Regulation of calcium channel expression in neonatal myocytes by catecholamines.

Expression of the dihydropyridine (DHP) receptor (alpha 1 subunit of L-type calcium channel) in heart is regulated by differentiation and innervation and is altered in congestive heart failure. We examined the transmembrane signaling pathways by which norepinephrine regulates DHP receptor expression in cultured neonatal rat ventricular myocytes. Using a 1.3-kb rat cardiac DHP receptor probe, and Northern analysis quantified by laser densitometry, we found that norepinephrine exposure produced a 2.2-fold increase in DHP receptor mRNA levels at 2 h followed by a decline to 50% of control at 4-48 h (P < 0.02). The alpha-adrenergic agonist phenylephrine and a phorbol ester produced a decline in mRNA levels (8-48 h). The beta-adrenergic agonist isoproterenol and 8-bromo-cAMP produced a transient increase in mRNA levels. After 24 h of exposure to isoproterenol, 3H-(+)PN200-110 binding sites increased from 410 +/- 8 to 539 +/- 39 fmol/mg (P < 0.05). The number of functional calcium channels, estimated by whole-cell voltage clamp experiments, was also increased after 24 h of exposure to isoproterenol. Peak current density (recordings performed in absence of isoproterenol) increased from -10.8 +/- 0.8 (n = 23) to -13.9 +/- 1.0 pA/pF (n = 27) (P < 0.01). Other characteristics of the calcium current (voltage for peak current, activation, and inactivation) were unchanged. Exposure for 48 h to phenylephrine produced a significant decline in peak current density (P < 0.01). We conclude that beta -adrenergic transmembrane signaling increases DHP receptor mRNA and number of functional calcium channels and that alpha - adrenergic transmembrane signaling produces a reciprocal effect. Regulation of cardiac calcium channel expression by adrenergic pathways may have physiological and pathophysiological importance.     

Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels.

To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.     

Biphasic Adrenergic Modulation of β-Adrenergic Receptors in Man: AGONIST-INDUCED EARLY INCREMENT AND LATE DECREMENT IN β-ADRENERGIC RECEPTOR NUMBER

beta-Adrenergic receptors in mononuclear leukocyte preparations were assessed with (-)[(3)H]-dihydroalprenolol binding studies during the infusion of adrenergic agonists into normal human subjects. During the infusion of isoproterenol into seven subjects, mean (+/-SE) (-)[(3)H]dihydroalprenolol binding increased from 25+/-3 fmol/mg protein to 47+/-8 fmol/mg protein (P < 0.02) at 0.5 h and 40+/-3 fmol/mg protein (P < 0.01) at 1 h and decreased to 12+/-1 fmol/mg protein (P < 0.01) at 4-6 h. During the infusion of epinephrine into three subjects, mean (-)[(3)H]dihydroalprenolol binding increased from 32+/-3 to 63+/-3 fmol/mg protein (P < 0.01) at 0.5-1 h. By Scatchard plot analysis, these changes were attributable to changes in the number of available binding sites rather than changes in binding affinity. The observed changes in the number of (-)[(3)H]dihydroalprenolol binding sites were not paralleled by changes in total mononuclear cell counts or in T lymphocyte, B lymphocyte, and monocyte distributions. Thus, we conclude that adrenergic agonists modulate the number of available beta-adrenergic receptors on circulating mononuclear cells in a biphasic manner, with an early increment and a late decrement, in man. Further, the finding that the increase in pulse rate in response to a "pulse" infusion of isoproterenol was significantly greater after 0.5-1 h of agonist infusion suggests that the observed early agonist-induced increment in beta-adrenergic receptor number on circulating cells is paralleled by increments in extra-vascular beta-adrenergic receptor sensitivity.     

