Determination of agonist affinity for cardiac beta-adrenoceptors during reserpine-induced supersensitivity

The positive inotropic responses to orciprenaline of paced left atria and papillary muscles and the positive chronotropic responses of spontaneously beating right atria of guinea-pigs were recorded. Tissues from guinea-pigs pretreated with reserpine exhibited supersensitivity to beta-adrenoceptor stimulation as demonstrated by the significantly lower EC50 values to orciprenaline. The antagonism of these responses by Ro 03-7894 was characteristic of irreversible beta-adrenoceptor blockade--depression of maximum response and resistant to washout. The dissociation constants (KA) of orciprenaline were determined from the concentration-response curves obtained before and after Ro 03-7894, and no difference was found between tissues from untreated and reserpine-pretreated animals. KA values were also determined from the functional antagonism of the responses by carbachol. The degree of antagonism was less after reserpine pretreatment, but there was no increase in KA value. The reserpine-induced supersensitivity does not therefore appear to be due to an increase in agonist affinity for the cardiac beta-adrenoceptor.     

Selective reserpine-induced supersensitivity of the positive inotropic and chronotropic responses to isoprenaline and salbutamol in guinea-pig isolated atria.

1 Dose-response curves for the positive inotropic and chronotropic responses to isoprenaline were obtained in atria from untreated guinea-pigs and those receiving various reserpine pretreatments. 2 Tension responses were unaffected, whereas rate responses were depressed by the lowest dose of reserpine (0.05 mg/kg i.p. at 24 hours). 3 With larger 24 h doses and a 3 day pretreatment, the rate and tension dose-response curves were progressively displaced to the left, indicating supersensitivity which was greater for tension at each pretreatment. 4 No supersensitivity to histamine or Ca2+ could be detected, leading to the conclusion that it was selective for the beta-adrenoceptor agonists possibly at the receptor level. 5 As an indication of the adrenergic neurone depleting effectiveness of each reserpine dosage, preparations were exposed to test doses of beta-phenylethylamine. 6 Salbutamol was a partial agonist in untreated atria, the maximum rate (63.3%) and tension (10.0%) responses being less than those for isoprenaline. In atria from reserpine pretreated animals the supersensitivity was revealed as an increase of this maximum compared with isoprenaline. 7 The significance of this observation in relation to the possible mechanism of the supersensitivity is discussed.     

Beta-adrenoceptor sensitivity of brown and white adipocytes after chronic pretreatment of rats with reserpine

The effect of pretreatment with reserpine (1.0 mg/kg i.p. daily for 7 days) on beta-adrenoceptor-mediated responses has been measured in epididymal white and interscapular brown adipocytes, left atria and vas deferens of rats in order to investigate the classification of the receptors and whether they are innervated. Lipolysis was measured in adipocytes, and from the same rats, the beta 1-adrenoceptor-mediated positive inotropic responses of isolated paced left atria and beta 2-adrenoceptor-mediated inhibition of field stimulation-induced contraction of the vas deferens were examined. The agonists used were isoprenaline, oxyfedrine (atria only) and (except in brown adipocytes) ritodrine, which was a partial agonist in white adipocytes, atria and vas deferens. Atria and brown adipocytes exhibited beta-adrenoceptor supersensitivity after reserpine pretreatment, whereas vas deferens and white adipocytes did not. Reserpine-induced reductions in food intake and body weight did not appear to influence beta-adrenoceptor-mediated lipolysis, since restriction of the diet equivalent to that of reserpine-treated rats produced no change in white adipocyte sensitivity. Responses mediated via beta 1-, but not beta 2-adrenoceptors, display supersensitivity after chronic depletion of neuronal catecholamines with reserpine and this is evidence for innervation of this receptor subtype. Thus, atrial beta 1-adrenoceptors are assumed to be innervated, whereas vas deferens beta 2-adrenoceptors are not. The present results are consistent with histochemical evidence that brown, but not white, adipocyte beta-adrenoceptors are innervated. However, they are not compatible with conventional receptor classification studies, which suggest that rat brown and white beta-adrenoceptors are similar--either both beta 1 or both atypical.     

