Differential localization of alpha 2-adrenergic receptor subtypes in brain.

The pharmacological identification and characterization of subtypes of alpha 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where alpha 2 agonist binding ([3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the alpha 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of alpha 2 receptors have overlooked some regions of binding due to the subtype selectivity of this ligand. A more complete localization of alpha 2-adrenergic receptors can be obtained using the tritiated antagonist rauwolscine, and the differential distribution of at least two subtypes of the alpha 2 receptor can be obtained by selective inhibition of this binding.     

Identification and structural characterization of alpha 1-adrenergic receptor subtypes

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.     

Functional α1-adrenergic receptor subtypes in human right gastroepiploic artery

AIM: To study the functional alpha1-adrenergic receptor (alpha1-AR) subtypes in human right gastroepiploic artery (RGA). METHODS: The effects of alpha2-AR, alpha1-AR, and alpha1-AR subtype selective antagonists on norepinephrine (NE)-induced vasoconstriction in isolated human RGA were observed by contractile function experiment. RESULTS: Cumulative concentration-response curves for NE were competitively antagonized in RGA by alpha2-AR selective antagonist yohimbine (pA2 6.82+/-0.28, slope 1.12+/-0.40),alpha1-AR selective antagonist prazosin (pA2 9.77+/-0.22, slope 0.90+/-0.22),alpha1A-AR selective antagonists RS17053 (pA2 8.42+/-0.20, slope 0.93+/-0.20) and 5-MU (pA2 8.42+/-0.22, slope 0.88+/-0.18),alpha1D-AR selective antagonist BMY7378 (pA2 6.84+/-0.32, slope 1.05+/-0.17), and alpha1A-,alpha1B-AR selective antagonist WB4101 (pA2 8.88+/-0.20, slope 1.15+/-0.16). The correlation coefficients between these pA2 values of alpha1-AR selective antagonists with pKi values of which obtained from alpha1A-, alpha1B- and alpha1D-AR cloned cells are 0.95, 0.82, and 0.42. After the vessels were pretreated by chlorethylclonidine (CEC), an alpha1B- and alpha1D-AR irreversible alkylating agent, the pD2 values were changed from 5.9+/-0.5 to 5.6+/-0.6 and the maximal contraction was changed from (8.9+/-3.2) g to (8.0+/-3.2) g, respectively. The difference was not significant. CONCLUSION: In human RGA, the contraction response is mainly mediated by alpha1-AR, of which alpha1A-AR plays an important role, whereas alpha1B- and alpha1D-AR are not involved in the contraction response.     

Expression of renal alpha 1-adrenergic receptor subtypes in established hypertension.

Radioligand binding studies were undertaken in renal membranes of normotensive and hypertensive rats in order to test the hypothesis that there are alterations in renal alpha 1-adrenergic subtypes of genetic hypertensive animals. The highly selective competitive compound, (+)-niguldipine, was used to distinguish high-affinity (alpha 1a) from low-affinity (alpha 1b) sites, after initial studies demonstrated that this compound had greater selectivity than 5-methylurapidil in distinguishing alpha 1a and alpha 1b sites in rat renal membranes. In contrast to the significant difference in the blood pressure of the spontaneously hypertensive rats (delta BP = 74 mm Hg), there was no difference in the renal alpha 1-adrenergic receptor density. Membranes from the whole kidneys of spontaneously hypertensive rats (SHRs) possessed 31% alpha 1a and 69% alpha 1b sites with -log(Ki) values of 10.0 +/- 0.3 and 7.1 +/- 0.1, respectively, for (+)-niguldipine. However, these values were not different from those obtained from renal membranes of the normotensive Wistar-Kyoto (WKY) rats. These results indicate that in spite of the elevated blood pressure during the established phase of hypertension, the number, the affinity, and the ratio of the alpha 1a and alpha 1b appear not to be responsible for the manifestation of hypertension during this phase.     

