α1B- but not α1A-adrenoceptors mediate inositol phosphate generation

Summary We used novel highly subtype-selective antagonists to study whether _1A- and/or _1B-adrenoceptors mediate the stimulation of inositol phosphate generation by noradrenaline in rat cerebral cortex. Phentolamine (10 M) and prazosin (100 nM) completely abolished the stimulated inositol phosphate generation. The _1A-selective antagonists 5-methyl-urapidil (100 nM) and (+)– and (–)-niguldipine (10 nM) caused only weak inhibition or none at all although these concentrations occupied _1A-adrenoceptors almost completely. In contrast, pretreatment with the irreversible _1B-selective chloroethylclonidine reduced the noradrenaline-stimulated inositol phosphate generation by 76 ± 8%. These data demonstrate that _1B-adrenoceptors couple to inositol phosphate generation; the signal transduction system of _1A-adrenoceptors remains unclear. Send offprint requests to G. Gross at the above address     

Comparison of α1A- and α1B-adrenoceptor coupling to inositol phosphate formation in rat kidney

We have compared the coupling mechanisms of rat renal _1A- and _1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where _1B-adrenoceptors had been inactivated by treatment with 10 mol/l chloroethylclonidine for 30 min at 37°C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca²⁺ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular G_i by 16–20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via _1B-like adrenoceptors may involve the influx of extracellular Ca²⁺ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast _1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca²⁺ or of G_i and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of ₁-adrenoceptor subtypes may depend on their cellular environment.     

Characterisation of α1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.     

Different α1-adrenoceptor-induced inositol phosphate formation in the two portions of rat vas deferens

The two portions of rat vas deferens differed in the postjunctional sensitivity to noradrenaline. Alpha1-adrenoceptor-linked phosphoinositide breakdown was analysed in this tissue. The noradrenaline-induced [3H]inositol phosphate accumulation was similar in both ends although the pEC50 was higher in the epididymal (5.97+/-0.07) than in the prostatic (5.47+/-0.15, P<0.01) portion. [3H]Prazosin showed similar density of binding sites in both portions. Tissue pretreated with pertussis toxin did not change [3H]inositol phosphate accumulation. Finally, Western blot analysis indicated a smaller concentration of Gq/11 protein in the prostatic half (-29+/-5%, P<0.01). These results suggest that the different sensitivity to noradrenaline could be due to the higher availability of this sort of G protein in the epididymal portion.     

α1- and α2-Adrenoceptors in rat cerebral cortex: Effect of frontal lobotomy

Summary Surgical noradrenergic denervation of the cortex via frontal lobotomy was used to destroy the noradrenergic nerve endings and thus give some insight into the distribution of alpha-adrenoceptors. Frontal lobotomy caused a reduction in noradrenaline content in rat cerebral cortex (2.1±0.4 ng/mg protein for lesioned side, 6.0±0.3 mg/mg protein for nonlesioned side), indicating an effective noradrenergic denervation. The differences in ³H-clonidine and ³H-prazosin binding observed following surgery were a significant decrease in the number of ₂-adrenoceptors (115.0±4.5 to 91.7±3.2 fmol/mg protein, n=7, P<0.001) and a smaller but significant increase in the number of ₁-adrenoceptors (119.7±2.5 to 131.6±5.4 fmol/mg protein, n=7, P<0.05) in the lesioned cortex. Results of this study indicate that ₂-adrenoceptors located on presynaptic noradrenergic terminals represent only a small proportion of the total ₂-adrenoceptors in rat cerebral cortex.     

