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Background: Alpha-adrenergic receptor (alphaAR)-stimulated hypertrophy in adult rat ventricular myocytes is mediated by reactive oxygen species-dependent activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. Because Ras is known to have redox-sensitive cysteine residues, we tested the hypothesis that alphaAR-stimulated hypertrophic signaling is mediated via oxidative modification of Ras thiols., Methods and Results: The effect of alphaAR stimulation on the number of free thiols on Ras was measured with biotinylated iodoacetamide labeling. alphaAR stimulation caused a 48% decrease in biotinylated iodoacetamide-labeled Ras that was reversed by dithiothreitol (10 mmol/L), indicating a decrease in the availability of free thiols on Ras as a result of an oxidative posttranslational modification. This effect was abolished by adenoviral overexpression of thioredoxin-1 (TRX1) and potentiated by the TRX reductase inhibitor azelaic acid. Likewise, alphaAR-stimulated Ras activation was abolished by TRX1 overexpression and potentiated by azelaic acid. TRX1 overexpression inhibited the alphaAR-stimulated phosphorylation of MEK1/2, ERK1/2, and p90RSK and prevented cellular hypertrophy, sarcomere reorganization, and protein synthesis (versus beta-galactosidase). Azelaic acid potentiated alphaAR-stimulated protein synthesis. Although TRX1 can directly reduce thiols, it also can scavenge ROS by increasing peroxidase activity. To examine this possibility, peroxidase activity was increased by transfection with catalase, and intracellular reactive oxygen species were measured with dichlorofluorescein diacetate fluorescence. Although catalase increased peroxidase activity approximately 20-fold, TRX1 had no effect. Likewise, the alphaAR-stimulated increase in dichlorofluorescein diacetate fluorescence was abolished with catalase but retained with TRX1., Conclusions: AlphaAR-stimulated hypertrophic signaling in adult rat ventricular myocytes is mediated via a TRX1-sensitive posttranslational oxidative modification of thiols on Ras.

Background: Heart failure is characterized by contractile dysfunction of the myocardium and elevated sympathetic activity. We tested the hypothesis that chronic alpha-adrenergic (alpha-ADR) stimulation modifies the molecular and contractile phenotype of cardiac myocytes., Methods and Results: Adult rat ventricular myocytes in culture were exposed to alpha-ADR stimulation (norepinephrine + propranolol) for 48 hours. alpha-ADR stimulation decreased the mRNAs for sarcoplasmic reticulum Ca(2+)-ATPase and Ca(2+) release channel by 56% and 52%, respectively, and increased mRNA and protein for the Na(+)-Ca(2+) exchanger by 70% and 39%, respectively. After washout of the alpha-ADR agonist, simultaneous measurement of [Ca(2+)](i) transients with fura 2 and myocyte shortening by video edge-detection showed that [Ca(2+)](i) amplitude and myocyte shortening were decreased in alpha-ADR-treated myocytes, and the time to peak and time from peak to 80% decline of both [Ca(2+)](i) and myocyte shortening were increased. The concentration-response curve for myocyte shortening by the Na(+) channel activator veratridine was shifted leftward in alpha-ADR-stimulated myocytes (EC(50), 21.6+/-4.6 versus 105.8+/-10.5 nmol/L, P:<0.001)., Conclusions: Chronic alpha-ADR stimulation of cardiac myocytes causes decreases in the expression of sarcoplasmic reticulum Ca(2+)-ATPase and the Ca(2+) release channel that are associated with decreases in [Ca(2+)](i) and contractility. alpha-ADR stimulation simultaneously increases Na(+)-Ca(2+) exchanger expression, thereby increasing sensitivity to intracellular Na(+).