Calcium channel receptor sites for (+)-[3H]PN 200-110 in coronary artery

The receptor sites for 1,4-dihydropyridine (DHP) Ca++ channel antagonists in porcine coronary artery were identified and characterized by a binding assay using (+)-[3H]PN 200-110 as a radioligand. Specific (+)-[3H]PN 200-110 binding in porcine coronary artery was saturable, reversible and of high affinity (Kd = 0.24 nM) and it showed a pharmacological specificity as well as stereoselectivity which characterized the receptor sites for DHP Ca++ channel antagonists. DHP antagonists competed for the (+)-[3H]PN 200-110 binding in order: PN 200-110 greater than mepirodipine greater than nisoldipine greater than nicardipine greater than nitrendipine greater than nimodipine greater than nifedipine greater than (-)-PN 200-110. (+)-PN 200-110 was approximately 140 times as potent as the (-)-isomer. The potencies (PKi) of these eight DHP Ca++ channel antagonists in competing for (+)-[3H]PN 200-110 binding sites in porcine coronary artery correlated well with their pharmacological potencies. Specific (+)-[3H]PN 200-110 binding in the coronary artery was enhanced by d-cis-diltiazem and was inhibited incompletely by verapamil and D-600. In EDTA-pretreated coronary artery, the maximal number of binding sites for specific (+)-[3H]PN 200-110 binding was reduced (80%) markedly, and it was restored to the untreated level by the addition of Ca++ and Mg++.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]BAY K 8644, a 1,4-dihydropyridine Ca++ channel activator: characteristics of binding to high and low affinity sites in cardiac membranes

The binding of [3H]BAY K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)- pyridine-5-carboxylate] to high and low affinity sites in rabbit ventricular membranes was characterized. Binding affinities were 0.66 and 138 nM at 15 degrees C and 9.1 and 72 nM at 37 degrees C, for the high and low affinity sites, respectively, and binding site densities were 0.3 and 14 pmol/mg at 15 degrees C and 0.41 and 1.4 pmol/mg at 37 degrees C, for the respective sites. The modification of high affinity [3H]BAY K 8644 binding by verapamil, diltiazem, tiapamil, Ca++ and EDTA appeared to be the same as that for nitrendipine binding, consistent with the hypothesis that the high affinity binding site for [3H]BAY K 8644 on isolated membranes is the same as the 1,4-dihydropyridine antagonist binding site. The binding of [3H]BAY K 8644 to a low affinity binding site was modified by temperature, Ca++ and diltiazem, but the lack of stereoselectivity, lack of denaturation by heat and the large number of sites indicated that most of the low affinity binding sites were not associated with Ca++ channels. It is concluded that the high affinity binding site for BAY K 8644 is associated with Ca++ channels, and is modified by at least some of the factors that modify the binding site for Ca++ channel antagonists, whereas many or all of the low affinity binding sites detected are not related to Ca++ channels.     

o-Isothiocyanate dihydropyridine (oNCS-DHP), a long-acting, reversible inhibitor of the Ca++ channel

The binding characteristics and pharmacological properties of o-isothiocyanate dihydropyridine [oNCS-DHP; 2,6-dimethyl-3,5-dicarbomethoxy-4-(2-isothiocyanatophenyl)-1, 4-dihydropyridine] were investigated in guinea pig heart and ileum. [3H]oNCS-DHP bound to a single population of high-affinity sites (Bmax = 107 fmol/mg of protein and Kd = 0.99 nM) in cardiac membranes, with a specificity characteristic of dihydropyridine receptors. After incubation of membranes with the tracer (0.5 nM), addition of excess nifedipine (1 microM) caused a dissociation of [3H]oNCS-DHP from its binding site. The reversibility of [3H]oNCS-DHP binding was confirmed by the lack of affinity labeling of cardiac membranes as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. oNCS-DHP inhibited the inward Ca++ current of isolated guinea pig cardiac myocytes as determined in voltage-clamp experiments. In isolated perfused guinea pig hearts, oNCS-DHP caused a concentration-dependent increase in coronary artery flow and a decrease in left ventricular pressure. The effects of the highest concentration (0.3 microM) were still near maximal after a 1-h washout. Suppression of K+ depolarization-induced contractures of isolated ileal longitudinal muscle strips by oNCS-DHP remained maximal even after 5 h of washout. In all of the three biological test systems investigated, the Ca++ channel activator Bay K 8644 caused a complete and rapid reversal of the inhibitory effects of oNCS-DHP. Thus, it can be concluded that oNCS-DHP does not bind irreversibly to Ca++ channel dihydropyridine receptors in guinea pig heart and ileum. However, the o-isothiocyanatophenyl substituent on the dihydropyridine molecule confers upon the compound a very long duration of Ca++ channel blocking activity.     

Upregulation of the rat cardiac sodium channel by in vivo treatment with a class I antiarrhythmic drug.