Presynaptic α2-autoreceptors in brain cortex: α2D in the rat and α2A in the rabbit

Summary Presynaptic ₂-autoreceptors in rat and rabbit brain cortex were compared by means of antagonists and agonists. Brain cortex slices were preincubated with [³H]-noradrenaline and then superfused and stimulated by 3 (rat) or 4 (rabbit) pulses at a frequency of 100 Hz.The ₂-adrenoceptor agonist bromoxidine (UK 14 304) reduced the electrically evoked overflow of tritium with EC₅₀ values of 4.5 nmol/l in the rat and 0.7 nmol/l in the rabbit. The antagonists phentolamine, 2-[2H-(1-methyl-1,3-dihydroisoindole)methyl]-4,5-dihydroimidazole (BRL 44408), rauwolscine, 1,2-dimethyl-2,3,9,13b-tetrahydro-1H-dibenzo(c,f)imidazo(1,5-a)azepine (BRL 41992), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), 6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H-2,3,4, 5-tetrahydro-3-benzazepine (SKF 104078), imiloxan, prazosin and corynanthine did not per se increase the evoked overflow of tritium but shifted the concentration-inhibition curve of bromoxidine to the right in a manner compatible with competitive antagonism. Up to 4 concentrations of each antagonist were used to determine its dissociation constant K_D. The K_D values correlated only weakly between the rat and the rabbit. Dissociation constants K_A of bromoxidine were calculated from equieffective concentrations in unpretreated brain slices and slices in which part of the ₂-adrenoceptors had been irreversibly blocked by phenoxybenzamine. The K_A value was 123 nmol/l in the rat and 7.2 nmol/l in the rabbit.The results confirm the species difference between rat and rabbit brain presynaptic ₂-autoreceptors. Comparison with data from the literature indicates that the rat brain autoreceptors can be equated with the _2D subtype as defined by radioligand binding, whereas the rabbit brain autoreceptors conform to the _2A subtype. For example, the antagonist affinities for the rat autoreceptors correlate with their binding affinities for the gene product of ₂-RG20, the putative rat _2D-adrenoceptor gene (r = 0.97; P<0.01), but not with their binding affinities for the gene product of ₂-C10, the putative human _2A-adrenoceptor gene. Conversely, the rabbit autoreceptors correlate with the ₂-C10 (r = 0.98; P<0.001) but not with the ₂-RG20 gene product. Since presynaptic ₂-autoreceptors are also _2D in rat submaxillary gland and perhaps vas deferens and _2A in rabbit pulmonary artery, the possibility arises that the majority of ₂-autoreceptors generally are _2D in the rat and _2A in the rabbit. Moreover, receptors of the _2A/D group generally may be the main mammalian ₂-autoreceptors.Correspondence to: N. Limberger at the above address     

Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs α1-adrenoceptor-mediated contractile responses and inositol phosphate formation in rat cardiomyocytes

The beneficial effects of n-3 polyunsaturated fatty acids of fish oil in the prevention of fatal arrhythmias in myocardial ischemia were suggested to be at least in part mediated by a modulation of dihydropyridine-sensitive L-type calcium channels. As cardiac ₁-adrenoceptor stimulation has been suggested to have no significant effect on L-type calcium channels, the aim of this study using cultured neonatal rat cardiomyocytes was to investigate whether chronic n-3 polyunsaturated fatty acid exposure may have an influence on ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias. Pretreatment of the rat cardiomyocytes for 3 days in the presence of the n-3 polyunsaturated fish oil-derived fatty acid docosahexaenoic acid (60 mol/l) markedly decreased ₁-adrenoceptor-stimulated increase in contraction velocity and induction of arrhythmias. The increase in contraction velocity of the cardiomyocytes induced by the -adrenoceptor agonist isoprenaline was also markedly reduced by the n-3 fatty acid pretreatment. Basal contractile amplitude and spontaneous beating frequency of the cardiomyocytes were not significantly altered by the docosahexaenoic acid exposure. The pretreatment of the rat cardiomyocytes for 3 days in the presence of docosahexaenoic acid (60 mol/l) decreased ₁-adrenoceptor-stimulated formation of the calcium-mobilizing second messenger IP₃ and its metabolites IP₂ and IP₁ by 55%. The depression of IP₃ formation by docosahexaenoic acid treatment was not mediated by a decreased uptake of myo-inositol into the cardiomyocytes nor by a decreased synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of phospholipase C. The level of glycerol-3-phosphate, an important substrate of the phosphoinositide cycle, was unaltered by the docosahexaenoic acid pretreatment. Receptor binding studies revealed that the dissociation constant and maximal binding capacity of the ₁-adrenoceptor antagonist (³H)prazosin was unchanged by the n-3 polyunsaturated fatty acid exposure. -Adrenoceptor-and forskolin-stimulated adenylyl cyclase activities were not diminished by the docosahexaenoic acid pretreatment. Chronic exposure of the cardiomyocytes to the n-6 polyunsaturated fatty acid arachidonic acid (60 mol/l) did neither significantly alter ₁-adrenoceptor-induced inositol phosphate formation nor ₁-adrenoceptor-stimulated increase in contraction velocity. The results presented show that chronic n-3 polyunsaturated fatty acid pretreatment of rat cardiomyocytes leads to a marked impairment of ₁-adrenoceptor-induced positive inotropic effects and induction of arrhythmias concomitant with a n-3 fatty acid-induced decrease in IP₃ formation. This derangement of the phosphonositide pathway by chronic n-3 fatty acid exposure may, thus, contribute to the beneficial effects of fish oil-derived fatty acids in the prevention of fatal arrhythmias in myocardial ischemia.     

