Characterization of 5-hydroxytryptamine release from isolated rabbit and rat trachea: the role of neuroendocrine epithelial cells and mast cells

Rabbit or rat isolated tracheae were incubated in vitro, and the release of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) was determined by HPLC with electrochemical detection.Release of 5-HT from rabbit tracheae could be evoked by the calcium ionophore A 23187 and, in a calcium-dependent manner, by depolarizing concentrations of potassium (45 mmol/1), but not by the mast cell degranulating drug compound 48/80. High potassium-and A 23187-evoked release of 5-HT was markedly higher from tracheae of newborn compared to adult rabbits. In rabbit tracheae, mechanical removal of the mucosa resulted in 80–90% reduction in tissue 5-HT and in a similar reduction in high potassium-evoked 5-HT release. 5-Hydroxytryptophan, but not tryptophan, caused a marked increase in the spontaneous outflow of 5-HT and 5-HIAA from tracheae of newborn rabbits, and the effect on 5-HT, but not that on 5-HIAA, required an intact mucosa. Furthermore, treatment with 5-hydroxytryptophan caused an increase in tissue 5-HT and 5-HIAA, and these effects required an intact mucosa. In tracheae of adult rabbits 5-hydroxytryptophan caused similar, although less profound, effects. Adrenaline (I mol/l) enhanced the release of 5-HT from newborn rabbit tracheae, and this effect was inhibited by 1 mol/l phentolamine or 1 mol/l prazosin, but not affected by 100 nmol/1 propranolol. In rat tracheae, compound 48/80 evoked a large release of 5-HT, whereas depolarizing concentrations of potassium (45 mmol/1) had only a very minor effect. In rat tracheae, 5-hydroxytryptophan had small effects on the outflow and tissue contents of 5-HT and 5-HIAA in comparison to the effects on rabbit tracheae; and removal of the mucosa resulted in only a minor reduction in tissue 5-HT.In conclusion, neuroendocrine epithelial (NEE) cells and mast cells are the major source of 5-HT in tracheae of the rabbit and rat, respectively. Isolated tracheae of newborn rabbits appear to be a useful model to study 5-HT secretion from NEE cells. 5-HT secretion from NEE cells is activated by a rise in intracellular calcium, and calcium influx through voltage-regulated channels appears to be one activating pathway. 5-HT secretion from NEE cells can be stimulated via -adrenoceptors.Dedicated to Prof. E. Mutschler on occasion of his 65th birthday     

Characterization of histamine H3 receptors inhibiting 5-HT release from porcine enterochromaffin cells: Further evidence for H3 receptor heterogeneity

The nature of the histamine receptor mediating inhibition of 5-HT release was investigated in strips of the porcine small intestine by investigating the effects of histamine ligands on the overflow of endogenous 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). The overflow was measured by HPLC, combined with electrochemical detection and represents calcium-sensitive 5-HT release from enterochromaffin cells, as reported previously. The histamine H₃ receptor selective agonists (R)--methyl-histamine and imetit inhibited the overflow of 5-HT maximally by 50–60%, with EC₅₀ values of 48 and 3.2 nmol/l, respectively. Effects on 5-HT overflow were always accompanied by similar effects on the overflow of 5-HIAA. Thioperamide (100 nmol/l) shifted the concentration response curve of (R)--methyl-histamine to the right (pK_B value 8.38). The inhibitory effect of 1 mol/l (R)--methyl-histamine was antagonized in a concentration-dependent manner by thioperamide (IC₅₀: 65 nmol/l) and dimaprit (IC₅₀: 8.6 mol/l); however, the effect of (R)--methyl-histamine was weakly antagonized by burimamide (by 38% at 100 mol/l) and not significantly affected by other H₃ receptor antagonists, such as impromidine, betahistine and phenylbutanoyl-histamine (each up to 100 mol/l). In conclusion, H₃ receptors mediating inhibition of 5-HT release from porcine enterochromaffin cells have a particular pharmacological profile indicating that heterogeneity of H₃ receptors may exist. The data suggest that histamine H₃ receptors modulating 5-HT release in pig small intestine do not belong to either H_3A or H_3B receptors as defined in rat tissue. Correspondence to: K. Racke at the above address     

