Similarity of Central and Peripheral Alpha-1 Adrenoceptors in Rat and Rabbit

Abstract

In order to examine species and tissue differences in α₁ adrenoceptors, binding experiments were performed using ³H-prazosin and membrane homogenates of central nervous and peripheral tissues of rabbit (cortex and spleen), and rat (cortex, spleen, and liver). Saturation studies indicated one binding site for ³H-prazosin, with apparent log molar dissociation constants (pK_D) ranging from 9.43 to 10.20. The rank orders of affinities of three competing antagonists (prazosin ? idazoxan > rauwolscine) and five agonists (cirazoline > clonidine ～ (-)-norepinephrine > (-)-phenylephrine > (+)-norepinephrine) were typical of α₁ receptors in all tissues. There were small but significant differences in the mean affinities of rauwolscine, idazoxan and cirazoline among the five tissues. No significant differences in pseudo-Hill coefficients were observed among tissues, although agonist binding curves were shallow (.7 to.85) and prazosin competition curves were significantly steeper (>.85). Guanine nucleotide did not affect the position or slope of the (-)-norepinephrine competition profile in rat cortex. These results demonstrate a qualitative similarity among central and peripheral α₁ receptors of the rat and rabbit, with small differences observed between central and peripheral sites in both species.     

Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha 1 adrenergic ligand, (3H) prazosin, were studied in blood vessel homogenates. No specific (3H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery 3H-prazosin binding was saturable with a Kd of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, [3H](-) quinuclidinyl benzilate (QNB) and one for alpha 2 adrenergic receptors, (3H) yohimbine. The results of these studies demonstrate a lack of alpha 1 adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine.     

5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition

Contractile responses to serotonin were examined $\text{in}$$\text{vitro}$ in the longitudinal portal vein to determine whether such responses were mediated by the interaction of serotonin with 5HT₁ receptors (those that preferentially bind [³H]serotonin) or 5HT₂ receptors (those that preferentially bind [³H]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the affinity for serotonin receptors in the rat portal vein and the ability to bind to 5HT₂, but no 5HT₁ receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED₅₀ value) relative to contractions produced by the non-cumulative addition of serotonin. Affinities of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentration-response curves compared to affinities obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT₂ receptor affinities of antagonists in the rat portal vein are best determined by the shift of non-cumulative responses to serotonin.     

Errors Introduced in Radioligand Binding Studies Due to Displaceable,Cation Dependent, [3H]prazosin Binding to Glass-Fibre Filters and Glass Surfaces

Abstract

[³H]prazosin not only specifically and homogeneously labels α₁-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled α-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [³H]prazosin binding experiments leads to erroneous results. Analysis of [³H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [³H]prazosin labels a homogeneous population of α₁-adrenoceptors (R_tot: 8.33 fmol˙mg^?1 wet tissue) with a dissociation constant of 1.28×10^?10 M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 μM phentolamine to the incubation medium) resulted in a best fit to a model in which [³H]prazosin labels two α₁-adrenoceptor subpopulations (R₁: 15.0 fmol˙mg^?1 and R₂: 14.6 fmol˙mg^?1 wet tissue) with dissociation constants of respectively 1.78×10^?10 and 5.63×10^?9 M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine.

Prazosin and oxymetazoline are also able to displace filter-bound [³H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.     

Effect of ketanserin and prazosin on blood pressure and cardiovascular reactivity to vasopressor agents during the development of two kidney-two clip renal hypertension in the conscious rat

The present experiment was performed in order to evaluate some of the actions of ketanserin, a blocking agent active at the serotonin 2 (S2) receptors. Male rats were divided into: 1. Two kidney-two clip (2K-2C) renal hypertensive: a silver clip (0.25 mm width) was placed in both renal arteries. 2. Sham-operated: a similar operation without placing the clip was performed. Blood pressure (BP), heart rate and pressor responses to tyramine, angiotensin II and norepinephrine (NE), and the hypotensive effect of prazosin (Pz) and ketanserin (Kt) were recorded in the conscious animals 8 weeks later. Results showed that Pz produced a similar decrease in BP in hypertensive and sham animals while Kt lowered BP much more in hypertensive than in normotensive rats. Prazosin abolished the pressor response to tyramine while ketanserin only diminished tyramine effect. Both hypotensive agents shifted the dose-response curve to NE to the right. Present data have shown that ketanserin and prazosin are effective hypotensive agents in 2K - 2C renovascular hypertension in the rat. They also suggest that both hypotensive compounds have an alpha 1-blocking effect, somehow they seem to have some differences in their pattern of pharmacological action.     