Background: There are alpha-adrenergic receptors on human myocardium that exert positive inotropic effects. The effect of alpha-adrenergic receptor blockade on human left ventricular (LV) performance has not been fully explored. Although alpha-adrenergic receptor blockade might have effects on LV function that are mediated via blockade of postsynaptic myocardial alpha-adrenergic receptors, it is also possible that blockade of presynaptic alpha 2-adrenergic receptors and subsequent increased release of norepinephrine would have effects on LV performance. In the present study, we explored the effects of nonselective alpha-adrenergic receptor blockade on LV performance and transcardiac norepinephrine concentrations in a group of patients with normal LV function and in a group of patients with congestive heart failure secondary to dilated cardiomyopathy., Methods and Results: Using an intracoronary drug infusion technique, we administered the nonselective alpha-adrenergic antagonist phentolamine to 13 patients with normal LV function and 19 patients with congestive heart failure secondary to dilated cardiomyopathy. With a high-fidelity LV catheter, the systolic (+dP/dt) and diastolic (-dP/dt and Tau) LV function responses to intracoronary infusion of phentolamine (0.2 mg/min x 5 minutes) were assessed. In 8 patients with normal ventricular function and 10 patients with congestive heart failure, arterial and coronary sinus blood samples were drawn to determine the effects of phentolamine on catecholamine concentrations. Phentolamine had no measurable effect on LV performance or catecholamine concentrations in the normal ventricular function group. In patients with congestive heart failure, intracoronary phentolamine caused a significant increase in +dP/dt and the rate of isovolumic LV relaxation (-dP/dt and Tau). These hemodynamic effects were accompanied by a significant increase in coronary sinus norepinephrine concentration but no change in arterial norepinephrine concentration., Conclusions: Myocardial alpha-adrenergic receptor blockade causes significant inotropic and lusitropic effects in the failing but not the nonfailing human LV. These effects appear to be mediated by increased release of norepinephrine from cardiac nerves secondary to blockade of presynaptic alpha 2-adrenergic receptors. Differences in the responses of the failing and nonfailing human LV appear to reflect the higher level of sympathetic activation that is seen in the group with congestive heart failure. This suggests that the presynaptic alpha 2-adrenergic receptor exerts a tonic inhibitory effect on the release of norepinephrine from cardiac nerves in patients with congestive heart failure.

Background: Although alpha-adrenergic receptors are present in both normal and failing human left ventricular myocardium and mediate a positive inotropic effect in several other species, it is not known whether stimulation of myocardial alpha-adrenergic receptors exerts a positive inotropic effect or contributes to basal contractile state in vivo in humans., Methods and Results: We studied 15 patients with angiographically normal coronary arteries (seven with normal left ventricular function and eight with left ventricular failure). To avoid the confounding effects of changes in ventricular loading conditions and systemic reflex mechanisms, the alpha-adrenergic receptor-selective antagonist phentolamine and agonist phenylephrine were infused directly into the left main coronary artery, and the change in contractile state was assessed by measuring left ventricular peak (+)dP/dt. Phentolamine alone had no effect on left ventricular contractility. Phenylephrine exerted a concentration-related positive inotropic effect in patients with normal as well as those with failing ventricles. The alpha-adrenergic effect of phenylephrine, defined as the component blocked by phentolamine, was significantly less in patients with ventricular failure (108 +/- 28 mm Hg/sec) than in normal subjects (248 +/- 54 mm Hg/sec; p less than 0.03)., Conclusions: Myocardial alpha-adrenergic receptors do not contribute to the maintenance of basal left ventricular contractile state in humans. However, stimulation of myocardial alpha-adrenergic receptors exerts a positive inotropic effect, the magnitude of which may be attenuated in patients with heart failure.