北京鸭红细胞膜β肾上腺素受体放射配基结合测定法

本文以[³H]dihydroalprenolol([³H]DHA)为放射配基。对北京鸭红细胞(RBC)膜β受体进行了系统研究。结果表明,[³H]DHA与北京鸭RBC结合具有饱和性,肾上腺素能激动剂与[³H]DHA竞争结合的强弱顺序与它们的生物活性一致,提示结合有结构特异性和立体特异性,[³H]DHA与鸭RBC结合迅速、可逆。可见北京鸭RBC膜存在β受体,用于放射配基结合测定有取材方便,受体较丰富,膜蛋白得率高,[³H]DHA非特异结合低和易于保存等优点,可以代替火鸡RBC。     

Cardiac postjunctional supersensitivity to β-agonists after chronic chemical sympathectomy with 6-hydroxydopamine

Summary The sensitivity to sympathomimetic amines of isolated atria removed from sham-injected and 6-hydroxydopamine-treated (6-OHDA) guinea-pigs was examined in the presence of an extraneuronal uptake blocker and an -adrenoceptor antagonist. Three weeks of pretreatment with 6-OHDA resulted in leftwards shifts of the dose-response curves for the positive chronotropic and inotropic responses of right and left atria to isoprenaline. The responses to the partial agonist salbutamol were also potentiated after 6-OHDA pretreatment, revealed as an increase in the maximum response relative to isoprenaline.The supersensitivity was post-synaptic in origin and independent of changes in disposition or metabolism, since it was observed with agonists immune to neuronal uptake and O-methylation, and in the presence of extraneuronal uptake inhibition by metanephrine. It was also specific for the -adrenoceptor, no supersensitivity to histamine being found. In the right atria, the supersensitivity was partially masked by an opposing depressant effect after 6-OHDA pretreatment which was observed with histamine.Dissociation constants (K _A) for the left atrial inotropic responses to orciprenaline were determined by use of the antagonist Ro 03-7894. Atria from 6-OHDA-pretreated animals were supersensitive to orciprenaline, but the K _A value did not differ from that after sham injection. It could therefore be concluded that the increase in sensitivity was not due to an increase in affinity for the -adrenoceptor.     

Phorbol ester does not mimic,but antagonizes the alpha-adrenoceptor-mediated positive inotropic effect in the rabbit papillary muscle

Summary The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) was used to examine the hypothesis that phosphoinositide turnover is involved in the regulation of myocardial contractility mediated by stimulation of alpha-adrenoceptors in the mammalian cardiac muscle. Exposure of the isolated rabbit papillary muscle electrically driven at a rate of 1 Hz at a temperature of 37°C to TPA in concentrations of 10–1000 nmol/l for 30 min did not affect the basal force of contraction. The concentration-response curve for the positive inotropic effect of (–)-phenylephrine mediated by stimulation of alpha-adrenoceptors in the presence of (±)-bupranolol (100 nmol/1) was shifted to the right and downward by TPA in concentrations of 30–1000 nmol/l, while the effect of (–)-phenylephrine mediated by stimulation of beta-adrenoceptors in the presence of prazosin (100 nmol/l) was not decreased, but slightly enhanced by exposure of the muscle to relatively low concentrations of TPA (10–100 nmol/l). Incubation of the membrane fraction isolated from the rabbit ventricular muscle with TPA in vitro under the same condition as employed in the physiological experiments decreased the specific binding of [³H]prazosin but not that of [³H]CGP-12177, while the non-tumor promoting phorbol ester, PDD, was ineffective. These results indicate that activation of protein kinase C by TPA does not mimic the positive inotropic effect of catecholamines mediated by activation of myocardial alpha-adrenoceptors. on the other hand, the specific interaction of alpha-adrenoceptor-mediated processes with TPA in the rabbit papillary muscle is in line with the view that the facilitation of phosphoinositide turnover and subsequent activation of protein kinase C may play a certain role in the coupling of alpha-adrenoceptor occupation by agonists to the process leading to the positive inotropic action. Send offprint requests to M. Endoh     

