Differential effects of adrenergic agonists and phorbol esters on the alpha 1-adrenoceptors of hepatocytes and aorta

Epinephrine, norepinephrine and phenylephrine stimulate phosphatidylinositol labeling with [32P]Pi in both rat hepatocytes and rabbit aorta. Methoxamine was a full agonist for this effect in rabbit aorta whereas cirazoline and oxymetazoline were partial agonists. In contrast, these three agents (methoxamine, cirazoline and oxymetazoline) were unable to stimulate phosphatidylinositol labeling in rat hepatocytes. Furthermore, cirazoline and oxymetazoline were able to displace the dose-response curve to epinephrine in rat hepatocytes, i.e., they behaved as antagonists. Binding competition curves of these agents with labeled adrenergic ligands indicate that the affinity of alpha 1-adrenergic receptors in these two tissues (aorta and liver) for the different agents tested was very similar. In addition it was observed that phorbol myristate-acetate inhibited in a dose-dependent fashion the epinephrine-mediated stimulation of phosphatidylinositol labeling in hepatocytes but was without effect on the action of the amine in aorta. Our data suggest that stereochemical differences for alpha 1-adrenergic activation in liver and aorta may exist and indicate that the ability of phorbol esters to inhibit alpha 1-adrenergic effects is not universal.     

Differences in the role of Na+/K+-ATPase during alpha 1-adrenergic events in rat and rabbit aorta

Transport activity of the Na+/K+-ATPase was studied in rat and rabbit aorta under basal and agonist-stimulated conditions. Basal ouabain-sensitive 86Rb+ uptake was 2.9-fold higher in rat tissues as compared to rabbit tissues. This higher uptake in the rat was associated with a greater sensitivity to the Na+/H+ exchange inhibitor amiloride. Stimulation of alpha 1-adrenergic receptors by norepinephrine (NE) or phenylephrine (PE) resulted in an increase in ouabain-sensitive 86Rb+ uptake, whose temporal pattern differed between arteries of the two species. In rat aorta the increase was maximal during the first 2 min of agonist exposure reaching approximately a 50% higher rate of uptake than controls while rabbit aorta exhibited a steady rise in 86Rb+ uptake. Removal of extracellular Ca2+ by EGTA (1 mM) for 10 min resulted in an activation of Na+/K+-ATPase-related 86Rb+ uptake after which PE was still capable of causing a further increase, suggesting that Ca2+ influx is not responsible for the receptor-induced stimulation. Removal of extracellular Na+ reduced the PE-induced stimulation, while amiloride did not block the agonist effect. To characterize the role of the Na+/K+-ATPase during contractile events, receptor-induced 45Ca2+ uptake, 45Ca2+ release and contraction were compared in rat and rabbit aorta following Na+/K+-ATPase inhibition. Rat responses to PE were readily inhibited by ouabain or K+-free conditions, while rabbit responses were relatively resistant. 45Ca2+ extrusion and relaxation following alpha 1-adrenergic receptor stimulation were both highly dependent on activity of the Na+/K+-ATPase in rat aorta. These species' differences in Na+/K+-ATPase transport activity and its role in the regulation of contractility illustrate an example of heterogeneity in the ionic control of arterial tone.     

Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells

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.     

Phenoxybenzamine is more potent in inactivating alpha 1- than alpha 2-adrenergic receptor binding sites

The effects of phenoxybenzamine on alpha 1- and alpha 2-adrenergic receptor binding sites were examined directly using radioligand binding assays of the antagonist drugs [125I]BE 2254 and [3H]rauwolscine, respectively, in homogenates of rat cerebral cortex. Treatment with phenoxybenzamine caused an irreversible, dose-dependent decrease in the density of both alpha 1- and alpha 2-adrenergic receptors. Phenoxybenzamine was approximately 250-fold more potent at decreasing alpha 1-adrenergic receptor density than it was at decreasing alpha 2-adrenergic receptor density.     

