Search

Background and Purpose: The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells., Experimental Approach: Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment., Key Results: Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325., Conclusions and Implications: The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

1. Rat foetal liver contains large amounts of alpha2-adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. 2. Binding experiments indicated that the density of alpha2-adrenoceptors in the foetal liver (embryonic day 18; 615+/-155 fmol mg(-1) of protein) is 18 fold higher than in newborn or adult (35.2+/-4.3 fmol mg(-1)). A high amount of receptor is also found in the placenta (443+/-53 fmol mg(-1)). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the alpha2B-adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. 3. Isolation of cell fractions from foetal liver showed that alpha2B-adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. 4. In foetal reticulocytes, a low proportion of the receptor population is coupled to G-protein. The alpha2-agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on alpha2B-knockout mice, the current finding strongly suggests a role for alpha2B-adrenoceptor during foetal haematopoiesis in rodents.

1. The effects of desipramine (3 mg kg(-1) i.p.) and clorgyline (1 mg kg(-1) i.p.) on extracellular noradrenaline (NA) in the locus coeruleus (LC) and cingulate cortex were assessed in freely-moving rats by dual-probe microdialysis. Functional activities of alpha(2)-adrenoceptors regulating NA release in the LC and cingulate cortex were determined by systemic (0.3 mg kg(-1) i.p.) or local (0.1 - 100 microM) clonidine administration. 2. Extracellular NA was increased in the LC and cingulate cortex following acute desipramine but not clorgyline treatment. Systemic clonidine decreased NA similarly in desipramine-, clorgyline-, and saline-treated animals, in both brain areas. 3. Long-term (twice daily, 14 days) but not short-term (twice daily, 7 days) desipramine, and long-term clorgyline (once daily, 21 days) treatments increased NA (3 fold) in cingulate cortex but not in the LC. Following long-term treatments, responses of NA to systemic clonidine were attenuated in the LC and cingulate cortex. 4. Clonidine perfusion by reverse dialysis into the cingulate cortex decreased local NA (-55 +/- 9%). The effect was attenuated by long-term desipramine (-31 +/- 9%) and clorgyline (-10 +/- 2%) treatments. 5. Clonidine perfusion by reverse dialysis into the LC decreased NA in the LC (-89 +/- 2%) and in cingulate cortex (-52 +/- 12%). This effect was attenuated in the LC following long-term desipramine (-72 +/- 4%) and clorgyline (-62 +/- 12%) treatments but it was not modified in the cingulate cortex (-57 +/- 10% and -68 +/- 6%, respectively). 6. These findings demonstrate that chronic desipramine or clorgyline treatments increase NA in noradrenergic terminal areas and desensitize alpha(2)-adrenoceptors modulating local NA release at somatodendritic and terminal levels. However, somatodendritic alpha(2)-adrenoceptors that control LC firing activity are not desensitized.

We examined the contribution of Ser203 of the human beta2-adrenoceptor (beta2-AR) to the interaction with isoprenaline. The affinity of (-)-isoprenaline was reduced by substitution of an alanine for Ser203, as well as for Ser204 and Ser207. An (-)-isoprenaline derivative with only one hydroxyl group, at the meta-position, showed reduced affinity for wild-type beta2-AR and S207A-beta2-AR and even lower affinities for S203A-beta2-AR and S204A-beta2-AR. By contrast, an (-)-isoprenaline derivative with only a para-hydroxyl group showed reduced affinity for wild-type beta2-AR but the serine to alanine mutations did not cause further decreases. The EC50 value for cyclic AMP generation in response to (-)-isoprenaline was increased, by about 120 fold, for each alanine-substituted beta2-AR mutant. These results suggest that Ser203 of the human beta2-AR is important for both ligand binding and receptor activation.