Dopamine D2 receptors selectively labeled by a benzamide neuroleptic: [3H]-YM-09151-2

Summary In order to label dopamine D₂ receptors selectively we tritiated the potent benzamide neuroleptic, YM-09151-2 (26.7 Ci/mmol). The binding of [³H]-YM-09151-2 to canine striatal membranes was saturable and specific with a K _D of 57 pmol/l and B _max of 36 pmol/g tissue as determined by Scatchard analysis. The K _D, but not the B _max, of [³H]-YM-09151-2 increased 6-fold in the absence of sodium chloride. [³H]-YM-09151-2 labeled 40% more sites than [³H]-spiperone in the same tissue homogenate. [³H]-YM-09151-2 binding was inhibited by dopaminergic drugs in a concentration and stereoselective manner with the appropriate dopamine D₂ receptor profile. Thus, dopamine agonists inhibited [³H]-YM-09151-2 binding to canine striatal membranes with the following rank order of potency: (–)-N-n-propylnorapomorphine > apomorphine > (±)-6,7-dihydroxy-2-aminotetralin > (+)-N-n-propylnorapomorphine > dopamine > (–)-noradrenaline > serotonin > (–)-isoprenaline. Dopaminergic antagonists competed for [³H]-YM-09151-2 binding with the following order of potency: spiperone > (+)-butaclamol > haloperidol > clebopride > (–)-sulpiride > SCH-23390 > (–)-butaclamol. Furthermore, dopamine agonists recognized 2 states of the receptor labeled by [³H]-YM-09151-2, D ₂ ^high and D ₂ ^low . The D ₂ ^high state of the receptor could be converted to D ₂ ^low by guanine nucleotides and sodium ions as is the case for [³H]-spiperone binding to D₂ receptors. [³H]-YM-09151-2 appears to be a more selective ligand for dopamine D₂ receptors than [³H]-spiperone, since YM-09151-2 displays approximately 9-fold lower affinity than spiperone for cortical serotonergic (S₂) receptors. [³H]-YM-09151-2 may become a useful tool for the selective characterization of dopamine D₂ receptors.Abbreviations used (±)ADTN (±)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene - NPA N-n-propylnorapomorphine - Gpp(NH)p 5-guanylylimidodiphosphate     

Bay K 8644 enhances the outflow of [3H]-noradrenaline and [3H]-DOPEG from isolated rat atria

Summary The effect of Bay K 8644 (a dihydropyridine Ca²⁺-channel activator), was examined on spontaneous and stimulus-evoked release of tritium from isolated rat atria prelabelled with [³H]-noradrenaline. Bay K8644 (3mol/l) significantly increased atrial rate from 206±7 to 259±9 beats·min^–1 (P<0.05) and also tritium outflow (expressed as fractional rate of loss in min^– × 103) from 6.49±0.35 to 8.61±0.74 (P<0.05). Neither the maximal rate nor the overflow of tritium induced by stimulation of sympathetic nerve terminals was changed by the compound. The increase in basal tritium outflow produced by Bay K 8644 was calcium-dependent. However, it could not be antagonized by nitrendipine. The overflow of tritium induced by Bay K 8644 consisted mainly of 3,4-dihydroxyphenylglycol ([³H]-DOPEG), indicating that the compound produces a leakage from the storage vesicles of sympathetic nerve terminals of the isolated rat atria.Members of Consejo Nacional de Investigaciones Científicas - Técnicas (CONICET), Argentina Send offprint requests to M. C. Camilión de Hurtado at the above address     