Class I antiarrhythmic drugs inhibit the sodium channel by binding to a drug receptor associated with the channel. In this report we show that in vivo administration of the class I antiarrhythmic drug mexiletine to rats induces sodium channel upregulation in isolated cardiac myocytes. The number of sodium channels was assessed with a radioligand assay using the sodium channel-specific toxin [3H]batrachotoxinin benzoate ([3H]BTXB). The administration of mexiletine to rats induced a dose-dependent increase in [3H]BTXB total specific binding (Bmax) on isolated cardiac myocytes. Sodium channel numbers were 15 +/- 5, 29 +/- 9, and 54 +/- 4 fmol/10(5) cells after 3 d treatment with 0, 50 mg/kg per d, and 150 mg/kg per d mexiletine (P less than 0.001, analysis of variance). Sodium channel number increased monoexponentially to a steady-state value within 3 d with a half-time of increase of 1.0 d. After cessation of treatment with mexiletine the number of sodium channels returned to normal within 12 d. Finally, treatment with mexiletine altered only sodium channel number; the Kd for [3H]BTXB and the IC50 for mexiletine were not different for myocytes prepared from control and mexiletine-treated rats.     

Competitive and cooperative effects of Bay K8644 on the L-type calcium channel current inhibition by calcium channel antagonists

Phenylalkylamines, benzothiazepines, and dihydropyridines bind noncompetitively to the L-type calcium channel. The molecular mechanisms of this interaction were investigated in enzymatically isolated rat ventricular myocytes using the whole-cell patch-clamp technique. When applied alone, felodipine, verapamil, and diltiazem inhibited the L-type calcium current with values of inhibitory constant (K(B)) of 11, 246, and 512 nM, respectively, whereas 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K8644) activated I(Ca) with activation constant (K(A)) of 33 nM. Maximal activation of I(Ca) by 300 nM Bay K8644 strongly reduced the inhibitory potency of felodipine (apparent K(B) of 165 nM), significantly reduced the inhibitory potency of verapamil (apparent K(B) of 737 nM), but significantly increased the inhibitory potency of diltiazem (apparent K(B) of 310 nM). In terms of a new pseudoequilibrium two-drug binding model, the interaction between the dihydropyridine agonist Bay K8644 and the antagonist felodipine was found purely competitive. The interaction between Bay K8644 and verapamil or diltiazem was found noncompetitive, and it could be described only by inclusion of a negative interaction factor nu = -0.60 for verapamil and a positive interaction factor nu = +0.24 for diltiazem. These results suggest that at physiological membrane potentials, the L-type calcium channel cannot be simultaneously occupied by a dihydropyridine agonist and antagonist, whereas it can simultaneously bind a dihydropyridine agonist and a nondihydropyridine antagonist. Generally, the effects of the drugs on the L-type calcium channel support a concept of a channel domain responsible for binding of calcium channel antagonists and agonists changing dynamically with the membrane voltage and occupancy of individual binding sites.     

Effect of thyroid hormone on slow calcium channel function in cultured chick ventricular cells.

The hyperthyroid state is associated with increased myocardial contractility. To clarify responsible mechanisms, we examined the effects of thyroid hormone on slow Ca channels, beta-adrenergic receptors, transsarcolemmal 45Ca flux and cytosolic free calcium in cultured chick ventricular cells. Compared with cells grown without triiodothyronine (T3), cells grown in 10 nM T3 possessed 67% (P less than 0.05) more dihydropyridine 3H-PN200-110 binding sites, 24% (P less than 0.05) more beta-adrenergic antagonist 3H-CGP12177 binding sites, a 57% (P less than 0.05) greater nifedipine-sensitive initial 45Ca uptake rate, and a 31% (P less than 0.05) greater nifedipine-sensitive 45Ca uptake rate in response to BAY k 8644. Time-averaged mean intracellular free Ca concentration ([Ca]i) measured with fura-2, total protein content, and dissociation constant values for 3H-PN200-110 or 3H-CGP12177 binding was not significantly different in the two groups of cells. BAY k 8644 (1 microM) increased mean [Ca]i 2.85- or 2.16-fold in cells grown with or without 10 nM T3, respectively. l-Isoproterenol (1 microM) increased [Ca]i 1.53- or 1.28-fold in cells grown with or without 10 nM T3, respectively. We conclude that thyroid hormone augments transsarcolemmal Ca influx, at least in part via slow Ca channels associated with increased numbers of these channels. T3-treated cells appear to be more responsive to the effects of BAY k 8644 or isoproterenol on [Ca]i.     

