Dystrophin associates with caveolae of rat cardiac myocytes: relationship to dystroglycan.

The possibility of an interaction between the cytoskeletal protein dystrophin and cell surface caveolae in the mammalian myocardium was investigated by several techniques. Caveolin (cav)-3-enriched, detergent-insoluble membranes isolated from purified ventricular sarcolemma by density-gradient fractionation were found to contain dystrophin and dystroglycan. Further purification of cav-3-containing membranes by immunoprecipitation using anti-cav-3-coated magnetic beads yielded dystrophin but not always dystroglycan. Electron microscopic analysis of precipitated material revealed caveola-sized vesicular profiles that could be double-labeled with anti-dystrophin and anti-cav-3 antibodies. In contrast, immunoprecipitation of membranes with anti-dystrophin-coated beads yielded both cav-3 and dystroglycan. Electron microscopic analysis of this material showed heterogeneous membrane profiles, some of which could be decorated with anti-cav-3 antibodies. To confirm that dystrophin and cav-3 were closely associated in cardiac myocytes, we verified that dystrophin was also present in immunoprecipitated cav-3-containing membranes from detergent extracts, as well as in sonicated extracts of purified ventricular myocytes. Confocal immunofluorescence microscopy of ventricular and atrial cardiac myocytes showed that the cellular distributions of cav-3 and dystrophin partially overlapped. Immuno-electron micrographs of thin sections of rat atrial myocytes revealed a fraction of dystrophin molecules that are in apparently close apposition to caveolae. These results suggest that a subpopulation of dystrophin molecules interacts with cardiac myocyte caveolae in vivo and that some of the dystrophin is engaged in linking cav-3 with the dystroglycan complex.

Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ hybridization to multiple parallel samples using single-stranded cDNA probes.

In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.

Regulation of atrial natriuretic peptide secretion by alpha 1-adrenergic receptors: the role of different second messenger pathways.

A critical problem in determining the intracellular mechanisms regulating atrial natriuretic peptide (ANP) secretion is the extrapolation of data obtained in cultures of atrial myocytes isolated from neonatal animals to that obtained in intact atria isolated from adult animals. We have therefore examined ANP secretory responses in primary cultures of atrial myocytes isolated from adult rats to more closely approach the adult phenotype. Activation of alpha 1-adrenergic receptors by norepinephrine (in the presence of propranolol) increased the rate of ANP secretion approximately two-fold (EC50 = 0.32 microM). This response was mediated predominantly by the alpha 1A-like subtype of alpha 1-receptors. Phorbol esters increased the rate of ANP secretion approximately 2.4-fold independently of alpha 1-receptor occupancy. Kinetic analysis showed that the secretory responses to either agonist did not appear to diminish within 2 h. The responses to both alpha 1-adrenergic stimulation and phorbol ester addition were inhibited by the protein kinase C inhibitor, H-7, but not by structurally related isoquinolines. Influx of extracellular Ca2+, independently of its effects on contraction of the myocytes, was also necessary for a full secretory response to alpha 1-receptor activation. Additionally, the secretory response to alpha 1-adrenergic agonists was attenuated by calmodulin inhibitors. In contrast to the response to alpha 1-adrenergic receptor activation, stimulation of beta-adrenergic receptors or addition of a membrane permeable cAMP analog reduced the rate of both basal and alpha 1-stimulated ANP secretion. These results show that activation of alpha 1-adrenergic receptors in adult rat atrial myocytes directly increases the rate of ANP secretion. This response is dependent upon protein kinase C and supported by extracellular Ca2+ influx. Conversely, activation of beta-adrenergic receptors, which increases intracellular cAMP, directly inhibits ANP secretion.

Mobilization of intracellular calcium by alpha 1-adrenergic receptor activation in muscle cell monolayers.