Calcium channels at the adrenergic neuroeffector junction in the rabbit ear artery

Summary Neurotransmitter release is dependent on influx of Ca²⁺ through voltage-operated calcium channels (VOCCs). These channels may be divided into L, N, T and P subtypes. To investigate the subtypes of VOCC involved in transmitter release from adrenergic nerves in the isolated rabbit ear artery, the effects of some subtype selective VOCC antagonists were examined on contractile responses induced by electrical field stimulation (EFS), and exposure to an isosmolar (low Na⁺, normal Cl^– content) or a hyperosmolar (normal Na^]+, high Cl^– or 60 mM K+ solution). Tetrodotoxin (TTX) and the L channel blocker nimodipine were present in the latter experiments to inhibit sodium-dependent action potential discharge and the direct contractile effect of K⁺ depolarization on the smooth muscle cells. Prazosin abolished the contractile effect of EFS, indicating that the response was elicited by activation of adrenergic nerves. The EFS-induced contractions were concentration-dependently inhibited by the N channel blocker -conotoxin (PIC₅₀ = 9.0) and the proposed L channel blocker T-cadinol (pIC₅₀ = 4.5), while nimodipine and the T channel blocker tetramethrin had no effect. The isosmolar and hyperosmolar K⁺ solutions induced a prazosin-sensitive contraction, amounting to 46% and 10% of the response to 10^–5 M noradrenaline (NA), respectively. -Conotoxin inhibited the contractile response to the hyperosmolar K⁺ solution, but not that to the isosmolar K⁺ solution. T-cadinol preferentially inhibited the response to the hyperosmolar K⁺ solution. Tetramethrin had no effect on contractions induced by either type of K⁺ solution. The contractile response to exogenous NA was unaffected by -conotoxin and tetramethrin, whereas the response was partially inhibited by both nimodipine and T-cadinol. These results suggest that NA release from adrenergic nerves in the rabbit ear artery, depend on Ca²⁺ influx through VOCCs of the N type, whereas L and T channels seem to be of minor importance. Calcium influx into the nerve terminals via a tentative Na⁺/Ca²⁺ exchange mechanism may explain the failure of -conotoxin to inhibit the adrenergic response to the isosmolar K⁺ solution.Correspondence to P. Zygmunt at the above address     

Inhibition of central neurotransmitter release by ω-conotoxin GVIA,a peptide modulator of the N-type voltage-sensitive calcium channel

Summary Tritium overflows, evoked by electrical stimulation (2 min; 2 ms, 3 Hz, 5 V/cm, and 24 mA) of [³H]dopamine-, [³H]-noradrenaline-, [³H]-5-hydroxytrypt-amine, and [³H]-acetylcholine-labeled slices prepared from discrete regions of the rabbit central nervous system, were inhibited 39–50% by co-conotoxin GVIA (-CT; 5 nmol/l), a peptide modulator of the N-type voltage-sensitive calcium channel (N-VSCC). Additional experiments using -CT (5 nmol/l) and [³H]-noradrenaline-labeled hippocampal slices indicated the time dependence of -CT-induced inhibition, the competitive antagonism between buffer calcium concentration and -CT, and the lack of effect of prolonged electrical stimulation (15 min; 0.4 Hz) on -CT-induced inhibition. These various results suggest that 1) -CT competes with calcium for the N-VSCC, 2) the inhibitory effects of -CT are independent of the gating state of the N-VSCC, and 3) the molecular nature of the N-VSCC may be similar or identical across central neurotransmitter systems. -CT appears to be a useful pharmacological tool in studying the involvement of the N-VSCC in neurotransmitter release.Abbreviations -CT -conotoxin GVIA - VSCC voltage-sensitive calcium channel Send offprint requests to D. Dooley     