Tumor necrosis factor α decreases inositol phosphate formation and phosphatidylinositol-bisphosphate (PIP2) synthesis in rat cardiomyocytes

Treatment of neonatal rat cardiomyocytes for 72 h in the presence of tumor necrosis factor (TNF) (10 U/ml) lead to a decrease in basal and ₁-adrenoceptor-induced formation of the calcium-mobilizing second messenger inositol trisphosphate (IP₃) and its metabolites, IP₂ and IP₁, by 35 and 26%, respectively. The synthesis of phosphatidylinositol bisphosphate (PIP₂), the substrate of PI-specific phospholipase C, was decreased by 45% following the TNF (10 U/ml) exposure. Time courses of TNF (10 U/ml)-induced alterations in rat cardiomyocytes showed a parallel decline of basal inositol phosphate formation and PIP₂ synthesis suggesting that the decrease in inositol phosphate formation was due to the reduction in PIP₂ synthesis. As the TNF-induced decrease of PIP₂ synthesis was associated with a decreased synthesis of the phospholipid phosphatidylinositol (PI), the precursor of PIP₂, by 33%, the decreased availability of PIP₂ is apparently, at least in part, the result of the decreased synthesis of PI. As an apparent functional consequence of the decrease in IP₃ formation following the TNF exposure, the ₁-adrenoceptor-mediated induction of arrhythmias by 100 mol/l noradrenaline + 10 mol/l timolol was abolished in TNF-pretreated rat cardiomyocytes.To investigate one of the possible mechanisms of the TNF-induced decrease of PIP₂ formation, the effect of TNF pretreatment on glycerol-3-phosphate dehydrogenase (GDH), a key enzyme of lipogenesis, was studied: Exposure of the rat cardiomyocytes for 72 h to TNF induced a concentration-dependent decrease in GDH activity by maximally 55%.The result presented are consistent with the hypothesis that the decreased basal and ₁-adrenoceptor-induced formation of the second messenger IP₃ observed in chronic endotoxinemia and sepsis may be mediated by a TNF-induced decrease in the synthesis of PIP₂, the substrate of PI-specific phospholipase C. This mechanism occurs following long-term exposure to low TNF/ha concentrations and is apparently distinct from the short-term cardiac effects induced by high concentrations of TNF. Correspondence to: C. Reithmann at the above address     

A quantitative analysis of antagonism and inverse agonism at wild-type and constitutively active hamster α1B-adrenoceptors

In order to characterize inverse agonism at alpha1B-adrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline alpha1-adrenoceptor antagonists at cloned hamster wild-type (WT) alpha1B-adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [3H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline alpha1-adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with Ki values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentration-dependent inhibition of basal IP formation; the maximum inhibition was approximately 55%, and the corresponding EC50 values were slightly smaller than the Ki values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that alpha1-adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.     