The fluorescent chelating agent quin 2 has been employed to monitor alterations of intracellular free Ca2+ concentrations ([Ca2+]i) in response to alpha 1-adrenergic receptor activation in adherent BC3H-1 cells. To correlate the kinetics of [Ca2+]i changes with transmembrane fluxes of this ion, continuous monitoring of [Ca2+]i has been undertaken on a monolayer of cells. Previous measurements of the transmembrane efflux of Ca2+ show a distinct lag in the response over a range of phenylephrine concentrations. By contrast, the elevation of [Ca2+]i is rapid (t1/2 approximately 2 s) and maintained for 30 s before it begins to decline to basal concentrations. The differences in kinetics indicate that the temporal delay in cellular Ca2+ efflux results from either activation of the transport system for Ca2+ extrusion or translocation of free Ca2+ to the transport site. The decline of [Ca2+]i with continued agonist exposure parallels both the efflux kinetics from the cell and the decline of total cellular Ca2+. At a time when free [Ca2+]i approaches the resting concentration, total cellular Ca2+ is reduced to a steady state value of 60% of that seen prior to stimulation. The Kact for phenylephrine-stimulated elevation in [Ca2+]i on the monolayer is 0.51 microM, which is similar to the Kact of 0.90 microM observed for phenylephrine-activated 45Ca2+ efflux. Addition of phentolamine subsequent to phenylephrine addition immediately reverses the agonist-stimulated Ca2+ mobilization, initiating a rapid return of [Ca2+]i to resting levels. A comparison of the kinetics of Ca2+ mobilization with its transmembrane flux suggests that the agonist augments the rate of recycling of intracellular Ca2+ between the free and bound states rather than causing release as a single bolus from the bound stores.

Genetic determinants of blood pressure regulation.

Blood pressure homeostasis in humans reflects the coordinate interactions of cardiac output, peripheral vascular resistance, renal volume control, and CNS integration in response to short- and long-term environmental stimuli. Variations in mean arterial pressure within the population include a significant hereditary component. The clearest examples of this genetic contribution occur in rare forms of monogenic hypertension (glucocorticoid remediable aldosteronism, apparent mineralocoid excess, Liddle's syndrome) or hypotension (pseudohypoaldosteronism type I, Bartter's syndrome, Gitelman's syndrome). Primary hypertension, which comprises approximately 95% of hypertensives and is a major risk factor for coronary heart disease, stroke, and renal disease in the U.S., represents a multifactorial and polygenic disease with incremental contributions from genetic and environmental determinants. Efforts to date have identified several candidate genes involved in primary hypertension, including angiotensinogen (AGT), a vasoactive peptide; alpha-adducin, a protein that regulates sodium transport; and the G protein beta 3 subunit, a protein involved in intracellular signal transduction. Advances in knowledge and technology associated with the Human Genome Project, combined with continuing basic research on the physiologic and biochemical causes of hypertension, offer promise for improved diagnosis and therapy of this prevalent disease.

Release of nonmitochondrial sequestered Ca2+ from permeabilized muscle cells in culture.

Activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells results in the rapid elevation of intracellular Ca2+, accompanied by an unusually slow and small increase in inositol 1,4,5-trisphosphate (IP3) formation [J. Biol. Chem. 263: 1952-1959 (1988); Mol. Pharmacol. 32: 376-383 (1987)]. To further assess the role of IP3 in receptor-stimulated Ca2+ mobilization, we have examined Ca2+ disposition in saponin-permeabilized BC3H-1 cells. Permeabilized cells loaded with tracer 45Ca2+ in a buffer containing 100 nM free Ca2+ accumulated greater than 75% of their Ca2+ into an ATP-sensitive compartment and were insensitive to inhibitors of mitochondrial Ca2+ uptake. Application of IP3 resulted in a rapid increase in 45Ca2+ efflux. Under isotopic equilibrium, approximately 90% of the total membrane-enclosed 45Ca2+ was released by 10 microM IP3 within 30 sec. Maximally and half-maximally effective concentrations of IP3 were 22 microM and 0.9 microM, respectively. Application of 10 microM GTP, but not guanine triphosphate-gamma-sulfate, resulted in a slight increase in 45Ca2+ efflux, which reflected a loss in total cellular Ca2+. The GTP-mediated response was slower and of far smaller magnitude than that mediated by IP3. A Ca2+-triggered Ca2+ release mechanism appears not to amplify the receptor response in BC3H-1 cells, inasmuch as 45Ca2+ efflux was not appreciably increased by elevated concentrations of free Ca2+. Furthermore, caffeine and ryanodine had no effect on basal, IP3-mediated, or alpha 1-adrenergic-stimulated Ca2+ release from intact or permeabilized cells. In conclusion, BC3H-1 cells, although showing small and slow increases in IP3 formation upon agonist stimulation, exhibit normal sensitivity to IP3-elicited release of Ca2+ and low sensitivity to other candidate Ca2+-mobilizing agents. The IP3-sensitive Ca2+ stores may be localized within specialized compartments and may play a greater role in the maintenance of elevated cytosolic Ca2+ than in the initial response to receptor activation.

Regulation of ANP secretion by endothelin-1 in cultured atrial myocytes: desensitization and receptor subtype.