Relaxant effect of dopamine on isolated rabbit pulmonary artery

In rabbit pulmonary artery, dopamine (10(-11)-10(-5) M) produced a concentration-dependent relaxation of the arterial strips contracted with prostaglandin F2 alpha (PGF2 alpha) in the presence of prazosin (10(-6) M), yohimbine (10(-6) M), propranolol (10(-6) M), and methysergide (10(-6) M). SKF38393, an agonist for D1 or DA1 dopamine receptor, mimicked partially the concentration-response curve for dopamine, whereas LY171555 and apomorphine did not. The order of potency of dopamine antagonists on the inhibitory effect was: cis-flupenthixol greater than bulbo-capnine greater than metoclopramide greater than haloperidol. Sulpiride was inactive. Cis-flupenthixol did not block the relaxation induced by acetylcholine, adenosine, and papaverine. In the arterial strips of the rabbits pretreated with 6-hydroxydopamine, the concentration-response curve for dopamine was similar to that in non-treated rabbits. Thus it is concluded that a specific dopamine receptor is located on the postsynaptic muscle membrane of the rabbit pulmonary artery.     

Propranolol withdrawal-induced supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit

The present study was undertaken to determine if chronic administration and abrupt withdrawal of propranolol would induce supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit. Eight rabbits were pretreated with propranolol (40 mg/kg/day, i.p.) for one week. Dose-response curves were obtained one week prior to pretreatment and 24, 48, 72 and 168 hours after the last dose of propranolol. Isoproterenol (0.01 to 10 ωg/kg) was injected via the marginal ear vein and heart rate was monitored continuously by recording the ECG. During the withdrawal period no significant rebound increase in basal heart rate was noted. Geometric mean ED₅₀ values and their corresponding 95% confidence intervals were calculated and used as an index of potency. Twenty-four hours after withdrawal of propranolol pretreatment, the ED₅₀ value for isoproterenol was significantly greater than control indicating continued surmountable β-adrenoceptor blockade. In contrast, 72 hours after withdrawal, the geometric mean ED₅₀ value was significantly less than control. The maximum chronotropic response to isoproterenol was not found to be different from control at any time during the withdrawal period indicating that a change in responsiveness was not induced. These data are interpreted as evidence of a true supersensitivity to the chronotropic effects of isoproterenol in the conscious rabbit following propranolol withdrawal.     

静脉桥狭窄家兔动物模型的建立

目的建立冠心病冠状动脉旁路移植术后移植静脉桥狭窄的动物模型。方法取3.0～3.5 kg普通新西兰兔8只,取同侧颈外静脉与颈总动脉进行端端吻合,吻合时采用间断缝合的方法。结果术后2周、4周取下静脉桥及对侧颈外静脉,光镜下见静脉桥新生内膜形成,中膜增厚,弹力纤维减少;胶原纤维不均匀性增厚。结论本模型能反映冠状动脉旁路移植术后静脉桥狭窄的情况,可满意模拟人冠状动脉旁路移植术后大隐静脉桥的病理变化。     

Characterization of alpha-adrenoceptor subtypes by [3H]prazosin and [3H]rauwolscine binding to canine venous smooth muscle membranes

Postsynaptic alpha-adrenoceptor subtypes were studied using [3H]prazosin and [3H]rauwolscine binding to plasmalemma-enriched microsomal fractions isolated from dog saphenous veins and mesenteric veins. Both radioligands showed saturable binding consistent with the presence of a single homogeneous binding site in each case, based on Scatchard analysis. The Kd values of [3H]prazosin and [3H]rauwolscine, calculated from kinetic studies were similar to those from equilibrium binding data in both venous muscle membranes. The microsomal membranes of dog saphenous vein and mesenteric vein contained about a fourfold higher density of the high affinity [3H]rauwolscine binding sites than those for [3H]prazosin binding. In competition studies, IC50 values for displacement of rauwolscine or prazosin suggested that the sites of interaction for the antagonists prazosin and rauwolscine were independent. Phenylephrine, a functionally selective alpha-adrenoceptor agonist, competed with a similar IC50 value for the specific binding sites of [3H]prazosin and [3H]rauwolscine; but B-HT 920, a functionally selective alpha 2-adrenoceptor agonist, competed for [3H]rauwolscine and [3H]prazosin binding with distinctly different IC50 values. Our data show the existence of two populations of alpha-adrenoceptor antagonist binding sites in the plasma membranes of dog saphenous vein and mesenteric vein, and raise the question whether agonist selectively depends on different affinities or on differential efficacies at one or two sites.     