Quantitative [3H] dipyridamole autoradiography: evidence for adenosine transporter heterogeneity in guinea pig brain

Summary [³H] Dipyridamole binding in guinea pig brain slices has been characterized. Binding of [³H] dipyridamole to guinea pig forebrian slices was found to be rapid, reversible and saturable. Saturation experiments revealed a class of high affinity binding sites with a B _max value of 592 ± 118 fmol/mg protein and K _d value of 10.8 nM ± 2.1 nM in the analysed concentration range. In competition experiments, the adenosine transport inhibitors hexobendine and dipyridamole itself were the most potent displacers (inhibition constants of 4.6 nM ± 1 nM and 11.5 nM ± 3 nM) with pseudo-Hill coefficients close to 1. Competition curves with nitrobenzylthioinosine, another adenosine transport inhibitor, however, showed a biphasic profile with a pseudo-Hill coefficient of 0.33 ± 0.04. Just 42% ± 4% of [³H] dipyridamole binding were inhibited by nanomolar concentrations of nitrobenzylthioinosine and only micromolar concentrations displaced the remainder. Subsequent quantitative autoradiography demonstrated regional differences in the inhibition of [³H] dipyridamole binding by submicromolar concentrations of nitrobenzylthioinosine. While in cortical areas of cerebrum and cerebellum 500 nM nitrobenzylthioinosine displaced binding of [³H] dipyridamole to only about one-third of its sites (in the Purkinje cell layer less than 10%), it showed similar potency as dipyridamole in various areas of the brainstem and hypothalamus. This biphasic and regionally heterogenous interaction of nitrobenzylthioinosine with [³H] dipyridamole binding sites in guinea pig brain slices strongly suggests heterogeneity of adenosine transporters.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Pharmacological comparison of the sigma recognition site labelled by [3H]haloperidol in human and rat cerebellum

Summary The radioligand binding characteristics of [³H]haloperidol (in the presence of spiperone, 25 nmolL^–1) were investigated in rat and human cerebellar membranes.In both rat and human cerebellar membrane preparations saturation studies with [³H]haloperidol (non-specific binding defined by pentazocine, 10 molL^–1) demonstrated high affinity saturable specific binding to a homogenous population of binding sites (rat, Bmax 6693 ± 1242 fmol mg^–1 protein, pK_D 8.33 ± 0.08; human, Bmax 2550 ± 437 fmol mg^–1 protein, pK_D 8.59 ± 0.11; mean ± SEM, n = 3–6). Competition studies employing a wide range of structurally diverse competing compounds displayed that the [³H]haloperidol binding site was pharmacologically similar in both preparations and comparable to sigma recognition sites previously identified in various tissues originating from different species. In addition, with reference to the potential subtypes of sigma recognition sites, the labelling of these sites by low nanomolar concentrations of [³H]haloperidol provides evidence that they belong to the sigma-1 recognition site subtype.The present findings suggest that the pharmacology of the rat and human cerebellar sigma recognition site are directly comparable and provides further supporting evidence towards the use of [³H]haloperidol radioligand binding studies in the rat to detect sigma receptor ligands with potential therapeutic activity. Send offprint requests to: N.M. Barnes at the above address     

Vanilloid receptors in the urinary bladder: regional distribution,localization on sensory nerves,and species-related differences