Expression of dihydropyridine receptor (Ca2+ channel) and calsequestrin genes in the myocardium of patients with end-stage heart failure.

Cytoplasmic free calcium ions (Ca2+) play a central role in excitation-contraction coupling of cardiac muscle. Abnormal Ca2+ handling has been implicated in systolic and diastolic dysfunction in patients with end-stage heart failure. The current study tests the hypothesis that expression of genes encoding proteins regulating myocardial Ca2+ homeostasis is altered in human heart failure. We analyzed RNA isolated from the left ventricular (LV) myocardium of 30 cardiac transplant recipients with end-stage heart failure (HF) and five organ donors (normal control), using cDNA probes specific for the cardiac dihydropyridine (DHP) receptor (the alpha 1 subunit of the DHP-sensitive Ca2+ channel) and cardiac calsequestrin of sarcoplasmic reticulum (SR). In addition, abundance of DHP binding sites was assessed by ligand binding techniques (n = 6 each for the patients and normal controls). There was no difference in the level of cardiac calsequestrin mRNA between the HF patients and normal controls. In contrast, the level of mRNA encoding the DHP receptor was decreased by 47% (P less than 0.001) in the LV myocardium from the patients with HF compared to the normal controls. The number of DHP binding sites was decreased by 35-48%. As reported previously, expression of the SR Ca(2+)-ATPase mRNA was also diminished by 50% (P less than 0.001) in the HF group. These data suggest that expression of the genes encoding the cardiac DHP receptor and SR Ca(2+)-ATPase is reduced in the LV myocardium from patients with HF. Altered expression of these genes may be related to abnormal Ca2+ handling in the failing myocardium, contributing to LV systolic and diastolic dysfunction in patients with end-stage heart failure.     

Ca²⁺ channel activators reveal differential L-type Ca²⁺ channel pharmacology between native and stem cell-derived cardiomyocytes

Human stem cell-derived cardiomyocytes provide new models for studying the ion channel pharmacology of human cardiac cells for both drug discovery and safety pharmacology purposes. However, detailed pharmacological characterization of ion channels in stem cell-derived cardiomyocytes is lacking. Therefore, we used patch-clamp electrophysiology to perform a pharmacological survey of the L-type Ca2? channel in induced pluripotent and embryonic stem cell-derived cardiomyocytes and compared the results with native guinea pig ventricular cells. Six structurally distinct antagonists [nifedipine, verapamil, diltiazem, lidoflazine, bepridil, and 2-[(cis-2-phenylcyclopentyl)imino]-azacyclotridecane hydrochloride (MDL 12330)] and two structurally distinct activators [methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate (Bay K8644) and 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176)] were used. The IC?? values for the six antagonists showed little variability between the three cell types. However, whereas Bay K8644 produced robust increases in Ca2? channel current in guinea pig myocytes, it failed to enhance current in the two stem cell lines. Furthermore, Ca2? channel current kinetics after addition of Bay K8644 differed in the stem cell-derived cardiomyocytes compared with native cells. FPL 64176 produced consistently large increases in Ca2? channel current in guinea pig myocytes but had a variable effect on current amplitude in the stem cell-derived myocytes. The effects of FPL 64176 on current kinetics were similar in all three cell types. We conclude that, in the stem cell-derived myocytes tested, L-type Ca2? channel antagonist pharmacology is preserved, but the pharmacology of activators is altered. The results highlight the need for extensive pharmacological characterization of ion channels in stem cell-derived cardiomyocytes because these complex proteins contain multiple sites of drug action.     

Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone.

