Protein kinase C and calmodulin kinase are required for endothelin-stimulated atrial natriuretic factor secretion from primary atrial myocytes.

Endothelin (ET), a potent stimulator of atrial natriuretic factor (ANF) secretion in atrial myocyte cultures, has been hypothesized to act via the stimulation of protein kinase C (PKC). This study was carried out in order to determine if ET activates PKC in atrial cultures and whether this activation fully accounts for the effects of ET on ANF secretion. By monitoring the phosphorylation of p80 upon exposure to phorbol ester or ET, it was shown that ET activated PKC in atrial cultures, but to a lesser extent than phorbol ester. In contrast, ET stimulated ANF secretion to a level five times greater than phorbol ester, indicating that PKC activation alone does not fully account for the effects of ET on ANF secretion. Down-regulation of PKC or exposure to the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) resulted in a 50% decrease in ET-stimulated ANF secretion. Interestingly, increasing calcium influx with BAY K 8644 stimulated ANF secretion but did not effect the phosphorylation of p80, indicating a PKC-independent pathway of ANF secretion. Similarly, a component of ET-stimulated secretion that required calcium influx was independent of PKC activation but was sensitive to the Ca2+/calmodulin kinase (CaMK) inhibitor KN-62. Complete inhibition of ET-mediated ANF secretion was obtained only in the presence of both H7 and KN-62. These results demonstrate that ET activates PKC in atrial myocyte cultures and that the full effects of ET on ANF secretion require both PKC and Ca2+/calmodulin kinase activities.     

The cosecretional maturation of atrial natriuretic factor by primary atrial myocytes.

Cardiac myocytes store the 126-amino acid precursor of atrial natriuretic factor (pro-ANF), yet the mature, bioactive 28-amino acid peptide, ANF-(99-126), and the resulting N-terminal product, ANF-(1-98), are the forms of the hormone that are released by the heart and found in the circulation. Although previous studies have shown that the maturation of ANF takes place in the heart, it is not known whether it occurs in or on the myocyte concurrently with secretion, or whether cleavage takes place postsecretionally on either the myocyte surface or the surface of a nonmuscle cardiac cell. To address these questions, experiments were carried out in the present study using primary atrial cultures that had been prepared such that greater than 90% of the cells were myocytes. Reversed-phase and ion-exchange HPLC, coupled with immunoprecipitation of biosynthetically labeled ANF, showed that the stored peptide, pro-ANF, was cleaved between residues 98 and 99 such that ANF-(1-98) and (99-126) accumulated in the medium. Coupling biosynthetic labeling with timed secretion experiments showed that the extent of ANF processing was not dependent on the time after secretion; maximal levels of processing were observed at all secretion times examined. Additionally, the processing-competent myocyte-enriched cultures were unable to cleave exogenously added pro-ANF. These results indicate that the myocyte is the cell type responsible for pro-ANF maturation and that this cleavage event takes place cosecretionally.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

Molecular biology of protein kinase C signaling in cardiac myocytes.

The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.     

Induction of atrial natriuretic factor and myosin light chain-2 gene expression in cultured ventricular myocytes by electrical stimulation of contraction.

While hormonal stimuli and mechanical stretch can induced cardiac-specific gene expression and in some cases cellular hypertrophy, the relationship between myocyte contraction frequency, gene expression, and myocyte growth has not been well characterized. In this study a new model system was developed in which cultures of neonatal rat ventricular myocytes were subjected to long term pacing of contractions with pulsatile electrical stimulation. Myocytes submitted to electrical stimulation for 3 days displayed dramatic increases in cellular size and myofibrillar organization, and a 5-10-fold increase in the expression of the cardiac genes atrial natriuretic factor and myosin light chain-2. Atrial natriuretic factor expression induced by electrical stimulation of contractions was inhibited by nifedipine or W7, indicating a dependence on calcium influx and calmodulin activity. Phosphoinositide hydrolysis and cAMP formation were not affected by electrical stimulation suggesting that gene induction occurred independently of the activation of protein kinases C or A above basal levels. These findings show that the cellular events associated with contraction, such as changes in cytoplasmic free calcium levels and/or cellular stretch, may serve as important determinants of myocyte growth and cardiac gene expression.     

Molecular biology of protein kinase C signaling in cardiac myocytes

The PKC family of serine/threonine kinases have been implicated in a diverse array of cellular responses. Adult cardiac myocytes express multiple PKC isozymes, which participate in the response of muscle cells to extracellular stimuli, modulate contractile properties, and promote cell growth and survival. Recently, the classification of this ubiquitous family of signaling molecules has been expanded from three to four subfamilies. This review will focus on the application of pharmacologic and molecular approaches to explore the biology of cardiac PKC isozymes. The availability of transgenic mice and peptide PKC modulators have been instrumental in identifying target substrates for activated cardiac PKC isozymes, as well as the identification of specific isozymes linked to distinct growth characteristics and cell phenotype. The rapid growth of knowledge in the area of PKC signaling and PKC substrate interactions, may result in the development of therapeutic modalities with the potential to arrest or reverse the progression of cardiovascular diseases.     

Extracellular signal-regulated protein kinase activation is required for the anti-hypertrophic effect of atrial natriuretic factor in neonatal rat ventricular myocytes.