Sex difference in adrenergic receptor-mediated glycogenolysis in rat livers

Catecholamine-induced stimulation of hepatic glycogenolysis in male and female rats was studied by detecting the cytosolic free Ca2+ concentration ([Ca2+]i), cAMP generation and adrenergic receptor function. Increase in alpha 1-adrenergic receptor-mediated [Ca2+]i and beta-adrenergic receptor-mediated cAMP generation were examined using isolated hepatocytes. No difference was found in the alpha 1-adrenergic receptor-mediated response of [Ca2+]i in fura-2-loaded hepatocytes between males and females, while epinephrine-induced cAMP accumulation in hepatocytes was about 3-fold higher in females. The alpha 1- and beta-adrenergic receptor properties of the plasma membrane were evaluated by ligand binding studies using [3H]prazosin (alpha 1-adrenergic antagonist) and [125I]iodocyanopindolol (beta-adrenergic antagonist); and little sex difference was found in either affinity or the number of binding sites of [3H]prazosin and [125I]iodocyanopindolol. Activation of adenylate cyclase by forskolin and GTP-gamma-S was also similar for both sexes. These results suggest that the sex difference of beta-adrenergic response is due to a difference in the guanine nucleotide regulatory binding proteins (G proteins) and/or beta-adrenergic receptor-Gs protein (the stimulatory G protein of adenylate cyclase) coupling ability.     

The relationships between receptor binding capacity for norepinephrine, angiotensin II, and vasopressin and release of inositol trisphosphate, Ca2+ mobilization, and phosphorylase activation in rat liver

Concentration-response relationships for norepinephrine-, angiotensin II-, and vasopressin-stimulated changes in cell Ca2+ content, phosphorylase activation, and cytosolic free Ca2+ and myo-inositol-P3 levels were examined in isolated hepatocytes. The specific binding of radioligands to alpha 1-adrenergic, vasopressin, and angiotensin II receptors was also examined in rat liver plasma membranes. Disparities occurred between the concentration-response curves for myo-inositol-P3 formation and the Ca2+ and phosphorylase responses, with the greatest difference being observed with vasopressin and the smallest with norepinephrine. It was also observed that all three agonists produced the same maximum changes in phosphorylase, cell Ca2+, and cytosolic Ca2+, but the maximum capacity of each agonist to generate myo-inositol-P3 varied greatly and was correlated with the maximum receptor binding capacity. The data indicated that a very small and submaximal elevation of myo-inositol-P3 was sufficient to maximally elevate cytosolic Ca2+ and activate phosphorylase. In addition, the relationship between the accumulation of myo-inositol-P3 and the elevation of cytosolic Ca2+ was similar, irrespective of whether the agonist was norepinephrine, angiotensin II, or vasopressin. It is proposed that the large differences between the concentration-response curves for myo-inositol-P3 formation and Ca2+ and phosphorylase changes observed with vasopressin and angiotensin II are due to the higher density of their receptors on liver cell plasma membranes compared with alpha 1-adrenergic receptors.     

The alpha 1C-adrenergic receptor: characterization of signal transduction pathways and mammalian tissue heterogeneity.