α1-Adrenoceptors in the rat cerebral cortex: New insights into the characterization of α1L- and α1D-adrenoceptors

Among the three α₁-adrenoceptor subtypes (α_1A, α_1B and α_1D) a peculiar intracellular localization and poor coupling to membrane signals of cloned α_1D-adrenoceptor have been reported. In addition, the α_1L-adrenoceptor (low affinity for prazosin), a functional phenotype of α_1A, has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of α₁-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (α_1D> α_1B ? α_1A). α₁-Adrenoceptors were also detected by immunoblotting, revealing α_1A- and α_1B-adrenoceptors to be predominantly expressed in the membrane fraction and the α_1D-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of α_1D-adrenoceptor in tissue homogenates, whereas only α_1A- and α_1B-subtypes were detected in membranes. The proportion of α_1A-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (α_1A/L)-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The α_1A/L-adrenoceptor was heavily implicated in the inositol phosphate response, while the α_1D-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of α_1D-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the α_1L-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the α_1A-adrenoceptor coupled to G-protein.     

Pharmacologically distinct phenotypes of α1B-adrenoceptors: variation in binding and functional affinities for antagonists

Background and Purpose

The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α_1B-adrenoceptor subtype in various tissue preparations and under various conditions.

Experimental Approach

[³H]-prazosin binding to α_1B-adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α_1B-adrenoceptors.

Key Results

In association and saturation-binding experiments with rat liver, binding affinity for [³H]-prazosin varied significantly between preparations (K_D value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α_1B-adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α_1B-adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α_1B-adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α_1B-adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α_1B-adrenoceptor profile.

Conclusions and Implications

In contrast to the consistent profile of recombinant α_1B-adrenoceptors, the pharmacological profile of native α_1B-adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional ‘subtype’ characterization, ‘phenotype’ pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.Table of Links

Characterization of sensory neurotransmission and its inhibition via α2B-adrenoceptors and via non-α2-receptors in rabbit iris

Summary To find out whether, and which type of, adrenoceptors mediate prejunctional inhibition of sensory neurotransmitter release from trigeminal fibres, the modulation of twitch response to electrical field stimulation on rabbit isolated iris was investigated. Evoked iris sphincter contractions consisted of a minor fast cholinergic and a large slow component. The latter was unaffected by atropine and guanethidine, hence nonadrenergic noncholinergic in nature (NANC), but nearly completely abolished by capsaicin pretreatment and by the neurokinin receptor antagonist spantide. The response was probably not mediated by NK₂ receptors as SR 48,968, an NK₂ selective nonpeptide antagonist, failed to reduce the response to the release of the endogenous neurokinin(s) (and exogenous substance P), but in part due to NK₁ receptor activation as shown by a reduction of response by CP 96,345, an NK₁ selective non-peptide antagonist, and in part perhaps mediated by NK₃ receptors. A small neurokinin receptor antagonist- and capsaicin-insensitive NANC contraction is probably not mediated by CGRP receptors.The ₂-adrenoceptor agonist oxymetazoline inhibited the evoked NANC response (22 nmol/1, IC₂₀; about 40%, maximum inhibition) without affecting the cholinergic response (up to 1 mol/1) or the postjunctional iris sensitivity to exogenous substance P. The inhibition was antagonized by rauwolscine (apparent -log K_B 8.04) and by the relatively _2B-adrenoceptor selective antagonist ARC-239 (-log K_B 8.51).The ₂- and imidazoline receptor agonist aganodine inhibited the evoked NANC response (0.25 mol/l, IC₂₀; about 30%, maximum inhibition) without affecting the postjunctional substance P responses. Rauwolscine 0.3 mol/l failed to antagonize this effect.It is concluded that the release of sensory neurotransmitter(s) from trigeminal fibres in the rabbit eye may be inhibited by _2B-adrenoceptors and by a non-₂-receptor, perhaps an imidazoline receptor.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/3)Correspondence to H. Fuder at the above address     