Interaction of neuronal nitric oxide synthase with alpha1-adrenergic receptor subtypes in transfected HEK-293 cells

Background

The effects of lindane, a gamma-isomer of hexachlorocyclohexane, were studied on transmembrane potentials and currents of frog atrial heart muscle using intracellular microelectrodes and the whole cell voltage-clamp technique.

Results

Lindane (0.34 microM to 6.8 microM) dose-dependently shortened the action potential duration (APD). Under voltage-clamp conditions, lindane (1.7 microM) increased the amplitude of the outward current (I_out) which developed in Ringer solution containing TTX (0.6 microM), Cd²⁺ (1 mM) and TEA (10 mM). The lindane-increased I_out was not sensitive to Sr²⁺ (5 mM). It was blocked by subsequent addition of quinidine (0.5 mM) or E-4031 (1 microM). E-4031 lengthened the APD; it prevented or blocked the lindane-induced APD shortening.

Conclusions

In conclusion, our data revealed that lindane increased the quinidine and E-4031-sensitive rapid delayed outward K⁺ current which contributed to the AP repolarization in frog atrial muscle.     

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.     

Expression of three alpha 2-adrenergic receptor subtypes in rat tissues: implications for alpha 2 receptor classification

Based on biochemical and ligand binding studies in various tissues and species, evidence for several alpha 2-adrenergic receptor subtypes has accumulated. The current alpha 2-adrenergic receptor classification (alpha 2A, alpha 2B, alpha 2C) is based exclusively on pharmacological criteria. The molecular cloning of three distinct genes for human alpha 2-adrenergic receptors has confirmed the existence of multiple alpha 2-adrenergic receptor subtypes. According to their localization on different human chromosomes, the receptor genes were termed alpha 2-C10, alpha 2-C4, and alpha 2-C2. The relationship, however, between the pharmacologically characterized alpha 2-adrenergic receptors and the isolated genes has yet to be clarified. Using Northern blot hybridization, we analyzed the expression of the three cloned alpha 2-adrenergic receptor genes in 13 rat tissues, as well as in cell lines previously described as model systems for the pharmacologically defined alpha 2-adrenergic receptor subtypes. The alpha 2-C10 receptor corresponds to the alpha 2A subtype and is expressed in rat brainstem, cerebral cortex, hippocampus, pituitary gland, cerebellum, kidney, aorta, skeletal muscle, spleen, and lung. Messenger RNA coding for the alpha 2-C4 receptor was detected only in brain regions, not in peripheral tissues, whereas the alpha 2-C2 message was found only in liver and kidney. Hybridization experiments with RNA derived from tissues and cells from which the pharmacological alpha 2-receptor classification has been developed lead to the conclusion that the alpha 2B subtype represents two distinct receptor molecules, the alpha 2-C4 and a subtype previously undetected by classical ligand binding approaches. Furthermore, our results suggest that the alpha 2C subtype characterized in opossum kidney cells is an interspecies variation of alpha 2-C4 rather than a separate subtype. Finally, the cloned alpha 2-C2 receptor was found to be "alpha 2B-like" and not covered by the current pharmacological classification.     

Recent progress in α1-adrenergic receptor research

Alpha1-Adrenergic receptors (AR) play an important role in the regulation of physiological responses mediated by norepinephrine and epinephrine, particularly in the cardiovascular system. The three cloned alpha1-AR subtypes (alpha1A, alpha1B, and alpha1D) are G protein-coupled receptors that signal through the Gq/11 signaling pathway, each showing distinct pharmacological properties and tissue distributions. However, due to the lack of highly subtype-selective drugs, the functional roles of individual subtypes are still not clear. Development of new subtype-specific drugs will greatly facilitate the identification of the functions of each subtype. Conopeptide rho-TIA has been found to be a new alpha1B-AR selective antagonist with different modes of inhibition at alpha1-AR subtypes. In addition, recent studies using genetically engineered mice have shed some light on alpha1-AR functions in vivo, especially in the cardiovascular system and brain. Several proteins have been shown to interact directly with particular alpha1-AR, and may be important in regulating receptor function. Receptor heterodimerization has been shown to be important for cell surface expression, signaling and internalization. These new observations are likely to help elucidate the functional roles of individual alpha1-AR subtypes.     