1. Electrical stimulation evoked release of 3H-noradrenaline (NA) and 14C-acetylcholine (ACh), as well as neurally evoked contractions were measured at various (1-40 Hz, 100 shocks) stimulation frequencies in bladder strips from neurally intact (NI) and spinal cord transected (SCT) rats. 2. The frequency response curves for ACh and NA release were shifted to the left in SCT bladder strips as compared to NI bladder strips. 3. Atropine (1 microM) depressed ACh release in NI bladder strips at high frequency stimulation (10 and 40 Hz) but not at low frequency stimulation (2-5 Hz). However, in SCT bladders, atropine depressed ACh release both at low and high frequencies of stimulation, indicating that muscarinic facilitation occurs at lower frequencies. 4. Atropine depressed the release of NA in NI bladders at only 40 Hz stimulation, but depressed release at all frequencies in SCT bladders. 5. The amplitude of neurally evoked contractions of bladder strips from NI rats was enhanced as the frequency of stimulation was increased from 1 to 40 Hz (80 shocks). The frequency response curve was shifted to the left in SCT bladders. Atropine blocked the neurally evoked contractions in SCT bladder strips to a greater extent than the contractions in NI strips indicating a cholinergic dominance in the SCT bladders. 6. Maximal contractile force of SCT bladder strips evoked by neural stimulation at 20 Hz 10 shocks and 80 shocks was significantly lower than that of NI bladder strips, whereas the release of ACh was significantly higher in SCT than NI bladders indicating a postjunctional defect in the SCT preparations. 7. It is suggested that presynaptic muscarinic facilitatory mechanisms are upregulated in the cholinergic and adrenergic nerve terminals in SCT bladders leading to a larger relative contractile response at lower frequencies of stimulation (2-5 Hz). Thus the hyperreflexic bladder occurring after spinal cord injury may be due in part to an enhancement of transmitter release at bladder postganglionic nerve terminals.

1 In segments of human right atrial appendages and pulmonary arteries preincubated with [3H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [3H]-noradrenaline release was investigated. 2 In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [3H]-noradrenaline release. The inhibition was not affected by blockade of alpha 2-adrenoceptors with 1 microM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 microM; apparent pA2 against aganodine and DTG: 5.55 and 5.21, respectively). 3 In the presence of 1 microM rauwolscine, [3H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 microM idazoxan and 30 microM cirazoline were abolished by 30 microM rauwolscine. 4 In the atrial appendages, the rank order of potency of all guidelines and imidazolines for their inhibitory effect on electrically-evoked [3H]-noradrenaline release in the presence of 1 microM rauwolscine was: aganodine > or = BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline] > DTG > or = clonidine > cirazoline > idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta. 5 When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed alpha 2-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [3H]-noradrenaline release was bell-shaped. In the presence of 1 microM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [3H]-noradrenaline release. 6 In human atrial appendages, non-adrenoceptor [3H]-idazoxan binding sites were identified and characterized. The binding of [3H]-idazoxan was specific, reversible, saturable and of high affinity (KD: 25.4 nM). The specific binding of [3H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model. 7 The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline = idazoxan > BDF 6143>DTG> or = clonidine) is compatible with the pharmacological properties of I2-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue. 8 It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I1 and I2 imidazoline binding sites.

1. In order to clarify one of the mechanisms involved in the analgesic effect of calcitonin, we have tested the in vitro modifications induced by calcitonin on the effect of opioids. 2. The inhibition of the contractions induced by opioids or clonidine, in guinea-pig ileum or in mouse vas deferens, were significantly reduced in tissues incubated with pertussis toxin (PTX). When tissues were incubated with PTX and calcitonin, the inhibitory effect was restored. 3. These results suggest that calcitonin is able to potentiate a non-PTX-sensitive mechanism of transduction and support the possibility of involvement of similar G-proteins in the effects of opioid and alpha 2-adrenoceptor agonists.

1. To investigate the structure-activity relationships of alpha-adrenoceptor agonists for the alpha 1-adrenoceptor subtypes, we have compared the imidazoline class of compounds, oxymetazoline and cirazoline, with the phenethylamine, noradrenaline, in their affinities and also in their intrinsic activities in Chinese hamster ovary (CHO) cells stably expressing the cloned human alpha 1-adrenoceptor subtypes (alpha 1a-, alpha 1b-, and alpha 1d-subtypes). 2. Radioligand binding studies with [125I]-HEAT showed that cirazoline and oxymetazoline had higher affinities at alpha 1a-subtype than at alpha 1b- and alpha 1d-subtypes, while noradrenaline had higher affinity at the alpha 1d-subtype than at alpha 1a- and alpha 1b-subtypes. 3. In functional studies, cirazoline caused transients of cytosolic Ca2+ concentrations ([Ca2+]i response) in a concentration-dependent manner and developed a maximal response similar to that to noradrenaline in CHO cells expressing the alpha 1a-subtype, while it acted as a partial agonist at alpha 1b- and alpha 1d-adrenoceptors. Oxymetazoline, on the other hand, was a weak agonist at alpha 1a-adrenoceptors, and has no intrinsic activity at the other subtypes. 4. Using the phenoxybenzamine inactivation method, the relationships between receptor occupancy and noradrenaline-induced [Ca2+]i response for alpha 1a- and alpha 1d-subtypes were found to be linear, whereas it was moderately hyperbolic for the alpha 1b-subtype, indicating the absence of receptor reserves in CHO cells expressing alpha 1a- and alpha 1d-subtypes while there exists a small receptor reserve for CHO cells expressing the alpha 1b-subtype. 5 In summary, our data obtained in cells exclusively expressing a single receptor subtype support the idea that the relative role of agonist affinity and intrinsic activity may vary depending on the subtype of alphal-adrenoceptor.