The effects of estrogen and progesterone on uterine alpha-adrenergic receptors were investigated by direct receptor-binding studies. Immature female rabbits were primed with estrogen by intramuscular injections for 4 days. Other rabbits were primed with progesterone by injections of estrogen for 4 days followed by injections of progesterone for 4 days. The alpha adrenergic antagonist, [3H]dihydroergocryptine, was used to directly assess the number and affinity of alpha adrenergic receptors in membranes derived from estrogen-and progesterone-primed uteri. Membranes from estrogen-primed uteri contained 257 +/- 52 fmol of [3H]dihydroergocryptine-binding sites per mg protein whereas membranes from progesterone-primed uteri contained 83 +/- 11 fmol of of binding sites per mg protein. This reduction of alpha adrenergic receptor-binding sites by progesterone was statistically significant (P less than 0.02). In contrast, no significant difference in the binding site affinity was observed between the estrogen- and progesterone-primed groups. The progesterone-induced decrease in the number of uterine alpha adrenergic receptors provides a potential explanation for the reduced alpha adrenergic contractile response to epinephrine in the progesterone-primed myometrium.     

Patch-clamp evidence for calcium channels in apical membranes of rabbit kidney connecting tubules.

To test the hypothesis that Ca channel plays a role in renal epithelial Ca transport, we exposed and patched apical membranes of freshly microdissected rabbit connecting tubules (CNTs). Single channel Ca currents were recorded with Ba as the charge carrier. In the cell-attached mode, 8-Br-cAMP increased the open-state probability (Po) to 0.6%. In excised, inside-out patches, Po was low spontaneously and remained low during either bath protein kinase A catalytic subunit (PKAcs) or Bay K 8644. Exposure to both agonists, however, unmasked Ca channels previously latent with only one, raising Po by 1.05% at membrane potential of -70 mV. Mean Po for 14 seals (2.57%) peaked at -70 mV, declining with either hyperpolarization or depolarization. The slope conductance was 25 pS. The extrapolated reversal potential (138 mV) agrees with the calculated equilibrium potential for Ca (158 mV). The Ca to Na permeability ratio exceeded 2,800. In four patches stimulated by Bay K 8644 and PKAcs, bath nifedipine reduced Po from 1.03 to 0.15% at -63 mV. These patch-clamp data demonstrate a selective, 25-pS, cAMP/PKAcs-sensitive Ca channel in apical membranes of CNT. Po is stimulated by PKAcs and dihydropyridine (DHP) agonist, but inhibited by DHP antagonist and by depolarization. The data are consistent with the potential role of apical membrane Ca channel in epithelial Ca transport.     

Beta-1 receptor is the predominant beta-adrenoreceptor on rat brown adipose tissue

A new in vitro radioligand binding assay is described for brown adipose tissue using the beta adrenergic antagonist [3H]CGP 12177 (4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one). Binding was saturable and stereoselectively inhibited by propranolol. There was 60 to 80% specific binding using either 30 microM l-isoproterenol or 10 microM l-propranolol to define nonsaturable binding. [3H]CGP 12177 was bound to partially purified membranes from collagenase-separated brown adipocytes with a Kd of 0.84 nM, as determined from kinetic studies, and 1.24 +/- 0.13 nM as found by equilibrium binding studies; maximum binding was 14.2 +/- 0.9 fmol/mg of protein. Membranes from whole-pad homogenates had a similar Kd of 1.17 +/- 0.14 nM but twice the maximum number of binding sites (28.5 +/- 4.4 fmol/mg of protein). Intact brown adipocytes had a Kd of 0.55 nM and a maximum binding of 29.4 +/- 1.5 fmol X 10(-6)/cell or 17,700 sites per cell. Competitive binding studies showed about 80% of the binding sites to be of the beta-1 and 20% of the beta-2 subtype. The pA2 values derived from inhibition of isoproterenol-stimulated in vitro oxygen consumption in intact brown adipocytes by the beta-1 selective antagonist metoprolol and beta-2 selective lCl 118551 were in close agreement with their respective K1 values at the beta-1 receptor as derived from competitive binding studies. These data strongly suggest that the beta-1 adrenoreceptor on brown adipose tissue is primarily responsible for the initiation of thermogenesis in this tissue.     

Comparative behavioral, neurochemical and pharmacological activities of dihydropyridine calcium channel activating drugs.