Summary Using selective surgical ablations we have investigated the localization of vanilloid receptors (specific [³H]resiniferatoxin binding sites) on terminals of the pelvic, hypogastric, and pudendal nerves in the rat urinary bladder. Pelvic and hypogastric nerve resections resulted in 90%6 and 25% loss of specific [³H]resiniferatoxin (RTX) binding sites, respectively, whilst pudendic nerve resection had no measurable effect on the binding. In control animals, the density of vanilloid receptors was 1.7-fold higher in the neck than in the dome of the urinary bladder; the B_max values were 57±8 and 34±7 fmol/mg protein, respectively. The binding characteristics of the vanilloid receptor were similar in the urinary bladder of the rat and mouse: K_d values were 87±15 and 61±11 pM, B_max values were 37±2 and 60±10 fmol/mg protein, respectively. In contrast to the findings for the rat and mouse, in the urinary bladder of the guinea pig and the hamster the low level of specific [³H]RTX binding prevented the detailed characterization of vanilloid receptors. Nonetheless, at a fixed (60 pM) concentration of [³H]RTX, specific binding both in the guinea pig and hamster urinary bladder was approximately 20% of that in the rat urinary bladder. In the urinary bladder of newborn rats, as in adults, a single class of specific [³H]RTX binding sites was found which bound RTX with an affinity of 110±20 pM and with a maximal binding capacity of 30±5 fmol/mg protein. We conclude that, in accord with the physiological findings, the majority of vanilloid receptors are located on terminals of the pelvic nerve in the rat urinary bladder with higher receptor density in the bladder neck as compared to the bladder dome. Whereas the comparably high density of vanilloid receptors in the rat and mouse urinary bladder and the low receptor density in the hamster are mirrored by the in vivo vanilloid-sensitivity of these species, the low level of vanilloid receptors in the urinary bladder of the guinea pig contrasts to the marked sensitivity of this species to capsaicin. We conclude that the level of vanilloid receptors is an important but not exclusive determinant of vanilloid-sensitivity.Correspondence to A. Szallasi at the above address     

A pharmacological comparison of [3H]-granisetron binding sites in brain and peripheral tissues of the mouse

The affinities of a range of structurally diverse 5-HT₃ receptor agonists and antagonists for [³H]-granisetron binding sites have been measured in membrane homogenates prepared from central and peripheral tissues of the mouse. By comparing the affinities of compounds across these tissues, the question of whether intea-species 5-HT₃ receptor subtypes exist in the mouse has been addressed.In entorhinal cortex and brainstem, [³H]-granisetron bound to a single high affinity saturable binding site (K_d 0.47 ± 0.14 and 0.60 ± 0.05 nM; B _max 20 ± 6 and 7 ± 2 fmol (mg protein)^–1 respectively; mean ±SEM; n = 3). In distal and proximal colon, the specific binding of [³H]-granisetron was best fitted to a 2-site model. K_d values obtained for the high affinity site were similar to those obtained in brain tissue (distal colon: 0.47 ± 0.09 nM, n = 4; proximal colon: 0.39 ± 0.09 nM, n = 4). In salivary gland, 2-sites were evident in 2 out of 4 experiments. The K_d value (calculated from the high affinity site in the 2-site model) was approximately 10-fold less than in brain or colon (3.3 ± 1.1 nM, n = 4). B _max values were 7 ± 2, 4 ± 1 and 71 ± 16 fmol (mg protein)^–1 for distal colon, proximal colon and salivary gland respectively. For all tissues the estimated affinity of the low affinity site was variable, and B _max values could not be reliably calculated.Extensive comparative studies performed with 17 different 5-HT₃ receptor agonists and antagonists in the five tissues did not reveal differences in affinity for any compound between the entorhinal cortex and the brainstem nor between the two regions of the colon. However, MDL72222, R-zacopride, d-tubocurarine, and GR80284 apparently had significantly lower affinity for colon than brain binding sites. Also, MDL72222, 2-methyl-5-HT, GR80284, 1-(m-chlorophenyl)-biguanide, metoclopramide, and granisetron had significantly lower affinity for the salivary gland binding sites than the brain binding sites. In an attempt to replicate these observations, we conducted a second study using the compounds which had shown the largest inter-tissue differences in affinity keeping as many variables as possible constant. Simultaneous comparative assays on entorhinal cortex, colon and salivary gland homogenates taken from the same mice showed that the differences that were apparent in the initial comparative study were not maintained. In conclusion, we can find no clear evidence for the existence of tissue-specific subtypes of the 5-HT₃ high affinity binding site for [³H]-granisetron in the mouse in the tissues tested. However, a low affinity binding site for [³H]-granisetron was detected in peripheral tissues.     