Differences in vascular responses to vasoactive agents of basilar artery and aorta from rabbits with alloxan-induced diabetes.

Responses of the basilar artery and aorta to vasoactive agents in alloxan-induced diabetic and age-matched control rabbits were examined. There were no significant differences in the reactivity of the basilar artery to norepinephrine (NE), 5-hydroxytryptamine (5-HT), and K+ between age-matched control and diabetic rabbits. The maximal contraction of the aorta with endothelium in response to NE was significantly enhanced in the case of the aorta from diabetic rabbits. Pretreatment with 10(-6) M methylene blue or removal of the endothelium enhanced the contractile response of aorta to NE from control rabbits and, after such treatment, the concentration-response curve to NE was almost identical to that of aorta from diabetic rabbits. Basal levels of cyclic GMP but not cyclic AMP in the diabetic aorta with endothelium were significantly lower than those in the control aorta with endothelium. These results demonstrate that the cerebral artery is resistant to diabetes mellitus within 10 weeks as compared with the peripheral artery. The enhancement in the contractile response of aorta to NE in diabetic rabbits is due to the attenuation of the spontaneous release of endothelium-derived relaxing factor, through an impairment of the function of endothelial cells.     

Increased sympathetic venoconstriction and reactivity to norepinephrine in mesenteric veins in anesthetized DOCA-salt hypertensive rats

Increased sympathetic nervous activity (SNA) elevates venomotor tone in deoxycorticosterone acetate (DOCA)-salt hypertension. We studied the mechanisms by which the SNA increases venomotor tone in DOCA-salt hypertension by making in situ intracellular recordings of venous smooth muscle cell (VSMC) membrane potential (E(m)) and measurement of outside diameter (OD) in mesenteric veins (MV) and mesenteric arteries (MA) of anesthetized rats. We also studied norepinephrine (NE)- and endothelin-1 (ET-1)-induced increases in MA or MV perfusion pressure (PP) in vitro. E(m) in DOCA-salt MV was depolarized compared with sham MV. Prazosin hyperpolarized VSMC E(m) in DOCA-salt but not in sham MV. NE concentration-response curves (CRCs) for OD decreases in MV from DOCA-salt rats were left-shifted with an increased maximum response (E(max)) compared with sham MV. NE CRCs for OD decreases in MA were right-shifted with reduced E(max) in DOCA-salt compared with sham rats. ET-1 CRCs were similar in DOCA-salt and sham MV but were right-shifted with reduced E(max) in DOCA-salt MA. NE CRCs for MAPP increases were left-shifted without a change in E(max) in DOCA-salt rats. NE did not change MVPP. MAPP and MVPP for ET-1 CRCs were similar in sham and DOCA-salt rats, but E(max) for MAPP was reduced in DOCA-salt rats. Hematoxylin staining revealed hypertrophy in DOCA-salt MA but not in MV. We conclude that there is increased reactivity to NE released from the sympathetic nervous system in DOCA-salt MV that causes VSMC depolarization and increased venomotor tone. In DOCA-salt rats, in vivo ET-1 reactivity is maintained in MV, but reduced in MA.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