We recently reported the cloning of a novel alpha 1-adrenergic receptor (AR), the alpha 1CAR. By transient and stable expression of the alpha 1CAR and the previously cloned alpha 1BAR in COS-7 and HeLa cells, respectively, we have now compared their ability to interact with major signal-transduction pathways (including polyphosphoinositide hydrolysis, intracellular calcium, and cAMP metabolism), as well as their mammalian tissue localization. Both alpha 1C- and alpha 1BARs primarily couple to phospholipase C via a pertussis toxin-insensitive GTP-binding protein, leading to the release of calcium from intracellular stores. Even though alpha 1C- and alpha 1BARs activate polyphosphoinositide hydrolysis by similar biochemical mechanisms, the alpha 1CAR couples to phospholipase C more efficiently than does the alpha 1BAR; activation of the alpha 1CAR results in a 2-3-fold greater increase in inositol phosphates, compared with the alpha 1BAR. Both alpha 1AR subtypes can also increase intracellular cAMP, by a mechanism that does not involve direct activation of adenylyl cyclase. In agreement with ligand binding data, the agonist methoxamine and the antagonist WB4101 are 10-fold more potent in activating or inhibiting, respectively, the ability of the alpha 1CAR to stimulate phospholipase C, compared with the alpha 1BAR. In addition, methoxamine is almost a full agonist at the alpha 1CAR, whereas it can only weakly activate the alpha 1BAR. Tissue localization, using Northern blot analysis of total and poly(A)+-selected RNA from rabbit tissues, revealed striking mammalian species heterogeneity. As previously described, the alpha 1BAR is present in several rat tissues, including heart, liver, brain, kidney, lung, and spleen, whereas the alpha 1CAR is not present in any rat tissue studied. The alpha 1BAR is also present in rabbit aorta, heart, spleen, and kidney (and absent in rabbit liver), whereas the alpha 1CAR is present in rabbit liver. Our results indicate that the cloning and expression of different alpha 1AR subtypes represents a valuable tool to elucidate functional correlates of alpha 1AR heterogeneity.     

1-(2,6-二甲氧基)-2-(3,4-二甲基苯乙氨基)丙烷盐酸盐(DDPH)拮抗α1肾上腺素受体的特性

目的　研究DDPH对α₁-肾上腺素受体(α₁-AR)及其亚型的拮抗作用。方法　放射配体结合实验和离体血管收缩功能实验。结果　DDPH对¹²⁵I-BE2254与大鼠脑皮质和脾脏α₁-AR结合呈竞争性拮抗作用。pK_I值在两者间无显著性差别, Hill系数均接近于1.0。在分别稳定表达α_1A,α_1B或α_1D-AR的克隆HEK293细胞中,其拮抗的pK_I值α_1A和α_1D比α_1B-AR高约2倍,Hill系数均接近于1.0。并拮抗去甲肾上腺素(NE)介导大鼠主动脉,肾动脉和脾脏收缩的pA₂值,在三者间无显著差别,斜率接近1.0。结论　DDPH对α₁-AR有竞争性拮抗作用,但其作用对α₁-AR亚型无选择性。     

Interaction of subtype-selective antagonists with alpha 1-adrenergic receptor binding sites in rat tissues.

(+)-Niguldipine inhibited specific 125I-BE 2254 binding more potently in membrane preparations from rat tissues enriched in the alpha 1A subtype (hippocampus and vas deferens) than those with the alpha 1B subtype (liver and spleen). Inhibition curves for (+)-niguldipine were better fit by a two-site model in most tissues, although Kl values for each site varied markedly between tissues. The potency of this lipophilic drug was highly dependent on tissue concentration, probably accounting for most of this variability. Pretreatment of membranes with chloroethylclonidine (CEC) to inactivate the alpha 1B subtype did not completely eliminate the low affinity sites for (+)-niguldipine, particularly in heart. Saturation analysis showed that (+)-niguldipine competitively inhibited both alpha 1A and alpha 1B subtypes. However, substantial non-competitive inhibition was also observed in several tissues. Analysis of inhibition curves for 5-methylurapidil gave similar proportions of alpha 1A and alpha 1B receptor sites as were calculated for (+)-niguldipine in various tissues. Although (+)-niguldipine and 5-methylurapidil revealed variable proportions of low affinity sites in CEC-pretreated hippocampus and heart, this was not observed with inhibition curves for WB 4101 and phentolamine. These results are generally consistent with the previously defined alpha 1A and alpha 1B subtypes. 5-Methylurapidil currently appears to be the best antagonist for discriminating these subtypes; (+)-niguldipine shows similar selectivity but is complicated by a high lipophilicity. However, the persistence of low affinity sites for 5-methylurapidil and (+)-niguldipine after CEC pretreatment and the noncompetitive effects of (+)-niguldipine in some tissues raise the possibility of an additional subtype(s) of alpha 1-adrenergic receptors in rat tissues.     