We examined endothelin-1 (ET-1) binding and ET-1-regulated atrial natriuretic peptide (ANP) secretion in primary cultures of adult rat atrial myocytes. ET-1 binding was analyzed as a reversible bimolecular reaction, with bimolecular association rate constant = 1.9 x 10(9) M-1.h-1, dissociation rate constant = 0.028 h-1, and a dissociation constant, calculated from these values = 0.015 nM. ET-1 increased ANP secretion with a one-half effective concentration (EC50) of 0.62 nM, which correlated with EC50 receptor occupancy under equivalent experimental conditions (0.75 nM). The secretory response rapidly desensitized (half-time = 10 min at 10 nM ET-1). The time courses for ET-1 binding, ET-1-stimulated secretion, and desensitization were all comparable. Recovery from desensitization was slow and paralleled the recovery of 125I-labeled ET-1 binding. The ETA receptor subtype-selective antagonist, BQ-123, inhibited 125I-ET-1 binding and ET-1-activated ANP secretion with high affinity, whereas the ETB-selective agonists, endothelin-3 and sarafotoxin S6c, inhibited binding with low affinity and did not effectively stimulate ANP secretion. We conclude that 1) ET-1 can stimulate ANP secretion by direct action on the atrial myocytes; 2) primary cultures of adult rat atrial myocytes express only the ETA receptor subtype; 3) the ANP secretory response to ET-1 desensitizes rapidly but recovers slowly; and 4) occupation of the ETA receptors by ET-1 initiates the unidirectional sequence of receptor activation, signal transduction, ANP secretion, and finally, desensitization.

The relationship between phosphatidylinositol metabolism and mobilization of intracellular calcium elicited by alpha1-adrenergic receptor stimulation in BC3H-1 muscle cells.

The BC3H-1 cell is a stable cell line of probable smooth muscle origin which expresses nicotinic acetylcholine, alpha1- and beta2-adrenergic receptors on its cell surface. Stimulation of the alpha1 receptor mobilizes 70% of the intracellular Ca2+ within a 2-3 minute interval. To delineate further the linkage between alpha1-receptor occupation and response, we have examined the quantitative relationship between fractional occupation of the receptor, the turnover of inositol-containing phospholipids, and the Ca2+ efflux. Alpha1-receptor activation stimulates the incorporation of [3H]inositol into phosphatidylinositol and the enhanced incorporation is linear over a 60-min interval. In contrast, agonist-elicited increases in hydrolysis of phosphatidylinositol and phosphatidylinositol mono- and bisphosphate develop more slowly, and a 5-min lag in enhanced formation of inositol trisphosphate, inositol bisphosphate, and inositol monophosphate is evident. The increased rate of Ca2+ efflux and enhanced rate of inositol incorporation into phosphatidylinositol elicited by phenylephrine exhibit virtually identical dependencies on agonist concentrations. Moreover, fractional inactivation of receptors with phenoxybenzamine shows equivalent increments in the reduction of the two intracellular responses. Both responses are linearly related to the residual receptor sites remaining after fractional inactivation. These findings indicate an absence of a receptor reserve in activating these intracellular events. Moreover, although alpha receptor occupation stimulates phosphatidylinositol hydrolysis, no evidence is provided that this event would precede Ca2+ release. Should inositol trisphosphate mediate intracellular Ca2+ mobilization in these cells, it would be active in extremely low concentrations or occur as a tightly coupled event in a microscopic environment.

Receptor-mediated inositol phosphate formation in relation to calcium mobilization: a comparison of two cell lines.