动脉壁脑啡肽的含量,存在部位及作用途径

用放射免疫法测得兔动脉的亮氨酸脑啡肽(L-ENK)和甲硫氨酸脑啡肽(M-ENK)样免疫活性物的含量(pg/mg组织湿重)分别为耳动脉38.99±17.29,134.67±8.11;肾动脉31.10±7.76,93.60±18.22,肠系膜动脉25.70 13.60,88.43±18.16。动物经注射交感神经末梢化学切割剂6-OHDA后,这三种动脉中L-ENK样免疫活性物的含量均明显下降(P<0.001);切除颈上神经节使支配耳动脉的交感神经溃变后,或离体耳动脉条受电场刺激后,脑啡肽(ENK)样免疫活性物的含量也都显著下降(P<0.001);但注射利血平则无明显影响,用荧光法测定培育动脉条的浴槽液中NE的含量,观察到ENK可抑制电场刺激所引起的NE的释放。结果提示,动脉壁的L-ENK和M-ENK存在于交感神经末梢中,它可因受电场刺激而释放,可能通过激活突触前膜阿片受体,减少交感神经末梢释放NE,从而抑制动脉的收缩活动。     

Rapid modulation of TRH and TRH-like peptide release in rat brain and peripheral tissues by prazosin

Hyperresponsiveness to norepinephrine contributes to post-traumatic stress disorder (PTSD). Prazosin, a brain-active blocker of α₁-adrenoceptors, originally used for the treatment of hypertension, has been reported to alleviate trauma nightmares, sleep disturbance and improve global clinical status in war veterans with PTSD. Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂) may play a role in the pathophysiology and treatment of neuropsychiatric disorders such as major depression, and PTSD (an anxiety disorder). To investigate whether TRH or TRH-like peptides (pGlu-X-Pro-NH₂, where “X” can be any amino acid residue) participate in the therapeutic effects of prazosin, male rats were injected with prazosin and these peptides then measured in brain and endocrine tissues. Prazosin stimulated TRH and TRH-like peptide release in those tissues with high α₁-adrenoceptor levels suggesting that these peptides may play a role in the therapeutic effects of prazosin.     

Comparative effects of endothelin (ET-1) and U46619 on human saphenous vein and gastroepiploic artery,sources of human autologous grafts

The effects of endothelin (ET-1) on smooth muscle contractile activity were investigated and compared in human saphenous vein and gastroepiploic artery, vessels frequently used in revascularization procedures. ET-1 contracted saphenous vein and gastroepiploic artery in a concentration-dependent manner. The peptide produced a greater maximal effect in the vein than in the artery and, in both preparations, ET-1 was less efficacious than U46619, an agent which mimics the actions of thromboxane A₂ at the thromboxane A₂/prostaglandin HZ receptor. The contractile response to ET-1 declined spontaneously at a more rapid rate in the artery than in the vein. The present data indicate that ET-1 has significant contractile activity in both vessels which are used for coronary arterial bypass surgery and suggest that although, a weaker vasoconstrictor than U46619, the peptide could induce vasospasm in both graft vessels.     

Renal adrenergic receptors: localization of [125I]prazosin binding sites along the microdissected rat nephron.

Norepinephrine stimulates renal tubular sodium reabsorption, probably through an alpha 1-adrenoceptor-mediated mechanism. Although the distribution of alpha 1-adrenoceptors in the kidney has been studied with autoradiography, the precise location of these receptors in isolated nephron segments is unclear. Using a microassay we determined the specific binding of [125I]iodoarylazidoprazosin ([125I]prazosin), a high specific radioactivity analog of the selective alpha 1-antagonist prazosin, to microdissected glomeruli and tubule segments. Specific binding of [125I]prazosin (3 nM) in the proximal convoluted tubule was time- and concentration-dependent, saturable, and reversible. In this segment the apparent KD by association and dissociation rate constants of [125I]prazosin binding was 0.47 nM, and the maximum receptor density was approximately 0.19 fmol/mm, or 720 fmol/mg protein. Binding specificity was verified in competition studies with excess (3 microM) unlabeled prazosin and probes for alpha 2- (yohimbine), beta- (propranolol), dopamine1- (SCH23390), and dopamine2- (S-sulpiride) receptors. [125I]Prazosin binding was inhibited significantly only by unlabeled prazosin. Mapping of prazosin binding along the nephron revealed that the highest density was in the proximal convoluted tubule, followed by the proximal straight tubule. Lesser binding was found in the thick ascending limb and in the distal convoluted tubule, whereas in the cortical and outer medullary collecting duct and in glomeruli, binding was not significantly different from zero.(ABSTRACT TRUNCATED AT 250 WORDS)     