1. In the present study we have evaluated whether alpha 2-adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non-catecholamine, clonidine-displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2. Saturation studies carried out in the presence and absence of noradrenaline, 10 mumol 1(-1), revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]-idazoxan and [3H]-RX-821002 were approximately 60-80% greater than those for [3H]-clonidine (62.6 fmol mg-1 protein). Rauwolscine, the selective alpha 2-adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective alpha 1-adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective alpha 1-adrenoceptor prazosin against each ligand was less than 6. 3. Adrenaline, UK-14034, rauwolscine, corynanthine, RX-811059 and prazosin produced concentration-dependent inhibition of binding of all three 3H-ligands. The agonists, adrenaline and UK-14304, were approximately 5 and 10 fold less potent against [3H]-idazoxan and [3H]-RX-821002, respectively, than against [3H]-clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX-811059 and prazosin exhibited a different profile, being approximately 2-3 fold more potent against sites labelled by [3H]-RX-821002 and [3H]-idazoxan compared to sites labelled by [3H]-clonidine. 4. Agmatine and histamine produced a concentration-dependent displacement of [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 binding to bovine cerebral cortex membranes. The pKi values for agmatine and histamine were independent of the 3H-ligand employed, approximately 4.8 and 4.5,respectively.5. Crude methanolic extracts of bovine brain and lung produced a concentration-dependent inhibition of [3H]-clonidine binding to bovine cerebral cortex membranes (>90%). Based on the volume of the extract that caused 50% inhibition of [3H]-clonidine binding, bovine lung contains 3 fold more CDS than bovine brain. Both extracts were at least 5 fold more potent against a2-adrenoceptor sites labelled by[3H]-clonidine than those labelled by [3H]-idazoxan and [3H]-RX-821002.6. All three 3H-ligands label the same population of alpha2-adrenoceptor binding sites on bovine cerebral cortex membranes, but [3H]-clonidine appears to label selectively the 'agonist' state of the sites: for which known agonists, adrenaline and UK-14304, exhibit a higher affinity. Our results indicate that neither agmatine nor histamine can account for the CDS activity present in crude extracts of bovine brain and lung. Moreover, these extracts appear to possess a binding profile similar to that of adrenaline and UK-14304, suggesting that they may possess agonist activity.

1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) approximately adrenaline (2) > noradrenaline (5) > phenylephrine (23) approximately amidephrine (31) > methoxamine (71) >> isoprenaline (1456) approximately dopamine (2210). 5. A high correlative association was shown between the affinity of antagonists obtained functionally in the isolated perfused kidney of rat and pKi values obtained from binding experiments with the cloned bovine alpha 1C-adrenoceptor (R2 = 0.85), native alpha 1A-adrenoceptors in submaxillary gland of rat (R2 = 0.79), and alpha 1A-adrenoceptors from several other tissues of rat (values taken from the literature, R2 = 0.89). 6. The present study demonstrates that the alpha 1A-adrenoceptor is the predominant alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, alpha 1A-adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovine alpha 1 c-adrenoceptor. Thus,definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