Affinities of full agonists for cardiac β-adrenoceptors calculated by use of in vitro desensitization

Summary Positive chronotropic and inotropic responses of guinea-pig isolated spontaneously beating right atria and paced left atria to -adrenoceptor agonists were recorded. Cumulative concentration-response curves for the rate and tension responses to isoprenaline, orciprenaline, terbutaline and fenoterol were obtained before incubation of the tissues with isoprenaline (10^–6 M), the tissues were then washed during 1 h to remove isoprenaline before constructing a post-incubation curve to the same agonist. Incubation with isoprenaline for 4 h caused parallel rightwards shifts to the curves, but extending the incubation to 8 h caused additional depression of the rate (85.2 ± 5.4%) and tension (68.9 ± 2.3%) maxima. Incubation with isoprenaline for 4 h only shifted the curves for orciprenaline to the right but depressed the post-incubation maximum rate and tension responses to terbutaline (74.8 ± 1.5 and 33.8 ± 2.5%) and fenoterol (85.6 ± 5.5 and 76.0 ± 5.1 %). Thus incubation with isoprenaline for 4 h induced desensitization of the cardiac -adrenoceptor which was revealed as a loss in sensitivity to both isoprenaline and other -adrenoceptor agonists. The reduced maxima were attributed to a loss of -adrenoceptors and exhaustion of the receptor reserve. Those experiments displaying the reduced maxima were used for calculation of dissociation constants (K_A) values by the method of Furchgott (1966) for irreversible antagonism. The values for the right atria were 57 (19–170)nM, 29 (8.4–100)M and 13 (1.9–8.4)M and for the left atria, 89 (15–510)nM, 25 (9.9–64)M and 18 (7.7–42)M for isoprenaline (8h incubation), terbutaline and fenoterol respectively. Each value was significantly greater than the corresponding EC₅₀ value and calculation of the fractional receptor occupancies revealed a substantial receptor reserve in all cases except terbutaline on left atria. Calculation of relative efficacies (e_r) showed greater values for fenoterol (6.7 and 2.9) than isoprenaline in spite of its weaker potency and affinity. Finally, the K_A values were not significantly different for the rate and tension responses of each agonist, although each was rate selective. This indicates that the -adrenoceptors mediating these responses are identical and that the rate selectivity is due to better occupancy-response coupling. The validity of the method is discussed with regard to criticism of the null method in general and by comparison with affinity values from binding data.Send offprint requests to K. J. Broadley at the above address     

Stimulus-evoked release of tritiated monoamines from rat periaqueductal gray slices in vitro and its receptor-mediated modulation

