Electrophysiological and pharmacological evidence that peripheral type benzodiazepine receptors are coupled to calcium channels in the heart

PK 11195, an antagonist of the peripheral type benzodiazepine receptor, does not affect either the duration of the action potential or the tension of the guinea pig papillary muscle. However, it antagonized the effects of the calcium channel blockers, nitrendipine, verapamil, diltiazem, and of BAY K8644, a calcium channel agonist in this heart preparation. On the other hand, PK 11195 does not change the increase in the action potential duration provoked by the potassium channel blocker tetraethylammonium. RO5-4864, an agonist of the peripheral type benzodiazepine receptor, decreased the tension of the guinea pig papillary muscle. The effect was reversed by increasing extracellular Ca2+ concentrations up to 4 mM. These results suggest that in the heart the peripheral type benzodiazepine receptors are coupled to calcium channels.     

Functional and autoradiographic characterization of dopamine D2-like receptors in the guinea pig heart

Dopamine receptors include the D1- (D1 and D5 subtypes) and D2-like (D2, D3, and D4 subtypes) families. D1-like receptors are positively and D2-like receptors negatively coupled to the adenylyl cyclase. Dopamine D2-like (D4 subtype) receptors have been identified in human and rat hearts. However the presence of D2 and D3 receptor subtypes is unclear. Furthermore, their role in cardiac functions is unknown. By autoradiographic studies of guinea pig hearts, we identified D3 and D4 receptors, using the selective radioligands [3H]-7-OH-DPAT and [3H]emonapride (YM-09151-2 plus raclopride). Western blot analysis confirmed D3 and D4 receptors in the right and left ventricle of the same species. Selective agonists of D3 and D4 receptors (+/-)-7-OH-DPAT and PD 168 077 (10(-9) to 10(-5) M, respectively) induced a significant negative chronotropic and inotropic effect in the isolated guinea pig heart preparation. Negative inotropic effect induced by PD 168 077 was associated with an inhibition in cyclase activity. No changes in cyclase activity were found with (+/-)-7-OH-DPAT. The aim of this study is to support the presence of D3 and D4 receptors in the heart. Although our results suggest that D3 and D4 receptors are functionally active in the heart, we need additional information with an antagonist and an agonist of improved potency and selectivity to understand the respective roles of D3 and D4 receptors in the cardiac functions.     

Cloning and characterization of the hamster and guinea pig nicotinic acid receptors

In this study, we present the identification and characterization of hamster and guinea pig nicotinic acid receptors. The hamster receptor shares approximately 80-90% identity with the nucleotide and amino acid sequences of human, mouse, and rat receptors. The guinea pig receptor shares 76-80% identity with the nucleotide and amino acid sequences of these other species. [(3)H]nicotinic acid binding affinity at guinea pig and hamster receptors is similar to that in human (dissociation constant = 121 nM for guinea pig, 72 nM for hamster, and 74 nM for human), as are potencies of nicotinic acid analogs in competition binding studies. Inhibition of forskolin-stimulated cAMP production by nicotinic acid and related analogs is also similar to the activity in the human receptor. Analysis of mRNA tissue distribution for the hamster and guinea pig nicotinic acid receptors shows expression across a number of tissues, with higher expression in adipose, lung, skeletal muscle, spleen, testis, and ovary.     

Pharmacological characterization and autoradiographic localization of substance P receptors in guinea pig brain

[3H]Substance P ([3H]SP) was used to characterize substance P (SP) receptor binding sites in guinea pig brain using membrane preparations and in vitro receptor autoradiography. Curvilinear Scatchard analysis shows that [3H]SP binds to a high affinity site (Kd = 0.5 nM) with a Bmax of 16.4 fmol/mg protein and a low affinity site (Kd = 29.6 nM) with a Bmax of 189.1 fmol/mg protein. Monovalent cations generally inhibit [3H]SP binding while divalent cations substantially increased it. The ligand selectivity pattern is generally similar to the one observed in rat brain membrane preparation with SP being more potent than SP fragments and other tachykinins. However, the potency of various nucleotides is different with GMP-PNP greater than GDP greater than GTP. The autoradiographic distribution of [3H]SP binding sites shows that high amounts of sites are present in the hippocampus, striatum, olfactory bulb, central nucleus of the amygdala, certain thalamic nuclei and superior colliculus. The cortex is moderately enriched in [3H]SP binding sites while the substantia nigra contains only very low amounts of sites. Thus, the autoradiographic distribution of SP binding sites is fairly similar in both rat and guinea pig brain.     