1. The aim of this study was to quantitate and compare the turnover of alpha 2-adrenoceptors in the cerebral cortex of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats, and its modulation during chronic treatment with the monoamine oxidase (MAO) inhibitor, clorgyline. 2. In SHR, the specific binding of the agonist [3H]-UK 14304 and of the antagonist [3H]-RX 821002 was significantly reduced in the brain (Bmax 15-19% lower) as compared to that in sex- and age-matched WKY rats. In contrast, no significant changes in the Kd values for both radioligands were found between WKY and SHR rats. Therefore, SHR rats offer a genetic model with a lower density of alpha 2-adrenoceptors in the brain. 3. Chronic treatment (21-35 days) with clorgyline (1 mg kg-1, i.p.) markedly decreased the density of brain alpha 2-adrenoceptors ([3H]-UK 14304 binding) in Sprague-Dawley (Bmax reduced by 50%) and in WKY (Bmax reduced by 30%) rats without any apparent change in the affinity of the radioligand. In contrast, the density of brain alpha 2-adrenoceptors in SHR was not down-regulated by chronic clorgyline treatment. 4. The recovery of [3H]-UK 14304 binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ; 1.6 mg kg-1) (an alkylating agent for the alpha 2-adrenoceptor) was assessed in control and clorgyline-treated (1 mg kg-1; i.p. for 7-21 days) WKY and SHR rats to study the process of alpha 2-adrenoceptor repopulation and to calculate receptor turnover parameters. 5. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain alpha2-adrenoceptors in SHR rats was accelerated (k = 0.141 day-1;t 1/2= 4.9 days; r/k =40 fmol mg-1 protein)compared to that in WKY rats (k = 0.085 day-1; tl/2= 8.1 days; r/k = 54 fmol mg-1 protein) and that the reduced density of cortical alpha2-adrenoceptors (Bmax or r/k values) in SHR was probably due to an abnormal higher receptor degradation (delta k = 66%) and not to a decreased receptor synthesis which in fact showed a slight increase (delta r = 24%).6. Treatment with clorgyline (1 mg kg-1, i.p. for 21 days) accelerated the turnover of brain alpha2-adrenoceptors in WKY rats (k = 0.328 days-1; tl/2= 2.1 days; r/k = 29 fmol mg-1 protein) and the greater increase in receptor degradation (delta k = 286%) over receptor synthesis (delta r = 109%) led to down-regulation of receptor density (r/k = 46% lower). In contrast, treatment with clorgyline did not modify significantly the turnover of brain M2-adrenoceptors in SHR (k = 0.192 days-1; t1/2 = 3.6 days;r/k = 39 fmol mg-1 protein), indicating that in this genetic model of hypertension, the desensitized alpha2-adrenoceptors cannot be further down-regulated by clorgyline treatment and that they lack the expected adaptative increase in receptor synthesis.

1. The alpha 2-adrenoceptors on human platelets and neonatal rat lung were characterized with the agonist and antagonist ligands [3H]-adrenaline and [3H]-RS-15385-197 respectively. A correlation of affinities for 3H-antagonist binding showed the receptors to be of the alpha 2A-(platelet) and alpha 2B-(neonatal rat lung) adrenoceptor subtypes, whereas a correlation of affinities for 3H-agonist binding showed the receptors to have similar characteristics (r = 0.88). 2. NaCl (100 mM) had no effect on [3H]-RS-15385-197 binding in the human platelet, but increased the density of sites labelled with [3H]-RS-15385-197 in neonatal rat lung by 52%. NaCl increased the density of sites labelled by [3H]-adrenaline in neonatal rat lung, but there was a consequent 3.5 fold decrease in affinity. In the human platelet, no specific [3H]-adrenaline binding was observed in the presence of 100 mM NaCl. 3. In the neonatal rat lung, NaCl had no significant effect on the affinity of prazosin for [3H]-RS-15385-197 binding; however, imiloxan affinity was increased 13 fold. The affinity of the catecholamines, adrenaline and noradrenaline was significantly decreased, whereas the imidazolines, oxymetazoline and UK-14,304 were much less affected. The affinity of prazosin and imiloxan for [3H]-adrenaline binding was significantly increased in the presence of 10 and 100 mM NaCl. Conversely, the affinity of adrenaline and noradrenaline was decreased in the presence of NaCl, and there was no change in the affinity of the imidazoline agonists. 4. In the human platelet, NaCl had no effect on the affinity of prazosin for [3H]-RS-15385-197 binding but the affinity of imiloxan was significantly increased. NaCl significantly decreased the affinity of the catecholamines adrenaline and noradrenaline, whereas the affinity of UK-14,304 and oxymatazoline was much less affected. Competition experiments with [3H]-adrenaline in the presence of NaCl in platelets were difficult to characterize as there was no specific binding under these conditions.5. The results show that both the alpha2A- and alpha2B-adrenoceptor subtypes are allosterically regulated by Na+, but only the alpha2B-subtype showed a significant increase in density. Interestingly, there is a differential regulation of imidazoline (unchanged) and catecholamine (decreased affinity) agonist interactions with these subtypes. Na+ may therefore critically affect receptor subtype selectivity of drugs. The implications for receptor subclassification are discussed.