The behavioral (deficits in motor function in mice), neurochemical (affinity for mouse brain membrane dihydropyridine receptors, effects on neurotransmitter/metabolite levels in mice) and pharmacologic (effect on the contractile activity of guinea pig ileal longitudinal smooth muscle) properties of the calcium channel activators (+/-)-BAY K 8644, (+/-)-202-791 (and their corresponding channel activating and antagonist enantiomers) and CGP-28392 were investigated and compared. The calcium channel activating enantiomers (-)-S-BAY K 8644, (+)-S-202-791 and (+/-)-BAY K 8644, (+/-)-202-791 and CGP-28392 produced a dose-dependent impairment of rotarod ability and decreases in motor activity in mice with the following order of potency: (-)-S-BAY K 8644 greater than (+/-)-BAY K 8644 much greater than (+)-S-202-791 greater than (+/-)-202-791 = CGP-28392. The calcium channel antagonists (+)-R-BAY K 8644 and (-)-R-202-791 were behaviorally inactive but blocked the behavioral effects of (-)-S-BAY K 8644. The binding of dihydropyridine calcium channel activator and antagonist enantiomers to mouse brain membranes was described by both one and two site models. (-)-S-BAY K 8644, (+/-)-BAY K 8644, (+)-S-202-791 and CGP-28392 produced contractions in partially depolarized (15 mM K+) strips of guinea pig ileal longitudinal smooth muscle which differed in the degree of maximum contraction obtained. (+)-R-BAY K 8644 and (-)-R-202-791 inhibited potassium-induced contractions (80 mM K+) in guinea pig ileal longitudinal smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary intake of sodium chloride in the rat influences [3H]nitrendipine binding to adrenal glomerulosa cell membranes but does not alter binding to vascular smooth muscle membranes.

Angiotensin II-stimulated secretion by adrenal glomerulosa cells and contraction by vascular smooth muscle (VSM) are dependent on calcium influx through membrane calcium channels. We have examined the hypothesis that the altered responsiveness of adrenal glomerulosa cells and VSM to angiotensin II during NaCl restriction may be associated with a change in membrane calcium channel number. To test this hypothesis, female rats were placed on a high or low NaCl diet. On the 14th day, membranes were prepared from the zona glomerulosa, aorta, mesenteric artery, and uterus. [3H]Nitrendipine binding was used to monitor calcium channel number. The [3H]nitrendipine binding capacity was observed to be higher in the zona glomerulosa during NaCl restriction than during high NaCl intake (83 +/- 18 vs. 49 +/- 9 fmol/mg protein, P less than 0.025, n = 6 paired experiments). The binding capacities of [3H]nitrendipine on the low and high NaCl diet were similar in the mesenteric artery (10 +/- 1 vs. 9 +/- 1 fmol/mg protein, n = 8), aorta (33 +/- 5 vs. 35 +/- 8 fmol/mg protein, n = 5), or uterus (87 +/- 15 vs. 85 +/- 16 fmol/mg protein, n = 4), respectively. The dissociation constants of [3H]nitrendipine binding did not differ on a low or high NaCl intake in the zona glomerulosa (0.84 +/- .12 vs. 0.79 +/- .10 nM), mesenteric artery (0.82 +/- .06 vs. 83 +/- .05 nM), aorta (0.90 +/- .11 vs. 0.92 +/- .12 nM), or uterus (0.55 +/- .12 vs. 0.56 +/- .10 nM), respectively. We conclude that the blunted response of VSM to angiotensin II during NaCl restriction is best explained by the previously reported lower number of angiotensin II receptors since calcium channel number does not change. In the adrenal glomerulosa cell, NaCl restriction is associated with a higher number of membrane calcium channels and angiotensin II receptors. The increase in calcium channel number may reflect the influence of an unknown factor(s) believed to be necessary for the full expression of the adrenal glomerulosa cell response to NaCl restriction.     

Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.     

Increase in human platelet alpha 2-adrenergic receptor affinity for agonist in unstable angina

