Platelet alpha-2 adrenergic receptor-mediated phosphoinositide responses in endogenous depression.

We have previously indicated that epinephrine stimulates phosphoinositide (PI) hydrolysis by activating alpha-2 adrenergic receptors in human platelets [H. Mori et. al. Life Sci., 741-747 44 (1989)]. This method involves the measurement of the accumulation of [3H]-inositol-1-phosphate (IP-1) as an index of PI hydrolysis; lithium is added to inhibit the metabolism of IP-1, thus giving an enhanced signal. In the present study, we assessed the platelet alpha-2 adrenergic receptor-mediated PI responses in samples from 15 unmedicated patients with endogenous depression and 15 age- and sex-matched control subjects. The responses to epinephrine (10 microM and 100 microM) in the depressed patients were significantly higher than those of the controls, whereas the basal values did not differ significantly. These results support the hypothesis that platelet alpha-2 adrenergic receptors may be supersensitive in patients with endogenous depression.     

Alpha-2 adrenergic regulation of norepinephrine release in the rat submandibular gland as measured by HPLC-EC

In many tissues, norepinephrine appears to inhibit its own release through an interaction at alpha adrenergic receptors. We have developed an assay for measuring the release of endogenous norepinephrine based on HPLC and have studied the regulation of release in the rat submandibular gland by alpha adrenergic antagonists. The method uses electrochemical detection to quantitate norepinephrine released from tissue slices and does not require preloading of the tissue with [3H]norepinephrine. Yohimbine, an alpha-2 adrenergic antagonist, potentiates by 50% the release caused by potassium induced depolarization with an EC50 of 0.14 microM. Prazosin, an alpha-1 antagonist, has a similar effect, but is less potent with an EC50 of 0.77 microM. Thus, the alpha adrenergic receptor mediating the regulation of norepinephrine release is of the alpha-2 subtype. The observed equal efficacies and lack of additivity of release potentiation by yohimbine and prazosin at maximal doses suggest that both drugs act at the same receptor. The five-fold difference in potency between prazosin and yohimbine is consistent with the recent observations indicating species differences between rodent and non-rodent alpha-2 adrenergic receptors.     

Absence of alpha-2 adrenergic effects on cAMP production in a genital tract smooth muscle cell line

This study sought to evaluate alpha-2 and beta adrenergic modulation of cAMP production in the DDT1 MF-2 transformed smooth muscle myocyte. After stimulation with forskolin or adrenergic agonists with or without subtype specific antagonists, cAMP production was determined. These experiments confirmed an increase of cAMP in response to forskolin, isoproterenol, epinephrine, and norepinephrine; the adrenergic stimulation was inhibited by propranolol. On the other hand, the alpha-2 agonist clonidine did not inhibit cAMP production. Likewise, alpha-2 receptor blockade did not increase cAMP production in response to epinephrine. These studies, therefore, suggest that the DDT1 MF-2 myocyte does not contain a significant population of functional alpha-2 adrenergic receptors.     

Effect of various factors on serotonin-induced Ca2+ response in human platelets

Serotonin (5-HT)-stimulated intracellular Ca2+ concentration change was studied in the platelets of healthy subjects, using fluorescent Ca indicator fura-2. 5-HT increased the Ca2+ response in a concentration-dependent manner. 10 microM of 5-HT induced the maximal response and its EC50 value was 0.4 microM. This response was potently inhibited by selective 5-HT2 antagonists, suggesting that 5-HT-induced Ca2+ mobilization in human platelets is mediated by 5-HT2 receptors. This 5-HT2-mediated Ca2+ response was not significantly affected by the time of blood sampling, gender, meal or exercise. However, this response declined with time after blood drawing, suggesting that it must be measured as soon as possible after sampling. These results indicate that 5-HT-stimulated Ca2+ response in human platelets is a stable parameter and that it will be suitable for assessing 5-HT2 receptor function in depressed patients.     