1. Rats received an intraperitoneal injection of 1.6 mg kg-1 N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to achieve an irreversible inactivation of presynaptic release-modulating alpha 2-autoreceptors. Cerebral cortex slices were prepared at different times after the injection (24, 48, 96, 192, 336, 774 h), incubated with [3H]-noradrenaline ([3H]-NA), superfused and stimulated electrically with 4 pulses at 100 Hz (= autoinhibition-free condition). Overflow of radioactivity was used to measure release. Furchgott analysis was used to estimate agonist dissociation constants (KA) and pool size of resynthesized receptors (q). 2. The KA values of the three alpha 2-autoreceptor agonists, bromoxidine (UK-14304), clonidine and noradrenaline (NA) were 187 nM, 72 nM, and 1202 nM, respectively. 3. The release-inhibiting effects of the agonists returned considerably faster than the receptor pool. The calculated half-lives for the recovery of the maximal release-inhibiting effects of bromoxidine, clonidine and NA were 30.7, 63.6 and 20.8 h, respectively, whereas the half-life for the recovery of the receptor pool was 445 h. 4. The data indicate a large receptor reserve at presynaptic alpha 2-autoreceptors for the agonists used and validate the use of EEDQ as a tool for the determination of agonist dissociation constants.

1. In agreement with the literature, correlation of the ability of a series of agonists and antagonists to displace [3H]-rauwolscine binding shows the alpha 2-adrenoceptors of HT29 cells, NG108-15 cells, OK cells and homogenates of rat sublingual gland to represent four distinct subtypes. 2. [3H]-rauwolscine also bound with high affinity (KD = 0.30 +/- 0.10 mM) to a human retinoblastoma cell line (Y79). Specific binding represents 73% of total binding, and a Bmax of 38 +/- 1 fmol mg-1 protein was determined. 3. Correlation of antagonist affinities against [3H]-rauwolscine with corresponding values in the other four tissue sources showed the Y79 cells to resemble most closely the OK cells, the prototype example of an alpha 2C-adrenoceptor, with a correlation coefficient of 0.90 and a regression slope of 1.01 being obtained for 10 antagonists in these two systems. 4. Comparison of KD values for [3H]-rauwolscine also showed a similarity between the OK cells (0.19 +/- 0.07 nM) and Y79 cells. 5. These data suggest that the human retinoblastoma cell line may represent an additional example of the alpha 2C-adrenoceptor subtype.

1. [3H]-prazosin homogeneously labels alpha 1-adrenoceptors in guinea-pig cerebral cortex and rat spleen membranes with dissociation constants of 1.28 and 1.49 x 10(-10) M respectively. 2. Phentolamine and WB 4101 displacement studies show that guinea-pig cerebral cortex contains 30% alpha 1A- and 70% alpha 1B-adrenoceptor subtypes, whereas rat spleen contains a virtually homogeneous alpha 1B-adrenoceptor subtype population. The alpha 1-adrenoceptor population of rat thoracic aorta is predominantly of the alpha 1A-adrenoceptor subtype, and in guinea-pig thoracic aorta it is mainly of the alpha 1B-adrenoceptor subtype. 3. Half of the compounds displacing [3H]-prazosin bound to guinea-pig cerebral cortex membranes display alpha 1A-adrenoceptor selectivity. Among these compounds, WB 4101 and methoxamine are most selective, displaying selectivity ratios of approximately 38 and approximately 26 respectively. 4. The affinity constants of the non-selective compounds for the alpha 1-adrenoceptor in guinea-pig cerebral cortex membranes correlate well with the affinity constants obtained for alpha 1B-adrenoceptors in rat spleen membranes. The affinities of selective compounds for the alpha 1B-adrenoceptor subtype in guinea-pig cerebral cortex correlate very well with their affinity for alpha 1B-adrenoceptor in the rat spleen homogenate. Both regression lines coincide with the line of identity. The affinity constants of selective compounds for the alpha 1A-adrenoceptors in guinea-pig cerebral cortex only apparently correlate with the affinity for either the alpha 1B-adrenoceptors in guinea-pig cerebral cortex or in the rat spleen. Regression analyses indicate a straight line relationship (r2>0.9) between pKEA and Pk1B but the regression lines deviate from the line of identity.