"Spare" alpha 1-adrenergic receptors and the potency of agonists in rat vas deferens

The existence of "spare" alpha 1-adrenergic receptors in rat vas deferens was examined directly using radioligand binding assays and contractility measurements. Alpha 1-adrenergic receptors in homogenates of rat vas deferens were labeled with [125I]BE 2254 (125IBE). Norepinephrine and other full alpha 1-adrenergic receptor agonists were much less potent in inhibiting 125IBE binding than in contracting the vas deferens in vitro. Treatment with 300 nM phenoxybenzamine for 10 min to irreversibly inactivate alpha 1-adrenergic receptors caused a large decrease in the potency of full agonists in causing contraction of this tissue and a 23-48% decrease in the maximal contraction observed. Using those data, equilibrium constants for activation (Kact values) of the receptors by agonists were calculated. These Kact values agreed well with the equilibrium binding constants (KD values) determined from displacement of 125IBE binding. The reduction in alpha 1-adrenergic receptor density following phenoxybenzamine treatment was determined by Scatchard analysis of specific 125IBE binding sites and compared with the expected reduction (q values) calculated from the agonist dose-response curves before and after phenoxybenzamine treatment. Exposure to 300 nM phenoxybenzamine for 10 min resulted in a 39% decrease in specific 125IBE binding sites, which did not agree with the 93% decrease expected from the calculated q values. Treatment of vas deferens with a dose of phenoxybenzamine (10 microM for 15 min) that completely abolished the contractile response to alpha 1-adrenergic agonists caused an 82% decrease in the density of 125IBE binding sites. Tissues exposed to 300 nM phenoxybenzamine in the presence of 100 microM phentolamine or 3 microM prazosin showed no change in the dose-response curves for agonist-induced contraction or in the density of 125IBE binding sites when compared with controls. This suggests that phenoxybenzamine functionally inactivates alpha 1-adrenergic receptors at or near the receptor binding site. These experiments suggest that the potencies of agonists in activating alpha 1-adrenergic receptors in rat vas deferens agree well with their potencies in binding to the receptors. The greater potency of agonists in causing contraction may be due to spare receptors in this tissue. The data also demonstrate that phenoxybenzamine irreversibly inactivates alpha 1-adrenergic receptors in rat vas deferens, but that the decrease in receptor density is much smaller than that predicted from receptor theory.     

Occupancy of alpha 1-adrenergic receptors and contraction of rat vas deferens

The interaction of agonists and antagonists with alpha 1-adrenergic receptors in rat vas deferens was examined using radioligand binding assays and contractility measurements. 125I-Labeled BE 2254 (125IBE) was found to bind rapidly and reversibly to a single class of high-affinity binding sites in homogenates of rat vas deferens. The k1 for association was 3.8 X 10(7) 1/mole-sec, the k-1 for dissociation was 2.3 X 10(-3) sec-1, and the KD was 105 pM. The order of potency for antagonists inhibiting 125IBE binding was prazosin greater than indoramin greater than phentolamine greater than yohimbine. Norepinephrine, phenylephrine, and other alpha-adrenergic agonists produced dose-dependent contractions of whole vas deferens in vitro. This contractile response was competitively inhibited by alpha-adrenergic blocking drugs with the same potency order observed for inhibition of specific 125IBE binding. Comparison of pA2 values for alpha 1- and alpha 2-selective antagonists competitively inhibiting contractile responses to norepinephrine, epinephrine, or phenylephrine suggested that these drugs caused their contractile effects solely through alpha 1-adrenergic receptors, and that there were no alpha 2-adrenergic receptors mediating contraction in this tissue. The pA2 values for antagonist inhibition of alpha-adrenergic receptor-mediated contractile responses were highly correlated (r = 0.995) with the KD values for antagonist inhibition of 125IBE binding in this tissue. The EC50 values for partial agonists were also highly correlated with the KD values for inhibition of 125IBE binding in vas deferens. However, the EC50 values of full agonists in causing contraction were in general 10- to 100-fold lower than the KD values for inhibiting 125IBE binding, possibly representing a substantial "spare receptor" population in this tissue. The results suggest that rat vas deferens contains a homogeneous population of alpha 1-adrenergic receptors mediating the contractile response to norepinephrine, that these receptors can be directly labeled with 125IBE, and that there may be a nonlinear relationship between agonist occupancy of alpha 1-adrenergic receptors and the functional response of this tissue.     

