binding of radio-labeled ligand at either 4 degrees C or 37 degrees C, only 50% of ligand is removed by acid washing. Permeabilization of the cells with Triton X-100 fails to release irreversibly bound ligand. Binding experiments in cell membranes mimic this irreversible binding. At 37 degrees C, the addition of mercaptoethanol or dithiothreitol inhibits concurrent ET binding but does not cause the dissociation of previously bound ligand or the reversal of previously activated signaling. We conclude that ET binds irreversibly to myocytes, that this irreversibility is reflected in the biochemical responses of the cells to ET and that the irreversibility is more complex than the formation of S-S bonds between surface receptors and ET or internalization of bound ET. We interpret these findings and others in the literature in light of a testable model of ET(A) receptor/G protein/effector interaction in which quasi-irreversible binding of ET to the ET(A) receptor occurs before the interaction of the ligand/receptor complex with G protein and in which irreversible binding contributes to the prolonged effects of ET and is a prelude to refractoriness and to the slow regeneration of free ET(A) receptor.

Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways.

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.

Small heat shock proteins and protection against ischemic injury in cardiac myocytes.

BACKGROUND: Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS: Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS: The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes.

We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5'-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.

Transmembrane mechanochemical coupling in cardiac myocytes: novel activation of Gi by hyposmotic swelling.

We have studied the effect of hyposmotic swelling on adenosin 3',5'-cyclic monophosphate (cAMP) metabolism in isolated cardiac myocytes. Decreasing extracellular osmolarity by 12.5-50% results in graded inhibition (10-40%) of isoproterenol-stimulated and forskolin-stimulated cAMP accumulation but does not affect basal and hormone-stimulated phosphoinositide hydrolysis or cellular ATP content. Treatment with pertussis toxin does not alter the swelling response but abolishes the inhibitory effect of swelling on cAMP accumulation. The response to swelling seems not to involve the release of effectors known to couple to inhibitory G protein (Gi) in myocytes: BQ-123, atropine, and adenosine deaminase do not alter the inhibitory effect of swelling on isoproterenol-stimulated cAMP accumulation; conditioned medium from swollen cells, with restored osmolarity, has no effect on cAMP accumulation when added to control myocytes. In distinction to these effects on myocytes, swelling enhances hormone-stimulated cAMP accumulation in cultured S49 lymphoma cells. We conclude that swelling of cardiac myocytes inhibits cAMP accumulation through a mechanism that involves activation of a pertussis toxin-sensitive Gi protein. Activation of Gi by this means may contribute to adrenergic hyporesponsiveness in hypoxic and ischemic myocardium.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol.

BACKGROUND: The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. RESULTS: As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. CONCLUSIONS: A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.

Coupling of the type A endothelin receptor to multiple responses in adult rat cardiac myocytes.

In adult rat cardiac myocytes, endothelin (ET) receptors couple to multiple signaling pathways, including stimulation of phosphoinositide hydrolysis (pertussis toxin insensitive) and inhibition of adenylyl cyclase via Gi. We have used ET-1 and congeners to characterize the subtypes of ET receptors on isolated rat myocytes. The rank orders of potency for stimulating phosphoinositide hydrolysis, inhibiting hormone-sensitive adenylyl cyclase, and competing with 125I-ET-1 for binding to myocytes are the same and show the pattern characteristic of an ETA receptor interaction, i.e., ET-1 approximately ET-2 > sarafotoxin 6b > ET-3; the corresponding EC50 values for the effects of ET on signal transduction are approximately 0.5 nM (ET-1), 0.7 nM (ET-2), 7 nM (sarafotoxin 6b), and 60 nM (ET-3). The ETA receptor antagonist BQ-123 abolishes the cellular responses to ET-1 and competes fully for 125I-ET-1 binding in a concentration-dependent manner. Sarafotoxin 6c, an ETB-specific agonist, does not diminish the responses to ET-1 or compete for 125I-ET-1 binding; no specific binding of the ETB-specific ligand 125I-IRL-1620 is detectable on myocytes. Myocytes express approximately 4 x 10(5) ET-1 binding sites/cell. The association of 125I-ET-1 with myocytes is largely irreversible, as are the biochemical responses to ET-1; thus, constants derived from analyses that assume reversible equilibria are in error. We conclude that the effects of ET on transmembrane signaling in rat ventricular myocytes result from occupation of ETA receptors and that the responses are likely to be long lived, compared with those of the readily dissociable neurotransmitters released by the autonomic nervous system.

Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes.

We have immunologically identified the isoforms of protein kinase C (PKC) present in neonatal and adult rat cardiomyocytes and examined their regulation by hormones and phorbol ester. Both cell types express the Ca(2+)-dependent alpha-PKC and the Ca(2+)-independent epsilon- and delta-PKC isoforms. The atypical zeta-PKC isoform is also expressed in neonatal, but only weakly in adult cells. Stimulation of the alpha 1-adrenergic or purinergic receptor with phenylephrine or ATP, respectively, increases membrane-associated immunoreactivity of both epsilon- and delta-PKC in neonatal and adult cells; endothelin and carbachol are also effective in adult cells. In contrast, none of the agonists leads to increases in membrane-associated alpha-PKC in cardiomyocytes. PKC zeta is also unaffected by receptor stimulation. The phorbol ester phorbol 12-myristate 13-acetate causes redistribution and subsequently down-regulation of alpha-, epsilon-, and delta- but not zeta-PKC. The three isoforms are down-regulated at distinctively different rates, with alpha-PKC being the most rapid and epsilon-PKC the slowest. We used selective down-regulation of alpha-, epsilon-, and delta-PKC to investigate the role of these isoforms in PKC phosphorylation-dependent events in neonatal myocytes. Our findings suggest that epsilon-PKC is responsible for the phenylephrine-induced phosphorylation of MARCKS, an endogenous PKC-specific substrate. In contrast, agonist-induced c-fos expression is unlikely to be mediated by epsilon-PKC since the response is rapidly down-regulated and apparently Ca(2+)-dependent. Our finding that the PKC isoforms are differentially responsive to neurohormones suggests that they play distinct and specific roles in cardiac function.