Molecular mechanisms of peripheral benzodiazepine receptors

Peripheral-type benzodiazepine receptors were identified initially as binding sites in peripheral tissues with markedly different drug specificity than the central type receptors. The density of peripheral receptors varies greatly among tissue with selective localization within organs. Steroid producing areas of glands, such as the adrenal, testes and ovary, are highly enriched in these receptors. Intracellular localizations provide further insight into function with peripheral receptors largely if not exclusively associated with outer membranes of mitochondria. Purification of the peripheral receptor protein from rat kidney mitochondria reveals two apparent subunits with molecular weights of about 30 and 18 kD respectively. This complex is functionally intact as determined by its ability to reversibly bink PK-11195 Ro5-4864, and PK-14105 with high affinity and specificity.Acknowledgements: Supported by USPHS grant DA-00266, Research Scientist Award DA-00074 to S.H.S. and a gift of the Bristol Myers Company.Special issue dedicated to Dr. Erminio Costa.     

Alterations in binding characteristics of peripheral benzodiazepine receptors in testes by vitamin A deficiency in guinea pigs

The correlation of vitamin A with the binding characteristics of peripheral benzodiazepine receptors (PBRs) in testes have been implicated on the basis of findings of involvement of vitamin A in testicular physiology and the abundance of PBRs in testicular tissue. Both vitamin A and PBRs are involved in the control of cell proliferation and differentiation but no data exists regarding the relationship between them. In the present study, we have examined the effects of vitamin A deficiency on the affinity and density of PBRs in testes of guinea pigs. Weanling guinea pigs were divided into three groups: control, pair-fed control and vitamin A deficient. They were fed a complete semipurified diet. The vitamin A deficient diet was similar to the control diet except vitamin A palmitate was omitted. Vitamin A deficiency status was achieved after 90 days of feeding. Binding of [³H]Ro 5-4864, a specific ligand for peripheral benzodiazepine receptors was determined in whole homogenate of testicular tissue. There was a 77% decrease in the receptor density (B_max) in vitamin A deficient group compared to control. The B_maxvalues for control, pair-fed control and vitamin A deficient groups were: 12.4 ± 0.4, 8.8 ± 0.2 and 3.0 ± 0.6 pmol/g, respectively. The equilibrium dissociation constant (K_D) values were also 86% decreased in the vitamin A deficient group compared to the other groups. The K_D values for control, pair-fed control and vitamin A deficient groups were: 3.4 ± 0.7, 2.8 ± 0.5 and 0.5 ± 0.01, respectively. The decrease in the binding characteristics of PBRs in testes due to vitamin A deficiency was accompanied with a corresponding decrease in the levels of testosterone in plasma. These results suggest a close functional relationship of vitamin A with PBRs in testes.     

Heterogeneity of muscarinic receptors coupled to phosphoinositide breakdown in guinea pig brain and peripheral tissues

Muscarinic receptors coupled to phosphoinositide hydrolysis (PI) are present in guinea pig bladder and colon. Compared to rat cerebral cortex, an extensively studied muscarinic/PI turnover system, all agonists were more potent and efficacious in both bladder and colon. The "M1-selective antagonists", pirenzepine and dicyclomine, were much more potent (Ki = 1-5 nM) and selective (300 to 500-fold) at both rat and guinea pig brain and guinea pig colon receptors, compared to PI-coupled receptors in guinea pig bladder. In contrast, "M2-selective antagonists", AF-DX 116 and HHSiD, were 2-6 fold more potent in bladder than in brain, while HHSiD was very potent in the colon (50 times more potent than in brain). These results suggest a pharmacological heterogeneity of PI-linked muscarinic receptors. If muscarinic receptors with a low affinity for pirenzepine are defined as M2, these results show that the guinea pig bladder contains PI-linked M2 muscarinic receptors, whereas the guinea pig colon contains PI-linked M1 receptors.     