The specific activity of retained and released norepinephrine in dog saphenous vein prelabeled with radiolabeled norepinephrine

The specific activities of released and retained norepinephrine (NE) in the isolated superfused dog saphenous vein preparation, prelabeled with [³H]NE, have been determined. Norepinephrine was isolated from extracts of vein superfusate and its concentrations were measured by high pressure liquid chromatography with electrochemical detection. The specific activity of NE in superfusate collected under basal conditions was lower than that of NE in veins after electrical stimulation or in parallel unstimulated veins. However, NE released during electrical stimulation had a specific activity 1.5 to 3 times higher than the NE in the veins. Thus, NE taken up by neuronal uptake in the prelabeling procedure enters a pool from which is preferentially released by electric stimulation. In addition, NE is released from different compartments during basal conditions and during electric stimulation.     

胶原/纤维蛋白止血效果观察

目的研究胶原/纤维蛋白对新西兰兔的止血作用,并与胶原蛋白海绵止血效果作比较。方法选用胶原/纤维蛋白止血贴,对新西兰兔耳部动、静脉出血、耳表创面、股动脉割伤、肝损伤、体表创面进行止血试验,同时与胶原蛋白海绵止血效果作比较,观察其止血时间、失血量、敷料与创面的粘合等情况,并定期观察创面愈合、体内吸收和抗炎情况。结果胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组新西兰兔耳动、静脉、耳表创面、股动脉割伤、标准肝创伤的止血时间与对照组比较,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组新西兰兔耳动、静脉、耳表创面的止血时间与胶原蛋白海绵组,差异显著（P〈0.05）。胶原/纤维蛋白复合止血贴组、胶原蛋白海绵组动物的耳表创面、标准肝创伤失血量与对照组比较,差异显著（P〈0.05）。体内标准肝创伤、体表创伤后期观察,胶原/纤维蛋白复合止血贴与胶原蛋白海绵均能在21d内完全吸收,未见炎症反应。结论胶原/纤维蛋白复合止血贴对新西兰兔耳动脉割伤、耳静脉割伤、耳标创伤、股动脉割伤和标准肝损伤模型都具有明显的止血作用,体表创面伤口恢复良好,体内吸收速度快,具有一定的抗炎作用,而且在新西兰兔耳动脉、耳静脉割伤和耳表创伤的止血效果明显优于胶原蛋白海绵。胶原/纤维蛋白复合止血贴是一种较安全有效的局部止血生物材料。     

Amplification of sumatriptan-induced contraction in rabbit saphenous vein but not in basilar artery

The modulation of serotonin (5-HT(1B/1D)) receptor-induced vascular contractility by histamine and U-46619 was compared in the rabbit basilar artery and saphenous vein. In the saphenous vein, histamine (5 x 10(-7) M) significantly increased the potency (from pEC(50) of 6.0 to 6.8) and efficacy (from 52.3% to 88.2%) of sumatriptan. Likewise, U-46619 (5 x 10(-9) M) also increased the potency (from pEC(50) of 5.9 to 6.6) and efficacy (from 51.8% to 92.1%) of sumatriptan in the saphenous vein. In contrast, equieffective concentrations of histamine (10(-7) M) and U-46619 (3 x 10(-9) M) failed to amplify contraction to sumatriptan in the basilar artery. Contraction to sumatriptan was inhibited by nitrendipine (10(-7) M) in the basilar artery but not in the saphenous vein, suggesting that different contractile signaling mechanisms are operating in these tissues. Furthermore, U-46619- and thrombin-induced contractility in the basilar artery were also not amplified by histamine, suggesting that lack of amplification of contraction in the basilar artery was not restricted to sumatriptan but was rather a characteristic of this cerebral vessel. These data suggest that in the in vivo plasma milieu sumatriptan will more markedly contract the peripheral saphenous vein than the basilar artery, a cerebral blood vessel.     

Suramin is a Competitive But Slowly-Equilibrating Antagonist at P2x-Receptors in the Rabbit Ear Artery

Abstract

Antagonist effects of suramin at the P_2x-receptor in the rabbit ear artery were associated with agonist curve depression and a steep Schild plot. Kinetic analysis revealed a problem of slow equilibration and established conditions under which suramin fulfilled all criteria for simple competition.