Two pharmacologically distinct alpha 1-adrenoceptor subtypes in the contraction of rabbit aorta: each subtype couples with a different Ca2+ signalling mechanism and plays a different physiological role

Using the alpha 1-adrenoceptor subtype-selective antagonists chlorethylclonidine (CEC), WB4101, and 5-methyl-urapidil, we have examined the possible heterogeneity in the alpha 1-adrenoceptor populations in rabbit aorta. The alpha 1-adrenoceptor alkylating agent CEC selectively inhibited the phasic component of the norepinephrine-induced contractile response, with little effect on the tonic component. The alpha 1-adrenoceptor occupancy-response relationship defined by the phenoxybenzamine inactivation method was rectangular hyperbolic for the tonic response, whereas that for the phasic response was linear, indicating the different degree of receptor reserve for the two responses. Radioligand binding studies with the nonselective alpha 1-adrenoceptor antagonist radioligand 125I-BE2254 showed that 73-87% of the binding sites in rabbit aorta are CEC sensitive and they are predominantly low affinity sites both for WB4101 (pKd = 8.1) and for 5-methylurapidil (pKd = 7.1). Moreover, alpha 1-adrenoceptor-mediated phosphatidylinositol (PI) hydrolysis was CEC sensitive, and fractional inactivation of alpha 1 receptors with CEC showed equivalent increments in the reduction of PI hydrolysis and phasic contractile response, suggesting that both responses are linearly related to the CEC-sensitive receptor sites. The Schild plots for the competitive antagonists WB4101 and 5-methyl-urapidil against alpha 1a-adrenoceptor-selective agonist methoxamine-induced contraction were linear and had slopes not significantly different from unity, with a pA2 of 9.07 +/- 0.07 (n = 5) for WB4101 and 9.09 +/- 0.05 (n = 3) for 5-methyl-urapidil. However, the Schilod plots for these antagonists against norepinephrine were curvilinear. Computer-assisted analysis of these curvilinear Schild plots in a two-receptor system indicated that alpha 1-adrenoceptor populations responsible for the constrictive response are predominantly (approximately 80-90%) low affinity sites for the two antagonists (pKd approximately 8.1 for WB4101 and pKd approximately 7.1 for 5-methyl-urapidil) and a small population (approximately 10-20%) are high affinity sites (pKd approximately 9.1 for both WB4101 and 5-methyl-urapidil), which was in good agreement with radioligand binding studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

Trifluoperazine and chlorpromazine antagonize alpha 1- but not alpha2- adrenergic effects

Trifluoperazine and chlorpromazine inhibited in a dose-dependent manner the stimulation of glycogenolysis, gluconeogenesis, and ureogenesis due to alpha 1-adrenergic stimulation in rat hepatocytes. In contrast, the antipsychotic agents were unable to block the inhibition of adenylate cyclase due to alpha 2-adrenergic activation in hamster adipocytes. Binding experiments showed that trifluoperazine and chlorpromazine at low concentrations displaced tritiated dihydroergocryptine binding from rat liver membranes (alpha 1-adrenergic sites), whereas very large concentrations of the phenothiazine derivatives were required to displace dihydroergocryptine from hamster adipocyte membranes (alpha 2-adrenergic sites). It is concluded that chlorpromazine and trifluoperazine are much more potent at alpha 1- than at alpha 2-adrenergic receptors. The use of rat hepatocytes and hamster adipocytes to study the alpha-adrenergic subtype selectivity of drugs is proposed.     