Alpha-2 and beta-adrenergic receptors mediate NE's biphasic effects on rat thick ascending limb chloride flux

The sympathetic neurotransmitter norepinephrine (NE) influences renal sodium excretion via activation of adrenergic receptors. The thick ascending limb (THAL) possesses both alpha-2 and beta-adrenergic receptors. However, the role(s) different adrenergic receptors play in how isolated THALs respond to NE are unclear. We tested the hypothesis that both alpha-2 and beta-adrenergic receptors are responsive to NE in the isolated THAL, with alpha-2 receptors inhibiting and beta-receptors stimulating chloride flux (J(Cl)). THALs from male Sprague-Dawley rats were perfused in vitro, and the effects of 1) incremental NE, 2) the alpha-2 agonist clonidine, and 3) the beta-agonist isoproterenol on J(Cl) were measured. Low concentrations (0.1 nM) of NE decreased J(Cl) from a rate of 114.2 +/- 8.1 to 93.5 +/- 14.6 pmol. mm(-1). min(-1) (P < 0.05), with the nadir occurring at 1 nM (67.7 +/- 8.8 pmol. mm(-1). min(-1); P < 0.05). In contrast, greater concentrations of NE significantly increased J(Cl) from the nadir to a maximal rate of 131.0 +/- 28.5 pmol. mm(-1). min(-1) at 10 microM (P < 0.05). To evaluate the adrenergic receptors mediating these responses, the THAL J(Cl) response to NE was measured in the presence of selective antagonists of beta- and alpha-2 receptors. A concentration of NE (1 microM), which alone tended to increase J(Cl), decreased THAL J(Cl) (from 148.9 +/- 16.4 to 76.2 +/- 13.6 pmol. mm(-1). min(-1); P < 0.01) in the presence of the beta-antagonist propranolol. In contrast, a concentration of NE (0.1 microM), which alone tended to decrease J(Cl), increased THAL J(Cl) (from 85.5 +/- 20.1 to 111.8 +/- 20.1 pmol. mm(-1). min(-1); P < 0.05) in the presence of the alpha-2 antagonist rauwolscine. To further clarify the role of different adrenergic receptors, selective adrenergic agonists were used. The alpha-2 agonist clonidine decreased J(Cl) from 102.4 +/- 9.9 to 54.0 +/- 15.7 pmol. mm(-1). min(-1), a reduction of 49.1 +/- 11.0% (P < 0.02). In contrast, the beta-agonist isoproterenol stimulated J(Cl) from 95.3 +/- 11.6 to 144.1 +/- 15.0 pmol. mm(-1). min(-1), an increase of 56 +/- 14% (P < 0.01). We conclude that 1) the sympathetic neurotransmitter NE exerts concentration-dependent effects on J(Cl) in the isolated rat THAL, 2) selective alpha-2 receptor activation inhibits THAL J(Cl), and 3) selective beta-receptor activation stimulates THAL J(Cl). These data indicate the response elicited by the isolated rat THAL to NE is dependent on the neurotransmitter concentration, such that application of NE in vitro biphasically modulates J(Cl) via differential activation of alpha-2 and beta-adrenergic receptors in a concentration-dependent manner.     

Alpha adrenergic modulation on effects of norepinephrine transporter inhibitor reboxetine in five-choice serial reaction time task

The study examined the effects of a norepinephrine transporter (NET) inhibitor reboxetine (RBX) on an attentional performance test. Adult SD rats trained with five-choice serial reaction time task (5-CSRTT) were administered with RBX (0, 3.0 and 10 mg/kg) in the testing day. Alpha-1 adrenergic receptor antagonist PRA and alpha-2 adrenergic receptor antagonist RX821002 were used to clarify the RBX effect. Results revealed that rat received RBX at 10 mg/kg had an increase in the percentage of the correct response and decreases in the numbers of premature response. Alpha-1 adrenergic receptor antagonist Prazosin (PRA) at 0.1 mg/kg reversed the RBX augmented correct responding rate. However, alpha-2 adrenergic receptor antagonist RX821002 at 0.05 and 0.1 mg/kg dose dependently reversed the RBX reduced impulsive responding. Our results suggested that RBX as a norepinephrine transporter inhibitor can be beneficial in both attentional accuracy and response control and alpha-1 and alpha-2 adrenergic receptors might be involved differently.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