Differential regulation on human skin fibroblast by alpha1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (alpha1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for alpha1 adrenergic [3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of alpha1-AR. The alpha1-AR agonists (from 1x10(-10) to 1x10(-4) M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1x10(-9) M and maximal inhibition at 1x10(-6) M. Prazosin, abolished the stimulatory (pA2: 9.24) and inhibitory (pA2: 8.80) actions of alpha1-AR agonists. The alpha1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (alpha1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of alpha1D-AR coupled to Gi protein. Non-functional alpha1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of alpha1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.     

Two alpha1-adrenergic receptor subtypes regulating the vasopressor response have differential roles in blood pressure regulation

To study the functional role of individual alpha1-adrenergic (AR) subtypes in blood pressure (BP) regulation, we used mice lacking the alpha1B-AR and/or alpha1D-AR with the same genetic background and further studied their hemodynamic and vasoconstrictive responses. Both the alpha1D-AR knockout and alpha1B-/alpha1D-AR double knockout mice, but not the alpha1B-AR knockout mice, had significantly (p < 0.05) lower levels of basal systolic and mean arterial BP than wild-type mice in nonanesthetized condition, and they showed no significant change in heart rate or in cardiac function, as assessed by echocardiogram. All mutants showed a significantly (p < 0.05) reduced catecholamine-induced pressor and vasoconstriction responses. It is noteworthy that the infusion of norepinephrine did not elicit any pressor response at all in alpha1B-/alpha1D-AR double knockout mice. In an attempt to further examine alpha1-AR subtype, which is involved in the genesis or maintenance of hypertension, BP after salt loading was monitored by tail-cuff readings and confirmed at the endpoint by direct intra-arterial recording. After salt loading, alpha1B-AR knockout mice developed a comparable level of hypertension to wild-type mice, whereas mice lacking alpha1D-AR had significantly (p < 0.05) attenuated BP and lower levels of circulating catecholamines. Our data indicated that alpha1B- and alpha1D-AR subtypes participate cooperatively in BP regulation; however, the deletion of the functional alpha1D-AR, not alpha1B-AR, leads to an antihypertensive effect. The study shows differential contributions of alpha1B- and alpha1D-ARs in BP regulation.     

一氧化氮及其衍生物导致血管平滑肌细胞α1-肾上腺素受体基因表达下调

目的观察炎症反应时血管平滑肌细胞α1-肾上腺素受体基因转录水平的改变。方法原代培养大鼠胸主动脉血管平滑肌细胞（VSMC）分为对照组（含10％FBS的DMEM培养液）、酪氨酸组（Tyr，250μmol／L）、3-硝基酪氨酸组（3-NT，250μmol／L）、硝普钠组（SNIP，500μmol／L）、炎性细胞因子组（Cyto，TNF-α100ng／ml＋IL1β50ng／ml）、炎性细胞因子组＋Nω-nitro-L-arginine methylester（Cyto＋L-NAME，TNF-α100ng／ml＋IL-1β50ng／ml＋L-NAME5mmol／L）和N-乙酰-5-甲氧基色胺＋炎性细胞因子组（Cyto＋MLT，TNF-α100ng／ml＋IL-1β50ng／ml＋MLT1 mmool／L），每组重复6次。上述各组细胞干预12h后，提取各组细胞的总RNA，逆转录聚合酶链反应（RT-PCR）法测定α1A-AR、α-1B-AR、a1D-ARmRNA的表达。结果与对照组相比，SNIP组和Cyto组血管平滑肌细胞细胞α1A-AR、α1D-A mRNA的表达显著下降（P〈0．05），Cyto＋L-NAME组、Cyto＋MLT组上述各受体亚型mRNA的表达比Cyto组显著升高（P〈0．01）。与对照组相比，SNIP组、3-NT组3-AR、α1B-D—AR,mRNA表达明显降低（P〈0．05）。结论NO及NO衍生物3-NT均显著抑制了血管平滑肌细胞细胞α1-肾上腺素受体mRNA的表达，可能为感染性休克时血管低反应性的病理机制之一。抑制NO的产生和抗氧化剂治疗可减轻这种病理反应。     