Previous studies indicated that activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells (S. K. Ambler and P. Taylor, J. Biol. Chem. 261:5866-5871, 1986) and muscarinic receptors in 1321N1 astrocytoma cells (S. B. Masters, T. K. Harden, and J. H. Brown, Mol. Pharmacol. 27:325-332, 1985) resulted in the rapid mobilization of Ca2+ from internal stores of both cell types. Paradoxically, alpha 1-adrenergic agonists did not rapidly increase inositol trisphosphate (Ins-P3) formation in BC3H-1 cells, in distinction to the rapid increase in Ins-P3 accumulation observed in 1321N1 cells after muscarinic stimulation. To determine whether the variations observed in the Ins-P3 response could be ascribed to differences in the relative amounts of inositol 1,4,5-trisphosphate, inositol 1,3,4-trisphosphate, and inositol tetrakisphosphate (respectively, Ins-1,4,5-P3, Ins-1,3,4-P3, and Ins-P4), we have separated the individual inositol phosphates by high-performance liquid chromatography and examined the rates of conversion of individual inositol phosphates in the two types of cells. Muscarinic stimulation of 1321N1 cells resulted in increased Ins-1,4,5-P3 production, as well as the rapid production of Ins-1,3,4-P3 and Ins-P4. Application of alpha 1-agonist to BC3H-1 cells produced a modest but delayed increase in accumulation of Ins-1,4,5-P3. Adrenergic stimulation also resulted in a smaller and even slower production of Ins-1,3,4-P3, and Ins-P4 could not be detected in BC3H-1 cells under any conditions employed. Thus, over a 30-sec interval in which Ca2+ is mobilized to a maximum extent, increases in Ins-1,4,5-P3, Ins-1,3,4-P3, or Ins-P4 amounted to less than 10% over basal values in BC3H-1 cells. These results indicate that the regulation of Ins-P3 isomer formation and conversion may vary substantially between different cell types. In addition, if inositol 1,4,5-trisphosphate is the sole mediator of intracellular Ca2+ release, it is necessary to propose that an increase in Ins-1,4,5-P3 sufficient to mobilize Ca2+ rapidly may occur only within discrete cellular localities in some cell types. According, it may not be possible to detect the increases in Ins-1,4,5-P3 over basal concentrations when measuring total cellular inositol phosphates.

Agonist-stimulated oscillations and cycling of intracellular free calcium in individual cultured muscle cells.

Receptor regulation of [Ca2+]i was monitored in individual BC3H-1 muscle cells with intracellularly trapped fura-2 using digital imaging analysis techniques. Activation of alpha 1-adrenergic or H1-histaminergic receptors resulted in multiple bursts, or oscillations, of elevated [Ca2+]i with an average interval frequency of approximately 1.8 min-1. The duration of oscillatory behavior was generally more prolonged in response to phenylephrine than in response to histamine. Additionally, a larger fraction of the cells responded with [Ca2+]i oscillations to phenylephrine (approximately 90%) than to histamine (approximately 60%), although the majority of cells produced oscillations in response to both agonists. In most cells, the receptor-mediated [Ca2+]i oscillations continued for several minutes in the absence of extracellular Ca2+, although the amplitude of the individual peaks gradually decreased. The activation of [Ca2+]i oscillations by H1-receptors was more dependent upon extracellular Ca2+ than those elicited by alpha 1-receptors, reflecting the greater dependency of the histaminergic response on Ca2+ influx. Readdition of Ca2+ to the incubation buffer resulted in the resumption of the [Ca2+]i oscillations. These results indicate that considerable cycling of Ca2+ between the cytoplasm and the endoplasmic reticulum must occur. Receptor-mediated [Ca2+]i oscillations were much more prevalent in subconfluent cells than in confluent cells, possibly due to increased coupling of the cells at higher densities. The cells were capable of responding independently of one another, since sister cells displayed unique temporal responses immediately following cell division. Thus, the linkage of receptor occupancy to [Ca2+]i elevation is a functionally unique property for each individual cell and can be influenced by epigenetic factors.

Type B atrial natriuretic peptide receptor in cardiac myocyte caveolae.

We have previously shown that atrial natriuretic peptide (ANP) is present in caveolae of in situ rat atrial myocytes. To investigate whether intracaveolar ANP of rat atrial myocytes exists within caveolae bound to type B ANP receptors (ANP-RB, a guanylyl cyclase), we have used confocal immunofluorescence microscopy applied to primary cultures of atrial myocytes from adult rats and to freshly dissociated rat atrial myocytes (not cultured). These experimental designs tested whether atrial myocyte ANP-RB colocalizes at the plasmalemma and elsewhere in the cell with the muscle-specific isoform of the caveolar coating protein caveolin-3, and with a fraction of cellular ANP. The experiments showed that cellular caveolin-3, a fraction of cellular ANP-RB, and a fraction of cellular ANP colocalize at the plasmalemma of cultured atrial myocytes and of freshly dissociated atrial myocytes. The observations support the hypothesis that in rat atrial myocytes, intracaveolar ANP is bound to ANP-RB, a protein whose cytosolic amino acid sequences are known to encode guanylyl cyclase activity. We suggest that among the (probably multiple) effects of the cGMP thus generated in the cytoplasmic microdomain underlying atrial myocyte caveolae may be the activation of cGMP-dependent protein kinase, which would thereby inhibit plasma membrane Ca2+ channel activity and contribute to a negative inotropic effect of ANP.