Alpha-adrenergic stimulation of phosphatidylinositol synthesis in human platelets as an alpha-2 effect secondary to platelet aggregation

Epinephrine and adenosine diphosphate (ADP) stimulated 3H-glycerol uptake into phosphatidylinositol of human platelets. Yohimbine, an alpha-2 adrenoceptor antagonist, markedly reduced epinephrine-stimulated 3H-glycerol uptake into phosphatidylinositol; while prazosin, an alpha-1 antagonist, was without effect. Likewise, yohimbine, but not prazosin, blocked epinephrine-induced platelet aggregation. Furthermore, clonidine, a specific agonist for alpha-2 adrenoceptors, stimulated incorporation of 3H-glycerol into phosphatidylinositol and promoted platelet aggregation in the presence of low concentrations of ADP. These studies indicate that the effects of epinephrine on platelet aggregation and phosphatidylinositol synthesis are mediated through alpha-2 adrenoceptors. Further, since the stimulation of phosphatidylinositol synthesis seen with epinephrine was also observed with ADP, this suggests that the increased 3H-glycerol labeling is an indirect result of platelet aggregation.     

Characterization of the alpha adrenergic receptor population in hippocampus up regulated by serotonergic raphe deafferentiation

Serotonergic raphe deafferentiation elicits an up regulation of a nM (3H)WB-4101 binding site in rat hippocampus for which norepinephrine displays high affinity and prazosin displays low affinity. Guanine nucleotide affects the nM binding to hippocampal alpha-1 adrenergic receptors. Firstly, Gpp(NH)p, a nonhydrolyzable analog of GTP, inhibits (3H)WB-4101 binding at 3 nM concentration of the radioligand, the ligand concentration labelling the lower affinity, nM, binding site. Secondly, the addition of Gpp(NH)p causes recovery of the heterogeneity of binding sites lost upon preincubation of the membranes with 100 microM epinephrine, apparently by decreasing the affinity of the nM (3H)WB-4101 binding site for the adrenergic receptors. The phenomenon was still observed in the presence of saturating concentrations of the alpha-2 antagonist, yohimbine, and the beta antagonist, propranolol. The results imply that Gpp(NH)p regulates ligand binding to hippocampal alpha-1 agonist sites. It is likely that agonist and antagonist binding sites for the alpha-1 receptor exist in hippocampus with the agonist site being modulated by serotonin.     

Activation of alpha-1 adrenergic receptors modulates the control of left/right sidedness in rat embryos.

Presomite stage rat embryos were cultured for 45-49 hr with medium containing various adrenergic agonists and antagonists. L-Norepinephrine but not D-norepinephrine (several orders of magnitude less potent than the L-isomer at alpha-1 adrenergic receptors) resulted in a dose-dependent increase of situs inversus similar to that found for phenylephrine, an alpha-1 adrenergic agonist. Prazosin, an alpha-1 adrenergic antagonist, inhibited phenylephrine-induced situs inversus in a dose-dependent manner. Neither dexmedetomidine, an alpha-2 adrenergic agonist, nor isoproterenol, a beta adrenergic agonist, caused situs inversus. These results provide pharmacological evidence that stimulation of alpha-1 but not of alpha-2 and beta adrenergic receptors modulates the control of left/right sidedness in rat embryos.     