Role of the postsynaptic alpha(2)-adrenergic receptor subtypes in catecholamine-induced vasoconstriction

Catecholamines induce direct vasoconstriction mediated by postsynaptic alpha-adrenergic receptors (alpha-ARs) of both the alpha(1) and alpha(2) type. To evaluate the contribution of each alpha(2)-AR subtype (alpha(2A), alpha(2B), and alpha(2C)) to this function, we used groups of genetically engineered mice deficient for the gene to each one of these subtypes and compared their blood pressure (BP) responses to their wild-type counterparts. Blood pressure responses to a bolus of norepinephrine (NE) were assessed before and after sequential blockade of alpha(1)-ARs with prazosin and alpha(2)-ARs with yohimbine. The first NE bolus elicited a brief 32 to 44 mm Hg BP rise (p < 0.001 from baseline) in all six groups. Prazosin decreased BP by 23 to 33 mm Hg in all groups, establishing a new lower baseline. Repeat NE at that point elicited lesser but still significant (p < 0.001) brief pressor responses between 32% and 45% of the previous BP rise in five of the six groups. Only the alpha(2A)-AR gene knockouts differed, responding instead with a 20-mm Hg fall in BP, a significant change from baseline (p < 0.001) and different from the pressor response of their wild-type counterparts (p < 0.001). The addition of yohimbine produced no further BP change in the five groups, but it did produce a small 7. 5-mm Hg fall (p < 0.05) in the alpha(2A)-AR knockouts. Norepinephrine bolus during concurrent alpha(1) and alpha(2)-AR blockade produced significant (p < 0.001) hypotensive responses in all subgroups, presumably attributable to unopposed stimulation of beta(2)-vascular wall ARs. We conclude that the alpha(2)-AR-mediated vasoconstriction induced by catecholamines is attributable to the alpha(2A)-AR subtype because mice deficient in any one of the other subtypes retained the capacity for normal vasoconstrictive responses. However, the alpha(1)-ARs account for the major part (as much as 68%) of catecholamine-induced vasoconstriction.     

Renal alpha 1-adrenergic receptor subtypes: MDCK-D1 cells, but not rat cortical membranes possess a single population of receptors.

Recent work has demonstrated that alpha 1-adrenergic receptors are composed of at least two subtypes, termed alpha 1a and alpha 1b. It has been proposed that these subtypes may be linked to distinct second messenger systems. In the current studies, we have compared the properties of alpha 1-adrenergic receptors in rat renal cortical membranes with those in MDCK-D1 cells, a clonal cell line derived from distal tubule/collecting duct. Competitive binding studies with [3H]prazosin and compounds [5-methylurapidil, (+)-niguldipine, WB4101, and oxymetazoline] that distinguish high affinity (alpha 1a) and low affinity (alpha 1b) sites indicated that rat renal cortical membranes contain about 50% of each class of site. In contrast, MDCK-D1 cells contained a single population of low affinity sites. 5-Methylurapidil, but not the other compounds, recognized binding sites in these cells with a substantially lower affinity than has been observed for the low affinity site in other tissues and in parallel studies with renal cortical membranes. [3H]Prazosin binding sites in these cells, as well as alpha 1-adrenergic receptor-mediated arachidonic acid release and phosphoinositide and phosphatidylcholine hydrolysis, were sensitive to inactivation by chloroethylclonidine (IC50 approximately 0.7 microM), as expected for alpha 1b receptors. However, alpha 1-adrenergic receptors of MDCK-D1 cells required extracellular calcium for biological response, unlike what has been hypothesized for the alpha 1b receptor subtype. These data indicate that the population of alpha 1-adrenergic receptors of distal tubule/collecting duct cells likely consists of receptors of the alpha 1b subtype. The low affinity binding of 5-methylurapidil and the requirement for extracellular calcium for biological response in these cells suggest that this receptor may not be identical to the alpha 1b receptor that has been observed in other systems.     