The pathogenesis of the myopathy occurring in the heart of the cardiomyopathic strain of the Syrian hamster is not well understood but is believed to be associated with abnormal calcium handling by myopathic cells. The purpose of this study was to determine whether the cardiomyopathy occurring in strain BIO 14.6 animals is associated with an enhanced alpha 1-adrenergic receptor-mediated rise in cytosolic calcium, whether a pertussis toxin-sensitive G protein is involved in coupling the alpha 1-adrenergic receptor to changes in intracellular calcium and whether enhanced alpha 1 responsiveness is associated with an increase in the level of expression of the alpha 1-adrenergic receptor or in the pertussis toxin-sensitive G protein or proteins. To test the hypothesis that the cardiomyopathic state is associated with a greater alpha 1-receptor-mediated rise in cytosolic calcium, we studied the effect of phenylephrine (in the presence of propranolol) on time-averaged cytosolic calcium concentration ([Ca2+]i) in isolated cardiac myocytes from cardiomyopathic and age-matched control hamsters. Phenylephrine caused a greater increase both in time-averaged [Ca2+]i (an increase of 48 +/- 8% versus 12 +/- 3%, p less than 0.01) and in contractility (+181 +/- 22% versus +35 +/- 9%, p less than 0.01) in cardiomyopathic than in normal cardiac myocytes. Exposure to pertussis toxin (200 ng/ml for 3 hours) attenuated the alpha 1-adrenergic receptor-mediated increase in contractility and time-averaged [Ca2+]i in both cardiomyopathic and normal cells. The level of pertussis toxin-sensitive G protein, as determined by pertussis toxin-mediated [32P]ADP-ribosylation, was 1.6-fold higher in cardiomyopathic versus normal hamster hearts. The density of alpha 1-adrenergic receptors, as measured by the antagonist radioligand [3H]prazosin and the affinity of the receptor for agonist and antagonist were similar in myopathic and normal heart membranes. Thus, in cardiac myocytes from hamsters, the alpha 1-adrenergic receptor-mediated effects on [Ca2+]i and contractility appear to be mediated by a pertussis toxin-sensitive G protein or proteins. In myocytes from cardiomyopathic hamsters, these alpha 1-adrenergic effects were increased in magnitude, as was the level of pertussis toxin-sensitive G protein, but there was no measurable alteration in the density or ligand binding properties of alpha 1-adrenergic receptors.

Prolonged agonist exposure results in a decrease in the density of alpha 1-adrenergic receptors in rabbit aortic smooth muscle cells. A cDNA for the alpha 1-adrenergic receptor was used to assess the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level in cultured vascular smooth muscle cells from the rabbit aorta. Norepinephrine caused a transient decrease (81% +/- 5%; n = 9) in alpha 1-adrenergic receptor mRNA. The effect was concentration dependent (EC50, approximately 0.3 microM; maximal effect, 10 microM). The maximum decrease occurred after 4 hr of exposure to norepinephrine and was followed by a gradual return to control levels by 24 hr. The decrease in mRNA level was blocked by prazosin, but not propranolol, and was mimicked by phenylephrine. These results indicate that the effect is mediated by stimulation of the alpha 1-adrenergic receptor and suggest that it involves one or more alpha 1-adrenergic-coupled second messenger pathways. The decrease in alpha 1-adrenergic receptor mRNA caused by norepinephrine exceeds that caused by actinomycin D, suggesting that norepinephrine may cause a decrease in the stability of alpha 1-adrenergic receptor mRNA. Actinomycin D also blocked the norepinephrine-induced decrease in mRNA level, further suggesting that the effect of norepinephrine requires induction of transcription, presumably leading to synthesis of a labile factor that is necessary for the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level.

The vascular postsynaptic alpha 1 receptor is the major locus through which adrenergically determined vascular tone is mediated. Therefore, blockade of this receptor is a particularly specific approach to the major pathophysiologic defect in hypertension--an elevation of peripheral vascular resistance. Although the mechanism responsible for this increase in peripheral vascular resistance in hypertension is not known, it is apparent that the sympathetic nervous system plays a key role. In addition, considerable evidence suggests that a common abnormality in hypertension is an increase in the sensitivity of vessels to alpha-adrenergic stimulation, a defect potentially located at the alpha-receptor level. Basic radioligand binding studies of vascular alpha 1 receptors, in fact, demonstrate that the affinity, or avidity, of binding of these sites is under the regulation of both neural and humoral factors. Although diuretics, and more recently, beta-adrenergic blocking agents, have been utilized for the initial treatment of hypertension, recent information about the adverse effects of these agents has led to a reappraisal of the role of alpha-receptor blockade as a rational approach to the initial treatment of hypertension.