Adrenergic receptors mediating prostaglandin production in the rabbit vas deferens

Contractile and prostaglandin E (PGE)-producing effects of adrenergic agonists were compared in the rabbit isolated vas deferens to determine which adrenergic receptor(s) potentially could mediate neural responses. Additionally, interactions among receptors were elucidated by comparing responses to norepinephrine, phenylephrine and isoproterenol to those in the presence of selective adrenergic agonists or antagonists. Norepinephrine increased the force of muscle contraction and the immunoassayable PGE concentrations in a concentration-dependent manner with EC50's of 55 +/- 8 and 112 +/- 39 microM, respectively. Propranolol (10 microM) enhanced the contractile effects of norepinephrine (p less than 0.01) whereas yohimbine (100 microM) or prazosin (1 microM) reduced norepinephrine-induced contractions and PGE production (p less than 0.01). Propranolol did not alter the PGE production induced by norepinephrine. Metoprolol (100 microM) also enhanced contractile effects of norepinephrine (p less than 0.05). The beta adrenergic agonist, isoproterenol (100 nM), decreased the contractile, but not the PGE-producing, effects of phenylephrine (p less than 0.001). Isoproterenol, given alone, increased PGE concentrations and inhibited electrically-induced force generation in a concentration-dependent manner. These results are consistent with the presence of alpha receptors on the vas deferens which mediate smooth muscle contraction and PGE generation. Beta receptors which mediate relaxation and PGE production also are present. Tentative identification of the beta receptor subtype revealed the presence of a beta 1 receptor.     

Necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and cholecystokinin2 receptors on parietal cells in isolated mouse stomach

The existence of a direct action of acetylcholine and gastrin on muscarinic M3 and cholecystokinin2 (CCK2) receptors on gastric parietal cells has not yet been convincingly established because these stimulated acid secretions are remarkably inhibited by histamine H2 receptor antagonists. In the present study, we investigated the necessity of intracellular cyclic AMP in inducing gastric acid secretion via muscarinic M3 and CCK2 receptors on parietal cells using an isolated mouse stomach preparation. Bethanechol (10-300 microM) produced a marked increase in acid output and this increase was completely blocked by famotidine (10 microM). In the presence of famotidine, bethanechol (1-30 microM) augmented the acid secretory response to dibutyryl AMP (200 microM) in a concentration-dependent manner. The augmentation was blocked by atropine (1 microM), 4-DAMP (0.1 microM), a muscarinic M3-selective antagonist, and by Ca2+ exclusion from the serosal nutrient solution. Pentagastrin (0.3-3 microM) also concentration-dependently stimulated gastric acid secretion, but the effect was completely inhibited by famotidine. In the presence of famotidine, pentagastrin (0.1-0.3 microM) elicited a definite potentiation of the acid secretory response to dibutyryl cyclic AMP (200 microM). This potentiation was inhibited by YM022 (1 microM), a CCK2 receptor antagonist, and by exclusion of Ca2+ from the serosal nutrient solution. The present results suggest that gastric acid secretion via the activation of muscarinic M3 and CCK2 receptors on the parietal cells is induced by activation of the cyclic AMP-dependent secretory pathway.     

Inhibition of dibutyryl cyclic AMP-induced insulin release by alpha-2 adrenergic stimulation

Effects of alpha adrenergic agonists and antagonists on dibutyryl cAMP (Bt2cAMP)-induced insulin release were investigated with isolated rat pancreatic islets. Bt2cAMP (4 mM) produced 2-fold stimulation of insulin release in all the concentrations of glucose examined (3.3-16.7 mM). Clonidine but not phenylephrine inhibited the Bt2cAMP-stimulated insulin release in a concentration-dependent manner with approximate EC50 of nanomolar range. Yohimbine but not prazosin antagonized the inhibitory effect of clonidine on the Bt2-cAMP-induced insulin release. These results suggest that alpha-2 adrenergic mechanisms are involved in a step distal to cAMP generation.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Activation of Rap1B by G(i) family members in platelets

It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.     

The P2Y1 receptor mediates ADP-induced p38 kinase-activating factor generation in human platelets.