α1- and α2-Adrenoreceptor antagonists differentially influence locomotor and stereotyped behaviour induced byd-amphetamine and apomorphine in the rat

The importance of dopamine (DA) in mediating locomotor, exploratory and stereotyped behaviour in rodents is well established. Evidence also indicates a modulatory role for noradrenaline (NA) although, due to non-specificity of previously available agents, a precise role remains undefined. The effects of the specific and selective -adrenoreceptor antagonists idazoxan (₂) and prazosin (₁) on behaviour induced by amphetamine and apomorphine have been investigated in the rat.d-Amphetamine (2 mg/kg) induced hyperactive locomotion and exploration. Pretreatment with prazosin (1 mg/kg) markedly reduced these responses. In contrast, pretreatment with idazoxan (20 mg/kg) only marginally alteredd-amphetamine hyperactivity. Apomorphine (0.5 mg/kg) induced biphasic locomotor and exploratory activity. Neither -antagonist affected the initial burst of activity (60 min), although prazosin inhibited whereas idazoxan potentiated the secondary phase (90–180 min). At higher dosage, amphetamine (6 mg/kg) and apomorphine (2 mg/kg) induced stereotyped behaviours. Prazosin pretreatment enhanced stereotyped gnawing and decreased sniffing and locomotion, whereas idazoxan increased locomotion and decreased amphetamine-induced mouth movements. These data indicate that DA-induced locomotor and stereotyped behaviours are differentially influenced (in opposite directions) by both ₁- and ₂-adrenoreceptor antagonists. NA may thus modulate the expression and character of behaviour by influencing DA function in certain brain areas.     

Mutual interaction of histamine H3-receptors and α2-adrenoceptors on noradrenergic terminals in mouse and rat rain cortex

Summary Brain cortex slices were preincubated with ³H-noradrenaline and superfused with physiological salt solution containing desipramine. We studied the inhibition of the electrically evoked tritium overflow caused by histamine in the presence of -adrenoceptor ligands (mouse and rat brain cortex), and the inhibition caused by talipexole (the former B-HT 920) in the presence of H₃-receptor ligands (mouse brain cortex).In mouse brain cortex slices, the inhibitory effect of histamine on the tritium overflow evoked by 36 pulses, 0.3 Hz was not changed by the ₁-adrenoceptor antagonist prazosin, but increased by the ₂-adrenoceptor antagonist rauwolscine. When the current strength or the duration of electrical pulses was reduced to compensate for the increase in evoked tritium overflow produced by rauwolscine, the latter still. enhanced the effect of histamine. The histamine-induced inhibition of tritium overflow evoked by 360 pulses, 3 Hz was not affected by the ₁-adrenoceptor agonist phenylephrine but attenuated by the ₂-adrenoceptor- agonist talipexole. Finally, the inhibition by histamine of the tritium overflow evoked by 3 pulses, 100 Hz was attenuated by talipexole but not affected by rauwolscine. Conversely,. the inhibitory effect of talipexole on tritium overflow elicited by 360 pulses, 3 Hz was slightly attenuated by the H₃-receptor agonist R-(–)--methylhistamine but not, affected by the H₃- receptor antagonist thioperamide. In rat brain cortex slices, histamine only tended to inhibit tritium overflow evoked by 360 pulses, 3 Hz, both in the absence of -adrenoceptor antagonists and in the presence of prazosin. However, histamine markedly inhibited the evoked overflow in the presence of rauwolscine. Again, enhancement of the histamine-induced inhibition also occurred when the current strength or the duration of pulses was reduced in order to compensate for the increase in evoked tritium overflow produced by rauwolscine.The results suggest that the ₂-autoreceptors and the H₃-heteroreceptors at the noradrenergic nerve endings in the brain of mouse and rat interact with each other. Activation of the ₂-autoreceptors decreases, whereas blockade of the activated (but not of the non-activated) ₂-autoreceptors increases, the inhibitory effect of histamine. Activation of the H3-heteroreceptors slightly decreases, whereas blockade of the H₃-receptors fails to affect, the inhibitory effect of talipexole.Send offprint requests to E. Schlicker at the above address     