Summary The periaqueductal gray is a brain region of considerable interest. It is innervated by monoamine-containing neurons as well as by a variety of peptidergic fiber systems, and it participates in the regulation of various functions. Virtually nothing is known about monoamine release in the periaqueductal gray and its receptor-mediated modulation. We therefore studied the release of radioactivity from periaqueductal gray slices preloaded with tritriated monoamines, using an in vitro superfusion method.The release of radioactivity from superfused periaqueductal gray slices after preloading of the tissue with [³H]noradrenaline increased upon electrical stimulation in a frequency-dependent manner. The stimulus-evoked release of radioactivity was Ca²⁺-dependent. Clonidine reduced and yohimbine enhanced the release. The inhibition curve for the effect of clonidine was shifted to the right in the presence of 10^–6 M yohimbine. While phenylephrine, isoprenaline, SK&F 38393, quinpirole, carbachol, [Arg⁸]vasopressin, -MSH and ACTH-(1-24), at a concentration of 10^–6 M, did not influence the electrically evoked release of radioactivity, [Leu⁵]enkephalin reduced it. The selective -opioid receptor agonists [d-Ala²,NMePhe⁴,Gly-ol⁵]enkephalin and [d-Arg²,Lys⁴]-dermorphin-(1–4)-amide reduced the release of radioactivity, whereas the selective opioid receptor agonist [d-Pen²,d-Pen⁵]enkephalin and the selective K opioid receptor agonist U-69593 had no effect. In the presence of naloxone, which by itself had no effect on the release of radioactivity, the effect of [d-Arg²,Lys⁴]dermorphin-(1–4)-amide was abolished. These results show that the release of noradrenaline from periaqueductal gray slices is via a Ca²⁺-dependent. exocytotic process, and that it is modulated through ₂-adrenoceptors as well as via -opioid receptors. Though the overflow of radioactivity from slices preloaded with [3H]dopamine in the presence of desipramine was measurable, there are reasons to assume that we are dealing here with the release of tritiated catecholamines from a population of nerve endings consisting of noradrenergic and dopaminergic terminals.The release of radioactivity from periaqueductal gray slices preloaded with [³H]5-hydroxytryptamine upon elevation of the K⁺ concentration in the superfusion medium was much more pronounced than that induced by electrical stimulation. The K⁺-evoked release of radioactivity was almost completely abolished in the absence of Cat²⁺; showing that the release is via a Ca²⁺-dependent process. 5-Hydrotryptamine reduced the K⁺-evoked release of radioactivity in a concentration-dependent manner.Some of these data were presented at the XIth International Congress of Pharmacology, 1–6 July 1990, Amsterdam, The Netherlands (Eur J Pharmacol 183:408) Send offprint requests to D. H. G. Versteeg at the above address     

[3H]MDL 100,907: a novel selective 5-HT2A receptor ligand

In studies using standard radioligands, unlabeled MDL 100,907 (R-(+)--(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4-piperidinemethanol) has been shown to have a high degree of selectivity for the 5-HT_2A receptor. The present study was undertaken to investigate the receptor binding characteristics of [³H]MDL 100,907 in rat cortical homogenates. [³H]MDL 100,907 was found to reach equilibrium at 37°C after 15 min. Saturation experiments indicated binding to a single site with a K_D of 0.56 nM, Hill slope of 1.15, and a B_max of 512 fmol/mg protein. In parallel experiments with the standard 5-HT_2A receptor radioligand, [³H]ketanserin, with prazosin added to block ₁ receptors, a similar Hill slope and B_max was noted but a two-fold higher K_D was found. In competition binding studies using 0.5 nM [³H]MDL 100,907, some 19 standard ligands to various receptors including the 5HT_1A, D₂, ₁, and receptors resulted in estimated K_I values that were consistent with [³H]MDL 100,907 selectively binding to the 5-HT_2A receptor. A comparison of the K_I values for 17 standard 5-HT_2A receptor agonists and antagonists displacing [³H]MDL 100,907 versus [³H]ketanserin resulted in a highly significant linear correlation (R² = 0.96, P<0.001). Taken together these results suggest that [³H]MDL 100,907 is binding to the 5-HT_2A receptor with a sub-nanomolar affinity without the use of secondary blocking agents.     

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

Summary The influence of the alpha_lb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha₁-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10^–5 mol/l chlorethylclonidine decreased the specific binding of [³H]prazosin (at a saturating concentration of 10^–9 mol/l) from the control value of 11.27±0.48 to 4.18±1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [³H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 × 10^–7 mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10^–7–10^–5 mol/l) and abolished by 10^–5 mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 × 10^–6 mol/l. The accumulation of [³H]inositol monophosphate and [³H]inositol trisphosphate induced by 10^–5 mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha₁-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha_1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis. Send offprint requests to M. Endoh at the above address     