To study the role of the alpha-adrenergic receptor (AAR) pathway in the regulation of catecholamine-induced vascular contraction, we developed methods for the direct evaluation of AAR and AAR-coupled calcium efflux in cultured vascular smooth muscle cells derived from the rabbit aorta by enzymatic dissociation. AAR were characterized by the binding of the alpha 1- and alpha 2-selective radioligands [3H]-prazosin and [3H]-yohimbine, respectively. Norepinephrine-stimulated efflux of 45calcium from preloaded cells was measured as an index of AAR-mediated calcium flux. The [3H]-prazosin binding was of high affinity (Kd = 0.15 nM), saturable (Bmax = 75-125 fmol/mg protein), and competed for by agonists and antagonists in the order expected for an alpha 1 receptor. There was no specific binding of [3H]-yohimbine. Norepinephrine-stimulated 45Ca efflux was concentration-dependent (EC50 congruent to 100 nM), and potently blocked by prazosin (IC50 congruent to 0.1 nM), but not yohimbine (IC50 greater than 100 nM), indicating, together with the binding data, that norepinephrine-stimulated 45Ca efflux in this system is alpha 1 mediated. Following treatment of cells with 1-norepinephrine (0.1 nM) for 24 hours, the density of AAR and maximal norepinephrine-stimulated 45Ca efflux were decreased by 72% +/- 14% and 91% +/- 9%, respectively. This cultured cell system offers several advantages for study of the mechanisms of coupling and regulation of alpha-adrenergic receptors in vascular smooth muscle.

The threshold sensitivity of cardiovascular tissues to alpha-adrenergic stimulation is determined largely by the affinity of alpha-adrenergic receptors for agonists. To determine whether changes in alpha-adrenergic receptor affinity could contribute to the regulation of cardiac and vascular responsiveness, we used the alpha-adrenergic-selective radioligands, [3H]prazosin, [3H]-WB-4101, and [3H]rauwalscine, to study and contrast the determinants of alpha-adrenergic receptor affinity in myocardium and vascular smooth muscle from the rat. In both tissues, l-epinephrine binding to the alpha 1-adrenergic receptor describes a shallow curve suggesting more than one affinity state. Computer analysis of binding to myocardial alpha 1-receptors indicates that 15% are of high affinity (Kd = 11 nM) and 85% are of low affinity (Kd = 400 nM). Expression of high affinity sites is magnesium dependent (maximum effect, 5-10 mM), and suppressed by the guanosine 5'-triphosphate analogue Gpp(NH)p (maximum effect, 1 mM) and sodium (maximum effect, 100-200 mM). In vascular smooth muscle, agonist-binding curves are also shallow and exhibit a similar response to that of Gpp(NH)p. Basal receptor affinity in myocardium is significantly higher (5.4-fold) than in vascular smooth muscle. Unlike vascular smooth muscle, in which alpha 1-adrenergic receptor affinity is increased by estrogen or reserpine treatment of the animal, the receptor in myocardium is unaffected by these treatments. In vascular smooth muscle, following reserpine-induced increase in alpha-adrenergic receptor affinity, the Gpp(NH)p effect is still present. Thus, alpha 1-adrenergic receptors in both myocardium and vascular smooth muscle exist in two affinity states and are subject to regulation by several factors, including guanine nucleotides, mono- and divalent cations, tissue of origin, sex hormones, and the level of sympathetic stimulation. Potentially, alterations in alpha 1-adrenergic receptor affinity, independent of a change in receptor number, may play an important role in the regulation of cardiovascular tissue responsiveness to catecholamines.