Melatonin potentiates phenylephrine-stimulated intracellular Ca2+ transient in smooth muscle cell of large arteries of chick embryo

In the current study we first characterized the properties of the 2-[125I]iodomelatonin binding in pulmonary artery and aorta of chick embryo, and then determined the location of the binding site with autoradiography. Receptor binding assays were used using 2-[125I]iodomelatonin as ligand. The binding was stable, saturable, specific and reversible. Scatchard anlaysis revealed an equilibrium binding constant (Kd) of 27.12 +/- 1.34 pM and a maximum binding capacity (Bmax) of 1.93 +/- 0.19 fmol/mg protein (n = 5). Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased the Kd, but reduced the Bmax, indicating the binding being coupled to a G-protein. Autoradiography revealed the binding sites in the smooth muscle layer. To delineate the physiological function of melatonin in the large artery of the chick embryo, we determined the intracellular calcium ([Ca2+]i) in smooth muscle cells of the aorta with spectrofluorometry, using fura 2-AM as calcium indicator. Melatonin at 10(-8) to 10(-5), which itself had no effect, potentiated the stimulating effect of 0.1 microM phenylephrine, a selective agonist of alpha -adrenoceptor, on [Ca2+]i. In conclusion, specific binding of 2-[125I]iodomelatonin and physiological response to melatonin suggest the existence of melatonin receptor in the smooth muscle of large arteries of the chick embryo. Melatonin potentiates the effects of alpha1-adrenergic stimulation.     

RX781094 a potent and selective alpha 2-adrenergic antagonist. Effects in adipocytes and hepatocytes

The potency and selectivity of RX781094, 2-(2-(1,4-benzodioxanyl]-2-imidazoline HCl, as alpha 1- and alpha 2-adrenergic antagonist was studied using rat hepatocytes and hamster adipocytes. The alpha 1-adrenergic stimulation of ureogenesis produced by epinephrine in rat hepatocytes was slightly diminished by 10(-4) M RX781094. On the contrary the alpha 2-adrenergic effect of epinephrine in hamster adipocytes (inhibition of adenylate cyclase) was antagonized dose-dependently by RX781094. This agent was approximately 10-fold more potent than yohimbine. Radioligand binding studies also showed that RX781094 was more potent and selective than yohimbine at alpha 2-adrenergic sites. It is concluded that RX781094 is a potent and selective alpha 2-adrenergic antagonist.     

Desensitization of alpha 1 adrenoceptor-stimulated glycogen phosphorylase activity in vascular smooth muscle

Desensitization of alpha 1-adrenoceptor-mediated activation of glycogen phosphorylase was investigated in rabbit aorta. Activation of glycogen phosphorylase by epinephrine was antagonized by the alpha 1-receptor selective antagonist prazosin but not by yohimbine (alpha 2-receptor selective) or by propranolol (beta-receptor antagonist). Preincubation of rabbit aortic ring segments for 5 h with norepinephrine (NE, 10(-5) M) led to a 30-fold loss in sensitivity and a 55% decrease in maximal activation of the enzyme by alpha agonists. Preincubation of aortic ring segments with phenylephrine (10(-5) M) in the presence of propranolol (10(-6) M) also caused desensitization of glycogen phosphorylase activation. The desensitization was heterologous since maximal activation of the enzyme by histamine or KCl was also markedly diminished in segments preincubated with NE. In contrast to these results, catecholamine-induced desensitization to alpha 1-adrenoceptor-mediated smooth muscle contraction in aortic ring segments resulted in loss in sensitivity but not maximal force of contraction on subsequent stimulation by alpha 1 agonists. These results suggest that the mechanism responsible for desensitization of glycogen phosphorylase is distal to receptor activation and may involve attenuation of responses to intracellular Ca2+-dependent enzymes which have limited reserve.     