Spontaneous increase in platelet activity and change in coronary vasomotor tone have been implicated in the pathogenesis of acute myocardial ischemia. To define the mechanism of platelet "hypersensitivity" in acute myocardial ischemia, we examined the status of platelet alpha 2-adrenergic receptors in patients hospitalized with severe unstable angina. With the use of the specific alpha 2-receptor antagonist 3H-yohimbine, we identified a 26% decrease in the receptor binding sites on platelet membranes from patients with unstable angina compared with controls (155 +/- 32 vs. 210 +/- 29 fmol/mg protein, P less than or equal to 0.005). The dissociation constants of 3H-yohimbine binding to platelet alpha 2-receptors were similar in both groups (3.3 +/- 1.1 and 4.1 +/- 1.6 nmol/L, P not significant). To study the alterations in the affinity of platelet alpha 2-receptors for the agonists, effects of 1-epinephrine on specific binding of 3H-yohimbine were examined. We observed a marked reduction in 1-epinephrine concentration for inhibition of antagonist binding by 50% in acute myocardial ischemia (IC50: 4.2 +/- 3.9 X 10(-8) vs. 6.7 +/- 3.4 X 10(-7) mol/L, P less than or equal to 0.01), indicating increase in platelet alpha 2-receptor affinity for the agonist. Platelet aggregation and thromboxane A2 generation in response to epinephrine were also significantly increased in the acute phase of myocardial ischemia. This study suggests enhanced affinity of platelet alpha 2-receptors to the agonist 1-epinephrine as a possible mechanism of platelet hypersensitivity in acute myocardial ischemia.     

Pharmacologic and radioligand binding analysis of the actions of 1,4-dihydropyridine activator-antagonist pairs in smooth muscle

The actions of the enantiomers of Bay K 8644 and 202-791 were studied in rat tail artery and guinea pig ileal longitudinal smooth muscle using pharmacologic and radioligand binding assays. (-)-(S)-Bay K 8644 (below 10(-7) M in rat tail artery and 3 X 10(-7) M in guinea pig ileum) and (+)-(S)-202-791 (below 10(-6) M) induced contractions and potentiated the responses to KCl depolarization in both smooth muscle preparations. In contrast, (+)-(R)-Bay K 8644 and (-)-(R)-202-791 inhibited the responses to KCl-induced depolarization. At higher concentrations, (-)-(S)-Bay K 8644 (10(-7) to 10(-6) M) and (+)-(S)-202-791 (10(-6) to 3 X 10(-5) M) in rat tail artery, and (-)-(S)-Bay K 8644 (3 X 10(-7) to 3 X 10(-6) M) in guinea pig ileal smooth muscle relaxed the tissues contracted maximally at lower concentrations of the same drug. Cross antagonism between 1,4-dihydropyridine activators was observed when (-)-(S)-Bay K 8644 (10(-7) to 10(-6) M) relaxed the maximum contraction in response to (+)-(S)-202-791. [3H]Nitrendipine bound in a tail arterial microsomal preparation to a single class of site, with KD of 3.63 X 10(-10) M and maximum binding of 552.7 fmol mg-1 protein. In both rat tail artery and guinea pig ileal smooth muscle, (-)-(S)-Bay K 8644, (+)-(R)-Bay K 8644, (+)-(S)-202-791 and (-)-(R)-202-791 inhibited specific [3H]nitrendipine binding competitively; the Kl values correlate well to the pharmacologic EC50 (for activators) or IC50 (for antagonists, measured against 80 mM KCl depolarization) values. The biphasic response to (-)-(S)-Bay K 8644 and (+)-(S)-202-791 suggests that the properties of Ca++ channel activation and antagonism may reside within a single 1,4-dihydropyridine molecule.     

Agonist-induced beta-adrenergic receptor internalization on intact human mononuclear leukocytes: effect of temperature of mononuclear leukocyte separation

The hydrophilic ligand 3H-CGP 12177 was used to measure beta-adrenergic receptors on intact human mononuclear leukocytes (MNLs). A single homogeneous class of receptor sites was found, with KD value of 0.71 +/- 0.04 nmol/L and Bmax of 3.0 +/- 0.4 fmol/10(6) cells (mean +/- SEM; n = 12). The receptor affinity (KD) and density (Bmax) were similar when measured on MNLs, purified lymphocytes, and a T-lymphocyte-enriched population from the same individual. Preincubation of intact MNLs with 1 mumol/L isoproterenol at 37 degrees C for 20 minutes reduced the number of surface receptors, measured by 3H-CGP 12177 binding at 4 degrees C for 20 hours, by approximately 70% (receptor internalization) without affecting KD. This effect was reversible, and surface receptors completely reappeared when binding was investigated at 37 degrees C for 40 minutes. Receptor internalization was similar when either isolated MNLs or whole blood was incubated with isoproterenol. Agonist-induced receptor internalization was stable during MNL isolation from whole blood at 4 degrees C but was partially or completely lost from MNLs prepared at 20 degrees C.