Rats were treated with reserpine (0.2 mg/kg) on days 1, 3, and 5. On day 6, binding parameters for alpha-1 adrenergic receptors (3H-prazosin) and putative voltage dependent calcium channels, VDCC (3H-nitrendipine), were determined. There was an increase in both the number (2.1 fold) and affinity (1.8 fold) of alpha-1 adrenergic receptors following reserpine treatment. In addition, there was a 2.7 fold increase in the number of VDCCs, but no change in VDCC binding affinity, following reserpine treatment. These data are consistant with the development of smooth muscle supersensitivity following reserpine treatment in a variety of tissues, and suggest that VDCC number may be modulated by the cell in response to tonic levels of catecholamines. Changes in the number of VDCCs may be an important regulatory mechanism for cell function in physiologic and pathologic states.

Cultured vascular smooth muscle cells derived by enzymatic dissociation of rabbit aortic media were used for study of alpha 1-adrenergic receptors (AAR) and receptor-coupled calcium flux. AAR were characterized by binding of the alpha 1-selective radioligand [3H]prazosin, and norepinephrine-stimulated 45calcium efflux was measured as an index of AAR-mediated calcium flux. The binding of [3H]prazosin to a crude cellular homogenate was to a single, saturable site of high affinity (Kd = 0.15 nM, Bmax = 75-125 fmol/mg protein), which was stereo-specific and exhibited the appropriate potency order for competition by agonists. Prazosin (Kd = 0.07 nM) was approximately 3000-fold more potent than the alpha 2-selective antagonist yohimbine (Kd = 222 nM), both of which exhibited Hill coefficients of one, indicative of binding to a single receptor type. In intact cells, norepinephrine caused a concentration-related (EC50 = 100 nM) increase in 45calcium efflux which was blocked by low concentrations of prazosin (IC50 approximately equal to 0.1 nM), but not yohimbine (IC50 greater than 100 nM). An alpha 1-selective concentration of prazosin (100 nM) fully blocked norepinephrine-stimulated 45calcium efflux, and therefore, it appears that this effect does not involve alpha 2-adrenergic receptors. Treatment of cultured cells with 1-norepinephrine for 48 h caused a concentration-related decrease in both AAR density and maximum norepinephrine-stimulated 45calcium efflux. This cultured vascular smooth muscle cell system provides several advantages for the study of alpha-adrenergic receptor coupling and regulation.

To determine whether alpha-adrenergic desensitization of vascular smooth muscle is due to an alteration in alpha 1-adrenergic receptor coupling, we determined the relationship between receptor occupancy and maximal receptor-coupled Ca2+ efflux in cultured rabbit aortic smooth muscle cells (i) under basal conditions as defined by receptor inactivation with phenoxybenzamine and (ii) after 48 hr of exposure to several concentrations of 1-norepinephrine (NE). Neither phenoxybenzamine nor NE exposure caused a change in binding affinity for [3H]prazosin or NE. Maximal [3H]prazosin binding capacity and maximal NE-stimulated 45Ca2+ efflux decreased progressively with exposure of incubated cells to increasing concentrations of phenoxybenzamine or NE. An approximately 80% decrease in maximal [3H]prazosin binding capacity caused by either phenoxybenzamine or NE resulted in complete loss of NE-stimulated 45Ca2+ efflux, indicating that under these conditions approximately 20% of alpha 1-adrenergic receptors are not coupled to the Ca2+ efflux. Under basal conditions, the relationship between maximal [3H]prazosin binding capacity and maximal NE-stimulated 45Ca2+ efflux was markedly nonlinear, so that a near maximal response could be elicited by occupancy of only approximately 40% of the receptors. In contrast, after a 48-hr incubation of cells with NE, occupancy-response coupling was considerably less efficient, so that even full occupancy of the 35% of receptors that remained after NE exposure resulted in only approximately 20% of maximal NE-stimulated 45Ca2+ efflux. Thus, an alteration in occupancy-response coupling at a step proximal to Ca2+ mobilization and/or influx, rather than a reduction in receptor number, is of primary importance in the process of agonist-induced alpha-adrenergic receptor desensitization of vascular smooth muscle cells.