Expression of both alpha 1- and alpha 2-adrenoceptors in an insulin-secreting cell line. Parallel studies of cytosolic free Ca2+ and insulin release

Changes in intracellular free Ca2+, [Ca2+]i, and immunoreactive insulin release in response to alpha-adrenergic agents were measured in RINm5F cell suspensions. Cells were loaded with the fluorescent indicator quin 2 for monitoring [Ca2+]i. Epinephrine (1 microM), which inhibited alanine-stimulated insulin release by 73%, evoked a transient rise in [Ca2+]i. This rise is in part due to Ca2+ mobilization, since it is still present in the absence of extracellular Ca2+. The alpha 2-adrenergic agonist clonidine (10 microM) mimicked the epinephrine effect on insulin release without any change in [Ca2+]i. In contrast, the alpha 1-adrenergic agonist phenylephrine (10 and 100 microM) raised [Ca2+]i, albeit to a lesser extent than epinephrine. Phenylephrine enhanced basal, but had no effect on alanine-stimulated insulin release. To examine further the nature of the effect of epinephrine, specific alpha-adrenergic blocking agents were employed. The epinephrine-induced increase in [Ca2+]i could be inhibited by the alpha 1-adrenergic antagonists BE2254 (0.1 microM) and prazosin (0.01 microM). In the presence of these blockers, epinephrine was still able to inhibit insulin release. When alpha 2-adrenergic receptors were blocked by the addition of idazoxan (0.1 and 1 microM), epinephrine still raised [Ca2+]i. At the higher concentration of idazoxan, the epinephrine inhibition of insulin release was completely overcome. The alpha-adrenergic agonists did not attenuate the alanine-induced rise in [Ca2+]i. This study shows that both subtypes of alpha-adrenergic receptors are present in the insulin-secreting cell line RINm5F. The activation of alpha 1-adrenergic receptors leads to an increase in [Ca2+]i. In contrast, the inhibition of insulin release due to epinephrine is mediated through alpha 2-adrenergic receptors. The alpha 2-adrenergic mechanism does not involve changes in [Ca2+]i, but is rather exerted at a later step in the secretory process.     

Comparison of alpha 1-adrenergic receptor subtypes distinguished by chlorethylclonidine and WB 4101

We showed previously that subtypes of alpha 1-adrenergic receptors can be differentiated by selective inactivation with chlorethylclonidine (CEC) [Mol. Pharmacol. 32:505-510 (1987)] or by their affinities for the competitive antagonist WB 4101 [Nature (Lond.) 329:333-335 (1987)]. Examining eight rat tissues, the proportions of 125IBE 2254-binding sites sensitive to inactivation by CEC correlated significantly (p less than 0.05) with the proportion having a low affinity for WB 4101. However, the proportion of CEC-sensitive sites was always smaller than the proportion of low affinity WB 4101 sites. Further experiments showed that repetitive pretreatment with CEC or pretreatment under hypotonic conditions caused a larger inactivation of binding sites, suggesting that CEC did not access all sites under the isotonic conditions used previously. The proportions of binding sites inactivated by 10 microM CEC under hypotonic conditions were quantitatively similar to and correlated significantly (p less than 0.01) with the proportion having a low affinity for WB 4101. Pretreatment of hippocampus and vas deferens with CEC caused a loss of all low affinity WB 4101-binding sites, leaving only high affinity sites. In vas deferens, CEC pretreatment decreased the potency of norepinephrine in stimulating 3H-inositol phosphate accumulation but not contractile responses. In rat liver slices, CEC inactivated norepinephrine-stimulated 3H-inositol phosphate accumulation in parallel with 125IBE-binding sites. These results suggest that: 1) the CEC-sensitive and -insensitive 125IBE 2254-binding sites are equivalent to those with a low and high affinity for WB 4101, respectively, and 2) the CEC-sensitive binding sites with a low affinity for WB 4101 are the alpha 1-adrenergic receptors linked to inositol phospholipid hydrolysis.     