1. The factors underlying the different responsiveness of the prostatic and epididymal portions of rat vas deferens to alpha 1-adrenoceptor stimulation were investigated. 2. The alpha 1-adrenoceptors in membranes of both halves of rat vas deferens were labelled with [3H]-prazosin and the affinities of agonists and antagonists for these receptors were determined. In saturation studies, the Bmax and KD values for [3H]-prazosin in membranes of both portions were the same. 3. In competition studies, the inhibition curves for phentolamine were biphasic and consistent with the presence of both alpha 1a- and alpha 1b-adrenoceptor subtypes. The proportions of binding sites with high and low affinity for phentolamine in both halves of rat vas deferens were similar and in good agreement with the percentages of binding sites for WB-4101 and phentolamine previously reported in the whole rat vas deferens. 4. The phenylethylamines displaced [3H]-prazosin with a shallow inhibition curve. The data are compatible with the assumption of two affinity states for the binding sites. For the imidazoline compounds no such distinct affinity states could be demonstrated. 5. The affinity for, and the relative intrinsic efficacy on postsynaptic alpha 1-adrenoceptors of both portions of rat vas deferens were studied for noradrenaline, phenylephrine and methoxamine by irreversible inactivation of the alpha 1-adrenoceptors by phenoxybenzamine. The parameters for partial agonists were determined by comparing the responses to the partial agonist to those of a full agonist in the same tissue. Homogeneous estimates of the equilibrium dissociation constants (Ka) were obtained, indicating that these agonists bind to the receptors of both tissues in an identical manner. Further, estimation of the intrinsic efficacy of agonists relative to noradrenaline, indicated no differences between the two halves of rat vas deferens. 6. Ka values for agonist activation of the functional alpha 1-adrenoceptors were compared with K, values for agonist inhibition of specific [3H]-prazosin binding. The K. values were well correlated with the low affinity K, values for phenylethylamines in both portions of rat vas deferens, suggesting that the initial event in signal transduction by alpha 1,-adrenoceptors is the binding to the low affinity state of the receptor. 7. There was a non-linear relationship between response and receptor occupancy in both halves of rat vas deferens but the occupancy-response coupling was more efficient in the epididymal than in the prostatic portion. This fact may account for the differences observed in the functional responses.

1. Saturation experiments indicated that [3H]-yohimbine binding was specific, saturable and labelled a single population of sites in rat cerebral cortex (Kd 5.3 +/- 0.9 nM, Bmax 121 +/- 10 fmol mg-1 protein) and human platelets (Kd 0.7 +/- 0.1 nM, Bmax 152 +/- 10 fmol mg-1 protein). 2. The alpha 2-adrenoceptor antagonists, yohimbine, rauwolscine, WY 26703, idazoxan and BDF 6143 displaced [3H]-yohimbine binding to each tissue in a simple manner, with high affinity and Hill slopes close to unity. 3. The alpha 1-adrenoceptor agonist, oxymetazoline and the antagonist prazosin inhibited the binding of [3H]-yohimbine to rat in a complex manner consistent with an interaction at more than one site. However, indoramin and WB 4101 only appeared to interact with one site. In contrast, in human platelets, all antagonists gave rise to monophasic displacement curves with Hill slopes close to unity suggesting a single site of interaction. 4. The 5-hydroxytryptamine (5-HT) receptor ligands, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), RU 24969, and methysergide inhibited the binding of [3H]-yohimbine to rat cortex with high and low affinity, consistent with an interaction with two populations of binding sites. However, inhibition of [3H]-yohimbine binding to human platelets suggested a single site of interaction. The low affinity of 5-HT, 5-carboxyamidotryptamine (5-CT) and dipropyl-5-CT indicated that [3H]-yohimbine was not labelling a 5-HT1-like site in rat cortex. 5. The ability of 8-OH-DPAT, RU 24969 and methysergide in addition to prazosin and oxymetazoline to differentiate [3H]-yohimbine binding provides additional pharmacological evidence for heterogeneity within rat cortical alpha 2-adrenoceptors. However, if the two sites in rat cortex that are differentiated by the 5-HT ligands represent (alpha 2A- and alpha 2B-adrenoceptor subtypes as defined by prazosin and oxymetazoline, then they do not correspond to the population of sites in human platelets. As receptor classification should be linked to affinity of drugs rather than tissue distribution, the current classification of alpha 2-adrenoceptor subtypes does not appear to be satisfactory.

