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Intracellular signaling mechanisms affected by endothelin (ET), a hypertrophic agonist, and platelet-derived growth factor (PDGF)-BB, a proliferative agonist, in vascular smooth muscle cells were examined. PDGF-BB was a potent mitogen compared with untreated cultures, stimulating both [3H]thymidine incorporation and cell number. In contrast, ET was a poor mitogen, enhancing [3H]thymidine incorporation but not cell number. Simultaneous ET and PDGF-BB treatment was significantly more effective than either agonist alone at stimulating both [3H]thymidine uptake and cell number. Although either ET or PDGF-BB alone stimulated arachidonic acid release, phosphoinositide hydrolysis, protein kinase C activation, PDGF receptor phosphorylation, and mitogen-activated protein kinase activity, of these effectors, only arachidonic acid release was further enhanced by simultaneous ET and PDGF-BB treatment. These results link proliferative and hypertrophic signal transduction pathways in these cells and suggest that arachidonic acid or its metabolites mediate the observed effects of ET on PDGF-BB-stimulated vascular smooth muscle cell proliferation.

Endothelin-1 (ET-1) is a potent stimulator of atrial natriuretic factor (ANF) secretion from myocardial cells. In heart tissue there are two ET receptor subtypes (ETA-R and ETB-R), which can be pharmacologically distinguished by the ET isopeptides ET-1 and ET-3. However, the identification of the ET-R subtype responsible for the rapid enhancement of ANF release, which occurs within minutes of exposing cardiac myocytes to ET, has not been investigated. In the present study ET-1 was about 100-fold more potent than ET-3 at stimulating membrane phosphoinositide hydrolysis, protein kinase C activation, and ANF release from purified primary atrial myocytes. These responses were completely abolished by BQ123, an ETA-R antagonist. Radioligand binding analyses showed that competitor peptides displaced 125I-ET-1 binding to atrial myocyte ET-Rs with a rank order of potency of ET-1 >> BQ123 > ET-3, a characteristic ETA-R pharmacological profile. While neither ET-1 or ET-3 altered forskolin-stimulated cAMP levels, suggesting the absence of the ETB-R, basal cAMP levels were also unaffected by the ETs. Northern analysis using ET-R subtype-specific probes demonstrated that the ETA-R transcript was present in the cultures at levels at least 50-fold greater than the ETB-R transcript. These findings demonstrate that the stimulation of the phosphatidylinositol/protein kinase C pathway, which is required for maximal ET-stimulated ANF release from primary atrial myocytes, is associated with the activation of only the ETA-R, thus defining a specific function for an endogenous ET-R in myocardial cells.

The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.

We have investigated endothelin (ET)-regulated secretion of atrial natriuretic factor (ANF) from primary neonatal rat ventricular myocytes, where hormone release is thought to be constitutive. In a dose-dependent, nifedipine-sensitive manner, ET acutely enhanced ANF release by two- to fivefold over control cultures within 15 min of agonist exposure, demonstrating that ventricular myocytes display a primary characteristic of a regulated secretory cell type. Unlike atrial cultures, ET enhanced ANF release during the first 30 min of exposure; thereafter, secretion rates returned to control levels. KCl, however, effectively enhanced ANF release only during the first 15 min of exposure. Subcellular fractionation of ventricular culture homogenates did not reveal atrial-type dense secretory granules, and pulse-chase labeling experiments showed that the transit time of newly synthesized ANF was short in ventricular myocytes [time required for half of labeled ANF to be released from cells (t1/2) = 0.5-1.5 h) compared with atrial myocytes (t1/2 = 4 h). These results suggest that, whereas ventricular myocytes possess some of the characteristics of a constitutively secreting cell type (e.g., few, if any, dense secretory granules and rapid transit time for newly synthesized hormone); however, they also display the capacity for regulated secretion of ANF in response to the physiological agonist ET.

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.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.

It has been shown recently that alpha-adrenergic agonists can stimulate atrial natriuretic factor (ANF) expression in ventricular cardiac myocytes; however, little is known about the intracellular signals mediating this activation. The present study focused on the potential roles of calcium-regulated kinases and calcium influx in the alpha-adrenergic stimulation of ANF gene expression in ventricular myocardial cell cultures. Myocardial cells maintained for 48 h in serum-free medium supplemented with phenylephrine (PE) possessed up to 15-fold higher levels of ANF peptide and ANF mRNA than control cells. The removal of PE, or the addition of nifedipine, resulted in a rapid decline in ANF expression, suggesting that the sustained elevation of some intracellular messenger (e.g. calcium and/or phospholipid hydrolysis products) was required for the adrenergic response. The calcium channel agonist BAY K 8644 was capable of increasing ANF expression in a nifedipine-sensitive manner; however, unlike PE, it did not stimulate phosphoinositide hydrolysis. The protein kinase C inhibitor, H7, caused an approximate 75% reduction in PE-stimulated ANF expression, but had no effect on BAY K-stimulated expression. W7, a calcium/calmodulin inhibitor, completely blocked the effects of both PE and BAY K 8644. The addition of either H7 or W7 24 h after the PE addition resulted in a decline of ANF expression. These results indicate that alpha-adrenergic agonists augment ANF gene expression through at least two pathways, one that is H7-sensitive, perhaps involving the sustained activation of protein kinase C, and the other that is W7-sensitive, perhaps involving the sustained activation of calmodulin-regulated kinases. Further, it appears that BAY K 8644-mediated increases in ANF expression are independent of protein kinase C activation and dependent on calmodulin-regulated events.