Receptor subtype involved in α1-adrenergic receptor-mediated Ca^2＋ signaling in cardiomyocytes

Aim： The enhancement of intracellular Ca^＋signaling in response to α1-adrenergic receptor （α1-AR） stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role（s） of the α1-AR subtype（s） in mediating this response. Methods： We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca^2＋ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results： After being cultured for 48 h, the myocytes exhibited spontaneous local Ca^2＋ release, sparks, and global Ca^2＋ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca^2＋ transients with an EC5o value of 2.3 larnol/L. Blocking the α1A-AR subtype with 5-methylurapidil （5-Mu） inhi- bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca^2＋ spark numbers were also increased by the activation of the α1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency （EC50 value =6.9 nmol/L） in the potentiation of Ca^2＋ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion： These results indicate that α1-adrenergic stimulation of intracellular Ca^2＋ activity is mediated selectively by the α1-AR.     

Synthesis and characterization of a high affinity radioiodinated probe for the alpha 2-adrenergic receptor

The availability of radioiodinated probes has facilitated the localization and molecular characterization of cell membrane receptors for hormones and neurotransmitters. However, such probes are not available for the study of the alpha 2-adrenergic receptor. This report describes the synthesis and characterization of functionalized derivatives of the selective alpha 2-adrenergic antagonists, rauwolscine and yohimbine, which can be radiolabeled to high specific activity with 125I. Following demethylation of rauwolscine or yohimbine, the resultant carboxylic acid derivatives were reacted with 4-aminophenethylamine to yield the respective 4-aminophenethyl carboxamides, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-4-amino-phenethyl]carboxamide (rau-pAPC) and 17 alpha-hydroxy-20 beta-yohimban-16 alpha-[N-4-aminophenethyl]carboxamide. In competitive inhibition studies using rat renal membranes and the radioligand [3H]rauwolscine, rau-pAPC (Ki = 11 +/- 1 nM) exhibited a 14-fold greater affinity than the corresponding yohimbine derivative (Ki = 136 +/- 45 nM). The higher affinity compound, rau-pAPC, was radioiodinated by the chloramine T method, and the product, 125I-rau-pAPC [17 alpha-hydroxy-20 alpha-yohimban-16 beta-(N-4-amino-3 -[125I]iodophenethyl)carboxamide], was purified by reverse phase HPLC to high specific activity (2175 Ci/mmol) and its binding characteristics were investigated in rat kidney membranes. Specific binding of 125I-rau-pAPC was saturable and of high affinity as determined by Scatchard analysis (KD = 1.8 +/- 0.3 nM) or from kinetic studies (KD = k2/k1 = 0.056 +/- 0.013 min-1)/4.3 +/- 0.2 X 10(7) M-1 min-1 = 1.3 +/- 0.3 nM). In competition studies, alpha-adrenergic antagonists and agonists inhibited the binding of 125I-rau-pAPC with a potency order consistent with an interaction at alpha 2-adrenergic receptors (rauwolscine greater than phentolamine greater than prazosin; clonidine greater than (-)-epinephrine greater than (-)-norepinephrine greater than dopamine greater than (+)-epinephrine). In rat liver and human platelet membranes, high affinity binding of 125I-rau-pAPC was also observed (liver, KD = 1.2 +/- 0.4 nM; platelet, KD = 3.2 +/- 1.5 nM). In addition, the density of alpha 2-adrenergic receptors identified from binding studies with 125I-rau-pAPC in kidney, liver, and platelet membranes was similar to that observed in parallel studies with [3H]rauwolscine. These findings indicate that 125I-rau-pAPC is a high affinity probe that selectively identifies alpha 2-adrenergic binding sites. Availability of this radioligand should facilitate the localization and biochemical characterization of this alpha-adrenergic receptor subtype.