Alpha 1-adrenergic receptor subtypes and formation of inositol phosphates in dispersed hepatocytes and renal cells

The ability of alpha 1a- and alpha 1b-adrenergic receptor subtypes to stimulate [3H]inositol phosphate [( 3H]InsP) formation was examined in collagenase-dispersed hepatocytes and renal cells. alpha 1-Adrenergic receptor binding sites were labeled with 125I-BE 2254, and the proportion of alpha 1a and alpha 1b subtypes was determined with chloroethylclonidine (CEC) and WB 4101. Hepatocytes contained only alpha 1b-adrenergic receptors, whereas renal cells had approximately equal proportions of both subtypes. Pretreatment of renal cells with CEC selectively inactivated the alpha 1b subtype, leaving a homogeneous population of alpha 1a receptors. Norepinephrine stimulated [3H]InsP accumulation to a similar extent in both hepatocytes and renal cells. Pretreatment with CEC inactivated this response completely in hepatocytes but only partially in renal cells. WB 4101 was 1000-fold more potent in inhibiting the [3H]InsP response in renal cells than hepatocytes; however, some of this difference was due to rapid metabolism of WB 4101 by hepatocytes. After correction for metabolism, WB 4101 was still 11-fold more potent in inhibiting norepinephrine-stimulated [3H]InsP formation in hepatocytes (alpha 1b) than in CEC-pretreated renal cells (alpha 1a). These results demonstrate that both alpha 1a- and alpha 1b-adrenergic receptor subtypes activate formation of [3H]InsP, although the molecular mechanisms by which these responses occur remain to be determined.     

脑微血管内皮细胞培养技术的研究进展

脑微血管内皮细胞作为构成血脑屏障(blood brain barrier,BBB)的主要结构基础,由于其特殊的细胞膜转运系统和细胞间的紧密连接,从而在中枢神经系统中的血管内外物质交换中发挥着重要的作用。因其与脑缺血、脑水肿等脑血管疾病的病理过程有着密切的关系,因此体外培养脑微血管内皮细胞(brain microvascular endothelial cells,BMECs)为研究脑血管疾病提供了一种新的方法。该文通过对国内外相关文献进行检索,综合了国内外近年来的研究,依次从大鼠脑微血管内皮细胞的分离、纯化、培养等几个方面,探讨了脑微血管内皮细胞的体外培养技术,为脑微血管内皮细胞的体外研究提供基础。     

大鼠血管中α_1肾上腺素受体的两种亚型

α_1肾上腺素受体(α_1受体)激动引起的大鼠离体血管收缩,在主动脉可为不可逆性α_1受体拮抗剂CEC大部分阻断,而不受钙离子拮抗剂硝苯吡啶的影响;在肾动脉不受CEC阻断,却可为硝苯吡啶大部分阻断;在肠系膜动脉与门静脉则介于两者之间。竞争性α_1受体拮抗剂WB4101的pA_2值,肾动脉>肠系膜动脉>主动脉。根据已知α_1受体两种亚型的药理特征,上述结果提示大鼠血管中的α_1受体存在两种亚型,在主动脉内以α_(1b)亚型为主,在肾动脉内以α_(1a)亚型为主,在肠系膜动脉与门静脉内两种亚型的含量较为均衡。     

The relationship between alpha 1-adrenergic receptor occupancy and response in BC3H-1 muscle cells