可乐定镇痛与中枢Ca2+的关系

用大鼠甩尾法和放射配基结合实验,探讨了可乐定镇痛与中枢Ca²⁺的关系。CaCl₂(1μmol/rat,icv)和EGTA(0.2μmol/rat,icv)分别拮抗和增强可乐定(1mg/kg,sc)的镇痛。戊脉安(0.1μmol/rat,icy)对可乐定(1 mg/kg,sc)镇痛无明显影响,但可部分翻转CaCl₂对可乐定镇痛的拮抗。CaCl₂(1×10^-3mol)对[³H]-可乐定结合无明显抑制。结果表明可乐定镇痛与脑室周围组织中Ca²⁺浓度变化密切相关,Ca²⁺至少部分需经对戊脉安敏感的钙通道进入细胞内方可拮抗可乐定镇痛。推沦:可乐定镇痛与神经元内Ca²⁺有关。     

Short-term lithium administration to healthy volunteers produces long-lasting pronounced changes in platelet serotonin uptake but not imipramine binding

Platelet [³H]-5HT uptake, [³H]-imipramine binding and endogenous 5HT levels were measured in healthy volunteers during short-term (20 days) administration of lithium, and following its withdrawal. The V _max of [³H]-5HT uptake was significantly decreased during lithium treatment. Following lithium withdrawal, platelet [³H]-5HT uptake (V _max) remained decreased and was followed by a pronounced rebound effect in some of the subjects for up to 3 months. The affinity constant (K _m) of [³H-5HT uptake was not modified. Binding of tritiated imipramine during the same period and platelet 5HT levels measured till 14 days after withdrawal was not affected by lithium treatment. As lithium is devoid of in vitro effects on both 5HT uptake and imipramine binding, it is concluded that the effects of lithium on the 5HT transporter do not reflect a direct effect on the transporter complex. Our results indicate that lithium-induced changes at the level of 5HT uptake in platelets are not correlated with concomitant variations in platelet 5HT content and can be dissociated from modifications at the level of imipramine binding sites within the macromolecular complex of the 5HT transporter. Moreover, platelet 5HT uptake is apparently modulated by lithium, with a similar pattern in healthy volunteers and in manic-depressive patients.     

β-Adrenoceptor ligand binding and supersensitivity to isoprenaline of ventricular muscle after chronic reserpine pretreatment

1. Isolated papillary muscles from guinea-pig hearts were paced at a constant frequency and isometric contractions reported. 2. Guinea-pigs were either untreated or pretreated with reserpine. Three pretreatment schedules were used; a) 0.5 mg kg-1 i.p. at 24 h, b) 5.0 mg kg-1 at 72 h and 3.0 mg kg-1 at 48 and 24 h, or c) 0.1 mg kg-1 daily for 7 days. 3. Cumulative concentration-response curves for the isoprenaline-induced increases in tension were obtained. The geometric mean EC50 values after the 3 and 7 day reserpine pretreatment schedules were significantly (P less than 0.05) less than for untreated guinea-pigs indicating a supersensitivity. 4. EC50 values for the positive inotropic responses to histamine and calcium in papillary muscles from reserpine-pretreated guinea-pigs did not differ significantly (P less than 0.05) from those from untreated animals. This suggests that the supersensitivity to isoprenaline is beta-adrenoceptor specific. 5. Membrane fractions were prepared from the ventricles of the untreated and reserpine-pretreated guinea-pigs from which papillary muscles had been removed. Binding of [3H]-dihydroalprenolol ([3H]-DHA) to beta-adrenoceptors of these membranes was determined. Equilibrium dissociation constants (KD) and total numbers of binding sites (Bmax) were determined by Scatchard analysis of the saturation curves for [3H]-DHA binding. 6. There was no increase in affinity (fall in KD value) or change in the total number of binding sites associated with reserpine-induced supersensitivity. The equilibrium inhibition constant (Ki) for the displacement of [3H]-DHA binding by isoprenaline was also identical in membranes from untreated and reserpine-pretreated animals. Thus reserpine-induced supersensitivity to isoprenaline does not appear to involve a change in affinity for the beta-adrenoceptor or in receptor numbers as determined by [3H]-DHA binding.