U46619, a thromboxane A2 mimetic, but not ADP, caused activation of p38 mitogen activated protein (MAP) kinase in aspirin-treated platelets. In nonaspirinated human platelets ADP activated p38 MAP kinase in both a time-and concentration-dependent manner, suggesting that ADP-induced p38 MAP kinase activation requires generation of thromboxane A2. However, neither a thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 and a thromboxane synthase inhibitor, furegrelate, either alone or together, nor indomethacin blocked ADP-induced p38 kinase activation in nonaspirinated platelets. Other cycloxygenase products, PGE2, PGD2, and PGF2alpha, failed to activate p38 kinase in aspirin-treated platelets. Hence, ADP must be generating an agonist, other than thromboxane A2, via an aspirin-sensitive pathway, which is capable of activating p38 kinase. AR-C66096, a P2TAC (platelet ADP receptor coupled to inhibition of adenylate cyclase) antagonist, did not inhibit ADP-induced p38 MAP kinase activation. The P2X receptor selective agonist, alpha, beta-methylene ATP, failed to activate p38 MAP kinase. On the other hand, the P2Y1 receptor selective antagonist, adenosine-2'-phosphate-5'-phosphate inhibited ADP-induced p38 kinase activation in a concentration-dependent manner, indicating that the P2Y1 receptor alone mediates ADP-induced generation of the p38 kinase-activating factor. These results demonstrate that ADP causes the generation of a factor in human platelets, which can activate p38 kinase, and that this response is mediated by the P2Y1 receptor. Neither the P2TAC receptor nor the P2X1 receptor has any significant role in this response.     

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M2 alpha and M2 beta muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF1 alpha was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M1 and M2 agonist) (1-10 microM) and arecaidine proparagyl ester (APE) (M2 selective agonist) (1-10 microM) enhanced 6-keto-PGF1 alpha output in a concentration-dependent manner. A selective M1 receptor agonist, McN-A-343, at 1 microM-1 mM did not alter 6-keto-PGF1 alpha output. ACh- and APE induced increases in 6-keto-PGF1 alpha output were attenuated by the M1/M2 antagonist atropine (0.1 microM), M2 alpha antagonist (AF-DX 116), (0.1-1.0 microM), and by selective M2 beta antagonist, hexahydro-sila-difendiol (HHSiD) (0.1-1.0 microM), but not by the M1 antagonist pirenzepine (1.0 microM). 6-Keto-PGF1 alpha output elicited by ACh- or APE was not altered by the adrenergic receptor antagonists phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF1 alpha accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF1 alpha output. Removal of the endothelium abolished the production of 6-keto-PGF1 alpha elicited by ACh, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K+ or norepinephrine. These data suggest that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M2 alpha and M2 beta but not M1 muscarinic receptors, which are most likely located on the endothelium.     

Characterization of alpha 2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[3H]Yohimbine, a potent alpha 2-adrenergic antagonist, was used to label the alpha-adrenergic receptors in membranes isolated from human platelets. Binding of [3H]yohimbine to platelet membranes appears to have all the characteristics of binding to alpha-adrenergic receptors. Binding reached a steady state in 2-3 min at 37 degrees C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10(-5) M; t1/2 = 2.37 min). [3H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [3H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (-) isomer was 11-times more potent that the (+) isomer. Catecholamine agonists competed for the occupancy of the [3H]yohimbine-binding sites with an order of potency: clonidine greater than (-)-epinephrine greater than (-)-norepinephrine much greater than (-)-isoproterenol. The potent alpha-adrenergic antagonist, phentolamine, competed for the sites whereas the beta-antagonist, (+/-)-propranolol, was very weak inhibitor. 0.1 mM GTP reduced the binding affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonin competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [3H]yohimbine binding. These results suggest that [3H]yohimbine binding to hunan platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label alpha 2-adrenergic receptors.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.