乳酸左氧氟沙星对豚鼠心肌细胞电生理的影响

目的了解乳酸左氧氟沙星(LVFX)对豚鼠心室肌细胞电生理的影响.方法经腹腔注射不同剂量的LVFX,记录并分析注药后5～360 min豚鼠Ⅱ导联心电图的QT间期,以及校正的QT间期(QTc).采用全细胞膜片钳技术,记录不同浓度LVFX对体外单个心室肌细胞的延迟整流钾电流(IK)的作用.结果①LVFX给药量为200 mg/kg时,心电图QT间期延长19.38%±3.15%(P＜0.05);在50 mg/kg和100 mg/kg等较低剂量时,QT间期延长不明显(P＞0.05).②LVFX抑制IK电流,且抑制作用呈现电压依赖性和浓度依赖性.结论LVFX可能通过抑制心肌细胞IK电流引起心脏QT间期延长,临床应谨慎使用.     

白藜芦醇对离体豚鼠乳头状肌的电生理效应

本文旨在应用标准玻璃微电极技术,观察白藜芦醇对离体豚鼠乳头状肌的电生理效应。结果显示：(1)白藜芦醇(30、60、120μmol／L)可剂量依赖性地缩短乳头状肌细胞的动作电位时程;(2)对部分去极化的乳头状肌,白藜芦醇(60μmol／L)不仅缩短动作电位时程,而且降低动作电位的幅值和超射值,减慢零期最大上升速度;(3)用无钙K-H液灌流标本可完全取消白藜芦醇对乳头状肌细胞的作用：(4)钾通道开放剂四乙基氯化铵(TEA,20mmol／L),不能阻断白藜芦醇的电生理效应;(5)预先应用一氧化氮合酶抑制剂L-NAME(1mmol／L),对白藜芦醇的上述效应无影响。以上结果表明,白藜芦醇可缩短正常乳头状肌细胞动作电位时程,这一效应可能与其抑制钙离子内流有关,但此作用机制中NO的作用并不显著。     

辣椒素对离体豚鼠乳头状肌的电生理效应

应用细胞内微电极技术,观察了辣椒素(capsaicin,CAP)对豚鼠乳头状肌细胞的电生理效应。结果表明：(1)CAP(30、60、120μmol／L)可浓度依赖地缩短正常乳头状肌的动作电位时程。(2)对部分去极化乳头状肌,CAP(60μmol／L)除缩短动作电位时程外,还使动作电位幅值和超射值降低,零相最大上升速度减慢。(3)预先应用L型钙通道开放剂Bay K8644(0．5μmol／L),则可阻断CAP(60μmol／L)的电生理效应。(4)预先应用辣椒素受体(va-nilloid receptor,VR)阻断剂钌红(20μmol／L),不影响CAP(60μnol／L)的电生理效应。以上结果提示,CAP可能通过非受体途径抑制Ca^2 内流,从而影响豚鼠乳头状肌电生理效应。     

Platelet peripheral benzodiazepine receptors in repeated stress.

[3H]PK 11195 binding to platelet membranes and plasma stress hormones were studied in soldiers at the beginning of a parachute training course, following 6 days of preparatory exercises, and after the fourth actual parachute jump. A slight reduction (15%; NS) in the number of peripheral benzodiazepine receptors (PBR) was detected at the end of the exercise period, prior to the first jump. Reduced (26%; P less than 0.05) density of PBR was observed immediately after the repeated actual jumps. Equilibrium dissociation constants were not affected by the stressful situation. Plasma cortisol and prolactin levels remained unaltered during the entire study period.     

胍基丁胺在离体豚鼠乳头肌的电生理效应

应用细胞内微电极技术,观察了胍基丁胺（ａｇｍａｔｉｎｅ,ＡＧＭ）对豚鼠乳头肌细胞的电生理效应。结果表明：（１）ＡＧＭ浓度依赖地缩短正常乳头肌动作电位的时程;（２）对部分去极化的乳头肌,ＡＧＭ（１ｍｍｏｌ／Ｌ）除缩短动作电位时程外,还抑制动作电位零相最大上升速度,并降低其幅值和超射值;（３）预先应用一氧化氮合酶抑制剂ＬＮＡＭＥ（０５ｍｍｏｌ／Ｌ）,不能影响ＡＧＭ（１ｍｍｏｌ／Ｌ）的电生理效应;（４）预先应用咪唑啉受体（ｉｍｉｄａｚｏｌｉｎｅｒｅｃｅｐｔｏｒ,ＩＲ）和α２肾上腺素能受体（ａｌｐｈａ２ａｄｒｅｎｅｒｇｉｃｒｅｃｅｐｔｏｒ,α２ＡＲ）拮抗剂ｉｄａｚｏｘａｎ（０１ｍｍｏｌ／Ｌ）,则可完全阻断ＡＧＭ（１ｍｍｏｌ／Ｌ）的电生理效应。以上结果提示,ＡＧＭ对乳头肌的电生理效应似由α２ＡＲ和ＩＲ介导,并与胞浆内Ｃａ２＋减少有关。     