1. The apparent Ki values of (-)-noradrenaline (NA), (+)- and (-)-adrenaline (Ad), phenylephrine and the mono-fluorinated NAs (in position 2, 5 or 6) for alpha 1-adrenoceptors of intact BC3H1 cells labelled with [3H]-prazosin were greatly dependent on the incubation temperature. 2. The EC50 values of these compounds for stimulation of the inositol phosphate (IP) accumulation at 37 degrees C were intermediate between their apparent dissociation constants at 2 degrees C (Ki2 degrees) and at 37 degrees C (Ki37 degrees). 3. The fact that an irreversible blockade of 46% +/- 6% (n = 3) of the [3H]-prazosin binding sites by phenoxybenzamine reduced the maximal IP-formation induced by NA by 57% +/- 5% (n = 3) shows that there is a direct coupling between alpha 1-adrenoceptors and phospholipase C in BC3H1 cells. 4. The Ki37 degrees s of all agonists tested were in the same range (0.1 to 1 mM) and showed no simple correlation with their EC50 values. 5. The Ki2 degrees values for all the agonist correlated linearly with their EC50 values but were about 20-100 times lower than the respective EC50 values (except for the partial agonist methoxamine). In order to explain this difference, we propose that the apparent high affinity in the cold could be due to an [3H]-prazosin-induced alteration of the active site of the alpha 1-adrenoceptor, increasing its apparent affinity for catecholamines.

1 The effects of alpha-adrenoceptor antagonists on contractile responses to noradrenaline and methoxamine were examined in isolated saphenous vein strips of the dog. 2 Phentolamine, labetalol and thymoxamine antagonized responses to methoxamine to a greater extent than responses to noradrenaline. 3 Cocaine (3.0 x 10(-5) mol/l) increased the potency of noradrenaline by about eight fold, but had little or no effect on the potency of methoxamine. 4 In the presence of cocaine (3.0 x 10(-5) mol/l) the potency of the antagonists against noradrenaline was increased such that the pA2 values were then similar to those obtained against methoxamine. 5 It is concluded that the lower potency of phentolamine, labetalol and thymoxamine as antagonists of noradrenaline than of methoxamine is due to the presence of an avid saturable uptake1 process in the saphenous vein.

[3H]-guanfacine (N-amidino-2-(2,6-dichloro 3[3H] phenyl) acetamide hydrochloride; 24.2 Ci/mmol) has been used as a radioligand in homogenates of rat cerebral cortex. Specific binding of [3H]-guanfacine was linear with respect to tissue concentration (2.5-15 mg/ml), saturable and not markedly affected in the pH range 6.5-8.0. Analysis of the saturation of [3H]-guanfacine binding using an iterative least squares fitting procedure gave best fits to a single site model. [3H]-guanfacine binding was of high affinity (Kd 1.77 +/- 0.24 nM; n = 8) to a population of non interacting sites (nH 0.99 +/- 0.02; n = 8) with a density of 118.2 +/- 8.4 fmol/mg protein (n = 8). Highest levels of binding were achieved in cerebral cortex followed by thalamus greater than hypothalamus greater than medulla/pons greater than spinal cord greater than striatum greater than cerebellum. Binding was stereoselective with regard to the (-)-isomer of noradrenaline and the order of potency for displacement of [3H]-guanfacine by agonists was naphazoline greater than clonidine greater than (-)-adrenaline greater than (-)-alpha methylnoradrenaline greater than (-)-noradrenaline greater than (+/-)-alpha-methylnoradrenaline greater than (+)-noradrenaline greater than methoxamine greater than (+)-adrenaline greater than phenylephrine and by antagonists was phentolamine greater than dihydroergocryptine greater than piperoxane greater than yohimbine greater than prazosin greater than labetalol greater than indoramin suggested binding to alpha 2-adrenoceptors. The monovalent cations Na+ and K+ and also guanosine 5'-triphosphate (GTP) produced concentration-dependent inhibition whereas the divalent cations Ca2+, Mg2+, and Mn2+ first enhanced, then inhibited [3H]-guanfacine binding. Na+ (150 mM) or GTP (100 microM) produced marked reductions and Mn2+ (5 mM) marked increases in the number of receptor sites labelled by [3H]-guanfacine. 9 It is concluded that [3H]-guanfacine preferentially labels a high affinity state of the alpha 2- adrenoceptor in homogenates of rat cerebral cortex.