The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate was used to probe the role of protein kinase-C in the modulation of alpha-adrenergic receptor-coupled calcium efflux in cultured vascular smooth muscle cells derived from rabbit aorta. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for 6 minutes caused a concentration-related (maximum effect at greater than or equal to 10 nM) increase in calcium-45 efflux from preloaded cells. Maximum calcium-45 efflux stimulated by 12-O-tetradecanoyl phorbol-13-acetate was equivalent to maximum norepinephrine-stimulated calcium-45 efflux, and maximally effective concentrations of 12-O-tetradecanoyl phorbol-13-acetate and norepinephrine together were no more potent than either drug alone. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for periods of 1-24 hours resulted in complete loss of norepinephrine-stimulated calcium-45 efflux and a much slower, concentration-related reduction in alpha-adrenergic receptor number, with a maximum reduction of 50-60% at 12-O-tetradecanoyl phorbol-13-acetate concentrations greater than or equal to 10 nM. Twenty-four hours of exposure to 12-O-tetradecanoyl phorbol-13-acetate (10 nM) and norepinephrine (10 microM) together caused no greater reduction in [3H]prazosin binding than did norepinephrine alone. 12-O-tetradecanoyl phorbol-13-acetate had no effect on [3H]prazosin-binding affinity. These data suggest an important role for protein kinase-C in both the acute excitation-contraction coupling of vascular smooth muscle alpha-adrenergic receptors, and in the long-term modulation of vascular alpha-adrenergic receptor responsiveness.

Alpha-adrenergic receptors are likely to be important determinants of the effects of catecholamines on vascular resistance. To study the alpha-adrenergic receptor in muscular arteries of the type that determine vascular resistance, we characterized and quantitated alpha-adrenergic receptors in a particulate fraction of the highly reactive, richly innervated arteries of the rat mesentery. With the ligand [3H]-dihydroergocryptine ([3H]-DHEC), specific binding (displaceable by 5 microM phentolamine) is saturable. There is a single class of binding sites with a dissociation constant (Kd) of 2.9 nM and a maximal binding capacity of 68 fmoles of [3H]-DHEC per mg of particulate fraction protein. Catecholamines compete for [3H]-DHEC binding stereospecifically and with the alpha-adrenergic potency series of (-)epinephrine greater than (-)norepinephrine greater than (-)isoproterenol. Binding is rapid (t 1/2 less than or equal to 2 mins) and rapidly reversible (t 1/2 less than or equal to 2 mins). Inhibition of [3H]-DHEC binding by the alpha-adrenergic antagonist phentolamine (Kd = 3.0 nM) is much greater than by the beta-adrenergic antagonist propranolol (Kd = 8200 nM). The alpha 1-selective antagonist prazosin (Kd = 63 nM) is 20 times more potent in competing for [3H]-DHEC binding than is the alpha 2-selective antagonist yohimbine (Kd = 1250 nM), thus suggesting that the alpha-adrenergic receptor identified is predominantly of the alpha 1 subtype that is responsible for vascular smooth muscle contraction. This extension of radioligand binding techniques to highly innervated muscular arteries of the type contributing to vascular resistance will allow the study of the role of the vascular alpha-adrenergic receptor in various physiologic states and models of hypertension.