Estimation ofpA2 values at presynaptic α2-autoreceptors in rabbit and rat brain cortex in the absence of autoinhibition

Summary An attempt was made to determinepA₂ values of antagonists at the presynaptic, release-inhibiting ₂-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA₂ values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with³H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The -adrenoceptor agonists clonidine, noradrenaline, and -methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and -methylnoradrenaline.pA₂ values were calculated either from linear regression of log [agonist concentration ratio – 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA₂ values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA₂ values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA₂ values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA₂ value of idazoxan in the rabbit. (about 7.1) was lower than itspA₂ value in the rat (about 8.0). The experiments confirm thatpA₂ values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA₂ values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that ₂-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex. Send offprint requests to E. A. Singer at the above address     

Release-inhibiting α2-adrenoceptors at serotonergic axons in rat and rabbit brain cortex: evidence for pharmacological identity with α2-autoreceptors

The pharmacological properties of the presynaptic ₂-adrenoceptors modulating the release of serotonin in rat and rabbit brain cortex (₂-heteroreceptors) were compared with the properties of presynaptic ₂-autoreceptors in the same brain area. Brain cortex slices were preincubated with [³H]-serotonin or [³H]-noradrenaline and then superfused and stimulated by brief high-frequency pulse trains.The ₂-adrenoceptor agonist bromoxidine reduced the electrically evoked overflow of tritium in experiments with both [³H]-noradrenaline and [³H]-serotonin and in brain slices from either species. The antagonists phentolamine, idazoxan, (+)-mianserin, rauwolscine, 5-chloro-4(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (ORG 20350), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), (–)-mianserin and corynanthine caused parallel shifts of the concentration-inhibition curves of bromoxidine to the right. Negative logarithms of antagonist dissociation constants pK_d were calculated from the shifts. In the rat, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, giving a close correlation, r = 0.97 (P<0.001). In the rabbit equally, the ₂-autoreceptor pK_d value of each single antagonist was similar to its ₂-heteroreceptor pK_d value, maximal difference 0.4, again yielding a close correlation, r = 0.96 (P < 0.001). However, antagonist pK_d values at rat ₂-autoreceptors differed from those at rabbit ₂-autoreceptors, r = 0.70 (P > 0.05), and antagonist pK_d values at rat ₂-heteroreceptors differed from those at rabbit ₂-heteroreceptors, r = 0.64 (P > 0.05). Comparison with radioligand binding experiments from the literature indicated that, in the rat, both auto- and heteroreceptors conformed best to the _2D subtype (r 0.97, P < 0.01 for pK_d correlation with binding sites in rat submaxillary gland) whereas, in the rabbit, they conformed best to the _2A subtype (r 0.93, P < 0.01 for pK_d correlation with binding sites in HT29 cells).It is concluded that, in both the rat and the rabbit, the ₂-adrenoceptors modulating the release of serotonin are pharmacologically identical with the presynaptic ₂-autoreceptors. However, rat ₂-autoreceptors and -heteroreceptors differ pharmacologically from rabbit ₂-autoreceptors and -heteroreceptors. Presynaptic ₂-auto-as well as -heteroreceptors are _2D in the rat and _2A in the rabbit. Correspondence to: N. Limberger at the above address     

Effects of long-term application of dopamine HCl on dopamine agonist-induced cAMP production in rat renal cortex