The relationship between alpha 1-adrenergic receptor occupancy by agonists or antagonists and the regulation of intracellular Ca2+ was examined. Receptor occupancy was measured using the antagonist [3H]prazosin and correlated with agonist-elicited 45Ca2+ fluxes. The agonists epinephrine (E), norepinephrine (NE), and phenylephrine (PE) coordinately activated Ca2+ efflux, reflecting a substantial mobilization of intracellular Ca2+, as well as a smaller 45Ca2+ influx. The agonist concentration dependences for influx and efflux were similar, with the order of potency expected for alpha 1 receptors (E greater than or equal to NE greater than PE). To determine the relationship between receptor occupancy and response, the slowly dissociating antagonist prazosin was used to inactivate specified fractions of the receptor population. A linear relationship was observed between the remaining activatable receptors and residual 45Ca2+ efflux elicited by E or NE, except at saturating agonist concentrations where some curvature was observed. Moreover, the concentration dependence for agonist-elicited 45Ca2+ efflux was shifted toward slightly higher concentrations of E or NE following prazosin inactivation. These results suggest the presence of a modest receptor reserve which is revealed by E or NE, but not by PE. Agonist occupation was measured over the same interval as receptor activation by competition with the initial rate of [3H]prazosin association. All three agonists exhibited the major fraction of receptor occupation over the same concentration ranges required for the functional response. Exposure of receptors to specified agonist concentrations for 30 min had little effect on the number of receptors or their ligand affinities, whereas a 2.5-hr exposure to agonist decreased apparent agonist affinity as well as the number of receptors recognized by [3H]prazosin.     

Differential antagonism by glibenclamide of the relaxant effects of cromakalim,pinacidil and nicorandil on canine large coronary arteries

Summary The relaxant mechanisms of action of cromakalim, pinacidil and nicorandil, potassium channel openers, on large epicardial coronary arteries were investigated in isolated canine left circumflex arteries contracted by 10^–7 mol/l U46619, a thromboxane A₂ analogue, or addition of 25 mmol/l KCl in comparison with nitroglycerin.Cromakalim (3 × 10^–8–3 × 10^–5 mol/l), pinacidil (10^–6–10^–4 mol/l), nicorandil (3 × 10^–6–10^–3 mol/l) and nitroglycerin (3 × 10^–8–10^–5 mol/l) all produced a concentration-dependent relaxation in both U46619- or KCl-contracted arteries. At their maximum effects pinacidil, nicorandil and nitroglycerin produced full relaxation in arteries contracted by either means. In contrast, cromakalim produced about a 73% relaxation in KCl-contracted arteries, although it caused full relaxation in U46619-contracted ones. In the presence of glibenclamide the concentration-relaxation curves for cromakalim in U46619- or KCl-contracted arteries underwent rightward parallel shifts. Schild regression had a slope of 1.00 and yielded a pA₂ of 7.47 for glibenclamide in U46619-contracted arteries, and corresponding values obtained in KCl-contracted arteries were 0.86 (not significantly different from unity) and 7.28. The concentration-relaxation curves for pinacidil in U46619-contracted arteries also underwent rightward parallel shifts in the presence of glibenclamide, however, Schild regression had a slope of 0.60. The concentration-relaxation curves for pinacidil in KCl-contracted arteries underwent rightward parallel shifts only to a limited extent in the presence of glibenclamide. The concentration-relaxation curves for nicorandil and nitroglycerin in U46619- or KCl-contracted arteries were not affected by glibenclamide in concentrations which antagonized cromakalim. The concentration-relaxation curves for nicorandil or nitroglycerin in U46619-contracted arteries were shifted by methylene blue (10^–5 mol/l) to the right without suppression of the their maximum effects. Similar curves for cromakalim were not affected at all by this concentration of methylene blue. The concentration-relaxation curves for nicorandil in U46619-contracted arteries determined in the presence of methylene blue (10^–5 mol/l) and glibenclamide (3 × 10^–7, 10^–6 and 3 × 10^–6 mol/l) were not significantly different from those in the presence of methylene blue alone.These results indicate the following: In canine large epicardial coronary arteries (1) cromakalim produced relaxation by the mechanism antagonized by glibenclamide, probably opening ATP-sensitive potassium channels, (2) pinacidil did so by the mechanism shared with cromakalim and by one not antagonized by glibenclamide as well, and (3) nicorandil did so exclusively by the mechanism of action as a nitrate. Send offprint requests to N. Taira at the above address