The short (5-60 min) and long (24 hrs) term effects of norepinephrine (10 uM) and the phorbol ester, 12-0-tetradecanoyl phorbol-13-acetate (10 nM), on total cellular and surface-accessible alpha-1 adrenergic receptor number were determined in DDT1 MF-2 smooth muscle cells. The density of alpha-1 adrenergic receptors was determined with [3H]-prazosin in a crude cellular homogenate (total cellular receptors) and in intact cells at 4 degrees C (surface-accessible receptors). Under basal conditions, all receptors were accessible to the cell surface at 4 degrees C. Short term norepinephrine exposure caused an approximately 40% decrease in surface-accessible binding without a change in total receptor number. Long term norepinephrine exposure caused a further decrease in surface-accessible binding, and an approximately 30% decrease in total receptor number. In contrast, phorbol ester had no effect on surface-accessible or total receptor number with either short or long term exposure. These data suggest that sequestration of cell surface alpha-1 adrenergic receptors is an early step in the process of agonist-mediated down-regulation. In DDT1 MF-2 cells, phorbol ester, alone, does not mimmick the effect of agonist on receptor sequestration or number.

The purpose of this study was to determine whether a cyclic adenosine 3',5'-monophosphate-dependent process can be involved in the regulation of vascular smooth muscle alpha 1-adrenergic receptor responsiveness. Experiments were performed in cultured rabbit aortic smooth muscle cells which were characterized previously according to alpha-adrenergic receptor-binding characteristics and receptor-coupled norepinephrine-stimulated 45Ca++ efflux. The addition of dibutyryl-cyclic adenosine monophosphate to the cell culture medium for 24 hours resulted in a concentration-related decrease in maximal [3H]prazosin-binding capacity (41 +/- 4% decrease with 1 mM dibutyryl-cyclic adenosine monophosphate) without an effect on [3H]prazosin-binding affinity. Prostaglandin E1 (10 microM) and forskolin (10 microM) caused similar decreases in maximal [3H]prazosin-binding capacity, whereas butyrate (1 mM) and dibutyryl-guanosine-3',5' cyclic-monophosphate (1 mM) had no effect. Dibutyryl-cyclic adenosine monophosphate (1 mM) caused significant potentiation of the decrease in [3H]prazosin-binding caused by a submaximal (10 nM) but not a maximal (10 microM) concentration of norepinephrine, suggesting that cyclic adenosine monophosphate may act at a distal step in common with norepinephrine to reduce alpha-adrenergic receptor number. Despite the approximately 41% reduction in alpha-adrenergic receptor number following 24-hour incubation of cells with dibutyryl-cyclic adenosine monophosphate, maximal norepinephrine-stimulated 45Ca++ efflux was not reduced, consistent with the markedly nonlinear relationship between alpha-adrenergic receptor occupancy and maximal norepinephrine-stimulated 45Ca++ efflux in this cell system. These data provide evidence for a novel mechanism by which hormones or drugs which increase cyclic adenosine monophosphate levels can modulate alpha-adrenergic responsiveness in vascular smooth muscle.

To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, we have studied [3H]prazosin binding and l-norepinephrine-induced 45Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, [3H]prazosin bound to a single high affinity site (Kd = 0.096 nM; Bmax = 105 +/- 15 fmol/mg of protein), whereas l-norepinephrine (NE) binding was best described by a two-site model with 43 +/- 8% of sites of high affinity (KH = 92 +/- 3 nM) and 57 +/- 7% of sites of low affinity (KL = 7460 +/- 4330 nM). NE-stimulated 45Ca efflux was concentration-dependent (EC50 = 108 nM) and potently inhibited by prazosin (IC50 = 0.15 nM), but not yohimbine (no inhibition at 10 microM). For the total receptor pool identified by [3H]prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated 45Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. Likewise, following irreversible inactivation of 69 +/- 5% of receptors by phenoxybenzamine, maximal NE-stimulated 45Ca efflux was decreased by only 8 +/- 2%. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1-adrenergic receptors in excess of those needed for maximum NE-stimulated 45Ca efflux. This evidence of "spare" receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.