目的：观察长期给予盐酸多巴胺对大鼠肾皮质多巴胺受体亚型所介导的腺苷酸环化酶活性的影响．方法：用放免分析法测定ｃＡＭＰ含量，作为反映多巴胺受体功能的指标．结果：长期给予盐酸多巴胺可显著减少肾皮质由非诺多泮引起的ｃＡＭＰ增加的量和在Ｓｃｈ２３３９０存在下由ＰＢＤＡ引起的ｃＡＭＰ降低的量，但其变量百分比则与对照组无显著差异．Ｓｃｈ２３３９０可阻断由非诺多泮和ＰＢＤＡ引起的ｃＡＭＰ的增加，而多潘立酮可阻断在Ｓｃｈ２３３９０存在下由ＰＢＤＡ引起的ｃＡＭＰ的降低．结论：长期应用多巴胺可使大鼠肾皮质的ＤＡ１和ＤＡ２受体均发生明显的“下调”，但余留受体的反应性不变．     

Phentolamine and hypoxia: modulation of contractility and α1-adrenoceptors in isolated rat atria

The hypoxia-induced effects on the binding sites and affinity constant of adrenoceptors, in the presence and absence of phentolamine, were determined for atrial membranes of hearts from normal and genetically hyperlipidaemic Yoshida (YOS) rats. Atrial function was also measured during normoxia and hypoxia, in the presence and absence of phentolamine.Hypoxia increased a1-adrenoceptor density in atrial membranes of normal rats (B_max 10.6 to 26.7 fmoles/mg protein). Phentolamine prevented the increase in the B_max of ₁-adrenoceptors and increased the equilibrium dissociation constant of these receptors (K _D 0.17 to 0.53 nmol/l). Beta-adrenoceptors did not change during hypoxia, but the B_max was slightly increased (26%) in the presence of phentolamine. Thus, the ₁/\ ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the ₁/\ ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of at-adrenoceptors in atrial membranes from YOS rats). This ratio was not modified by hypoxia (0.84), but decreased when phentolamine was present (0.30).Hypoxia reduced the force of contraction and increased diastolic tension of atria of normal rats, while the sinus rate was not significantly modified. Phentolamine abolished the increase in diastolic tension and reduced the negative effect of hypoxia on contractile force. In YOS rat atria, functional parameters were modified by hypoxia in a qualitatively similar way to that of normal rat atria.The observed increase in ₁-adrenoceptor density during hypoxia is in accordance with the results of experiments with animal models of the ischaemic heart and with findings in human heart failure. The possible therapeutic significance of these data is considered. Correspondence to: G. Fassina at the above address     

α1B-Adrenoceptors mediate adrenergically-induced renal vasoconstrictions in rats with renal impairment

AIM: This study examined whether alpha1B-adrenoceptors are involved in mediating adrenergically-induced renal vasoconstrictor responses in rats with pathophysiological and normal physiological states. METHODS: Male Wistar Kyoto and spontaneously hypertensive rats were induced with acute renal failure or experimental early diabetic nephropathy by cisplatin or streptozotocin, respectively. Cisplatin-induced renal failure was confirmed by impaired renal function and pronounced tubular damage. Experimental early diabetic nephropathy was confirmed by hyperglycemia, changes in physiological parameters, and renal function. The hemodynamic study was conducted on anesthetized rats after 7 d of cisplatin (renal failure) and 4 weeks of streptozotocin (experimental early diabetic nephropathy). RESULTS: In the rats with renal failure and experimental early diabetic nephropathy, there were marked reductions in their baseline renal blood flow (P<0.01). The baseline mean arterial blood pressure was either unaltered or lower (all P>0.05) in the renal failure and experimental early diabetic nephropathy rats, respectively, as compared to their non-renal failure and non-diabetic nephropathy controls. In the rats with renal impairment, chloroethylclonidine caused either accentuation or attenuation (all P<0.01) of the renal vasoconstrictor responses elicited by the adrenergic stimuli. However, in the non-renal failure and in the non-diabetic nephropathy rats, chloroethylclonidine did not cause any alteration in such responses (P>0.05). CONCLUSION: This study demonstrated the presence of functional alpha1B-adrenoceptors that mediated the adrenergically-induced renal vasoconstrictions in rats with renal impairment, but not in rats with normal renal function.