PK 11195, an antagonist of peripheral type benzodiazepine receptors, modulates Bay K8644 sensitive but not beta- or H2-receptor sensitive voltage operated calcium channels in the guinea pig heart

In a partially depolarized guinea pig papillary muscle preparation, BAY K8644 stimulated voltage-operated calcium channels, promoting slow action potentials; this effect was dose-dependent over a concentration range of 3 X 10(-7) M to 3 X 10(-6) M. Isoproterenol and histamine also induced slow action potentials by stimulating beta or H2 receptors, respectively. PK 11195, the antagonist of peripheral type benzodiazepine receptors, inhibited the effect of BAY K8644, but not those of histamine or isoproterenol. Moreover, PK 11195 "dose-dependently" antagonized the ability of RO5-4864 to inhibit the slow action potentials elicited by barium chloride. Thus, in the heart, PK 11195, an antagonist of peripheral type benzodiazepine receptors, can modulate voltage-operated calcium channels when they are activated directly, but not when they are activated by stimulation of neurotransmitter receptors.     

植物性雌激素genistein对豚鼠乳头肌的电生理效应

应用细胞内微电极技术,观察了genistein(GST)对豚鼠乳头肌的电生理效应。结果显示：（1）GST（10-100μmol/L)浓度依赖地缩短正常乳头肌动作电位时程;（2）对部分去极化乳头肌,GST（50μmol/L)除缩短动作电位时程外,还使动作电位幅值和超射值降低,零相最大上升速度减慢;（3）预先应用一氧化氮合酶抑制剂L-NNA（5mmol/L)。不影响GST（50μmol/L)的电生理效应;（4）单独应用17β-雌二醇（E2,5μmol/L)或GST（10μmol/L)时,动作电位各参数无明显变化。而预先应用同剂量的GST再加入E2,则动作电位时程缩短,结果提示,GST可能通过非NO途径抑制Ca^2 内流,从而影响豚鼠乳头肌电生理效应,并与E2有加强或协同效应。     

Interactions between nitrogen oxide-containing compounds and peripheral benzodiazepine receptors

Nitrogen oxide-containing compounds displaced the peripheral benzodiazepine ligand [³H]Ro5-4864 from guinea pig membrane preparations. Sodium nitroprusside (SNP) was the most potent (IC₅₀ = 5.61 ± 1.72 × 10⁻⁵ M). Moreover, its ability to bind to these receptors showed marked tissue variability (heart> kidney cerebral cortex). When tested on rat atrium, SNP by itself had no effect on basal inotropy or the increase in inotropy induced by (−)-S-BAY K 8644. In contrast, Ro5-4864 potentiated the marked increase in inotropy induced by (−)-S-Bay K 8644, and SNP completely abolished the potentiation of inotropy observed with Ro5-4864. Since peripheral benzodiazepine receptors are associated with calcium mobilization in the heart, these findings may indicate that some of the clinical effects of nitric oxide-generating drugs could be mediated by these receptors.     

Benzilylcholine mustard and spare receptors in guinea pig ileum

A comparison was made of muscarinic receptor occupancy by the irreversible antagonist benzilylcholine mustard (BCM) as determined from shifts in the dose-response curve to a muscarinic agonist and from 3H-QNB binding to homogenates of BCM-treated tissue. Major discrepancies were found. A low concentration of BCM (3x10-8M/15 min.) produced a parallel dose-response curve shift corresponding to 98-99% receptor occupancy by BCM, whereas 3H-QNB binding revealed only 48% receptor occupancy. Possible origins of this discrepancy are discussed. High concentrations of BCM (5x10-5M, 15 min.) fail to completely alkylate all 3H-QNB binding sites even though response is completely lost. Although significant (64%) recovery of response occurs after prolonged tissue washing (240 min.) this is not accompanied by an increase in 3H-QNB binding. The small fraction (approximately 5%) of sites inaccessible to BCM and with reduced affinity for 3H-QNB may represent a subpopulation of muscarinic receptors.