Atrial natriuretic factor (ANF) inhibits proliferation in non-myocardial cells and is thought to be anti-hypertrophic in cardiomyocytes. We investigated the possibility that the anti-hypertrophic actions of ANF involved the mitogen-activated protein kinase signal transduction cascade. Cultured neonatal rat ventricular myocytes treated for 48 h with the alpha(1)-adrenergic agonist phenylephrine (PE) had an 80% increase in cross-sectional area (CSA). ANF alone had no effect but inhibited PE-induced increases in CSA by approximately 50%. The mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD098059 minimally inhibited PE-induced increases in CSA, but it completely abolished ANF-induced inhibition of PE-induced increases. ANF-induced extracellular signal-regulated protein kinase (ERK) nuclear translocation was also eliminated by PD098059. ANF treatment caused MEK phosphorylation and activation but failed to activate any of the Raf isoforms. ANF induced a rapid increase in ERK phosphorylation and in vitro kinase activity. PE also increased ERK activity, and the combined effect of ANF and PE appeared to be additive. ANF-induced ERK phosphorylation was eliminated by PD098059. ANF induced minimal phosphorylation of JNK or p38, indicating that its effect on ERK was specific. ANF-induced activation of ERK was mimicked by cGMP analogs, suggesting that ANF-induced ERK activation involves the guanylyl cyclase activity of the ANF receptor. These data suggest that there is an important linkage between cGMP signaling and the mitogen-activated protein kinase cascade and that selective ANF activation of ERK is required for the anti-hypertrophic action of ANF. Thus, ANF expression might function as the natural defense of the heart against maladaptive hypertrophy through its ability to activate ERK.     

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.     

Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue

Elements controlling tissue-specific expression of the human atrial natriuretic factor gene have been examined in primary cultures of neonatal rat cardiocytes. When a 68-base pair fragment from human atrial natriuretic factor (hANF) 5'-flanking sequence (positions -400 to -333) was placed upstream from the herpes simplex thymidine kinase promoter linked to a bacterial reporter gene (chloramphenicol acetyltransferase), a tissue-specific positive regulatory effect was observed in atrial as well as ventricular cardiocytes but not in nonmyocardial cells. The cis-acting element in this fragment was orientation- and position-dependent. Examination of nuclear protein extracts for the presence of factors capable of interacting with the 5'-flanking sequence of the hANF gene revealed a cardiocyte-specific factor which bound to the 68-base pair fragment. This association was both tissue- and sequence-specific. These findings indicate that a cis-acting element present in the proximal 5'-flanking sequence confers tissue-specific expression upon the hANF gene, possibly through association with a cardiac-specific nuclear protein.     

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin-binding EGF-like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca²⁺ionophore, ionomycin, suggesting the involvement of extracellular Ca²⁺ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca²⁺ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca²⁺ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998. © 1998 Wiley-Liss, Inc.     

Dissociation and enrichment of rat atrial myocytes containing atrial natriuretic factor (ANF)

Methodologies developed for the dissociation and subsequent enrichment of muscle and nonmuscle cells from atrial myocardium were used to evaluate the contribution of these cell populations to the natriuretic, diuretic and vasoactive properties of crude atrial tissue extracts. Suspensions of single cells, which contained approximately 34% myocytes, were prepared from atrial tissue blocks with a collagenase-trypsin digestion followed by gentle mechanical disruption. Differential centrifugation and unit gravity sedimentation techniques were employed to enrich the 'muscle' and 'nonmuscle' cell suspensions to a purity of approximately 91 and 95%, respectively. Cell extracts were bioassayed for natriuretic activity in saline-expanded, pentobarbital-anesthetized, female rats. Extracts obtained from 'initial' and 'muscle' cell suspensions significantly enhanced sodium and chloride excretion as well as urine flow while extracts from 'nonmuscle' cell suspensions had no effect on renal function. Sodium excretion was dose-dependent and increased linearly with increasing numbers of extracted and infused myocytes. This simple two-step centrifugation and sedimentation protocol can be utilized to obtain enriched atrial myocyte populations for subsequent physiologic and biochemical studies.     

A Raf-induced, MEK-independent signaling pathway regulates atrial natriuretic factor gene expression in cardiac muscle cells

The atrial natriuretic factor (ANF) gene is activated in cardiac myocytes by Ras and its effector Raf. However, MEK, the best-characterized Raf substrate, cannot efficiently activate ANF suggesting that Raf uses a MEK-independent pathway to activate ANF. By manipulating MEK and Raf activities so that they are equally effective at activating ERK, we now demonstrate that Raf activates at least two signaling pathways in cardiac myocytes that regulate the ANF promoter; the MEK-->ERK pathway inhibits ANF gene expression while a Raf-induced, MEK-independent pathway activates expression. This mechanism may provide increased ability to regulate ANF expression in response to hypertrophic stimuli.     

Involvement of protein kinase C and protein kinase A in the muscarinic receptor signalling pathways mediating phospholipase C activation, arachidonic acid release and calcium mobilisation.

The involvement of protein kinase C (PKC) and protein kinase A (PKA) in cholinergic signalling in CHO cells expressing the M3 subtype of the muscarinic acetylcholine receptor was examined. Muscarinic signalling was assessed by measuring carbachol-induced activation of phospholipase C (PLC), arachidonic acid release, and calcium mobilisation. Carbachol activation of PLC was not altered by inhibition of PKC with chelerythrine chloride, bisindolylmaleimide or chronic treatment with phorbol myristate acetate (PMA). Activation of PKC by acute treatment with PMA was similarly without effect. In contrast, inhibition of PKC blocked carbachol stimulation of arachidonic acid release. Likewise, PKC inhibition resulted in a decreased ability of carbachol to mobilise calcium, whereas PKC activation potentiated calcium mobilisation. Inhibition of PKA with H89 or Rp-cAMP did not alter the ability of carbachol to activate PLC. Similarly, PKA activation with Sp-cAMP or forskolin had no effect on PLC stimulation by carbachol. Carbachol-mediated release of arachidonic acid was decreased by H89 but only slightly increased by forskolin. Forskolin also increased calcium mobilisation by carbachol. These results suggest a function for PKC and PKA in M3 stimulation of arachidonic acid release and calcium mobilisation but not in PLC activation.