Different effects of lipolytic hormones and phosphodiesterase inhibitors on cyclic 3′,5′-AMP levels in isolated fat cells

Summary Cyclic AMP levels of isolated fat cells of rats were increased about 50-fold by noradrenaline (1 M) and isoprenaline (1 M) within 4 min of incubation and had declined markedly after 10 min. The effect of a variety of lipolytic hormones on cyclic AMP levels was dose-dependent over a wide range of concentrations, ACTH and glucagon being most potent on a molar basis. Among the adrenergic compounds tested isoprenaline elicited the grea test response. Glucagon, in maximally effective concentrations, caused only half the increase in cyclic AMP values produced by other hormones. This pattern was largely paralleled by the lipolytic effects of the hormones as measured by glycerol production. Various inhibitors of cyclic AMP phosphodiesterase had only small effects on cyclic AMP accumulation. Methylxanthines caused only a 3- to 5-fold elevation of cyclic AMP levels at concentrations which induced maximal lipolytic effects. Papaverine (0.5 mM) and phentolamine (0.1 mM) had virtually no effect and did not potentiate hormone effects on cyclic AMP production. However, theophylline (1 mM) caused a nearly tenfold increase in the effects of isoprenaline when added to 100 000 cells per ml in the medium. At a concentration of 20 000 fat cells per ml isoprenaline alone increased cyclic AMP levels 300-fold and the addition of theophylline had no further stimulatory effect. Our results suggest that lipolysis induced by hormones is mainly mediated by an accumulation of cyclic AMP, whereas methylxanthines must have additional effects. The potentiation of lipolytic hormones by methylxanthines cannot be attributed to an inhibition of phosphodiesterase alone, but seems to be due mainly to their antagonism with an inhibitory factor, which is produced by fat cells and released into the incubation medium.     

A comparative study of the effects of cyclocytidine and norepinephrine on renin and esterase release from mouse submandibular gland

Summary The effects of cyclocytidine and norepinephrine on the release of renin and esterase from mouse submandibular gland were compared in in vivo and in vitro experiments. Cyclocytidine (150 mg/kg i.p.) produced the depletion of renin and esterase in in vivo experiments as did the -adrenoceptor agonist norepinephrine (1 mg/kg i.v.). Cyclocytidine was more effective in depleting renin and esterase than norepinephrine. The -adrenoceptor antagonist phenoxybenzamine (10 mg/kg i.p.), but not the -adrenoceptor antagonist propranolol (10 mg/kg i.p.), attenuated the depletion of renin and esterase by both cyclocytidine and norepinephrine. These results suggest that the mechanism of action of cyclocytidine on the release of renin and esterase in mouse submandibular gland could be similar to that of norepinephrine, and the secretory process for both cyclocytidine and norepinephrine could be initiated by activation of -adrenoceptors. In in vitro experiments using dispersed cells, cyclocytidine (10^–7 M–10^–2 M) had no effect on renin and esterase release, but dose-dependent release of both was clearly observed in response to norepinephrine (10^–7 M–10^–4 M). Immunocytochemical studies of renin in both in vivo and in vitro preparations showed similar enzymatic activity as measured by radioimmunoassay. The results of in vitro experiments did not correspond to those of in vivo experiments, which may suggest that the site of action of cyclocytidine in mouse submandibular gland may be different than that of norepinephrine. Pretreatment with bretylium (20 mg/kg, i.p.) prior to i.p. injection of cyclocytidine (150 mg/kg) completely blocked the esterase depletion induced by cyclocytidine. From these results, it seems possible to conclude that the in vivo effect of cyclocytidine on mouse submandibular gland is an indirect one and is mediated via reflex activation of sympathetic nerves.     

The effect of thiorphan and epithelium removal on contractions and tachykinin release produced by activation of capsaicin-sensitive afferents in the guinea-pig isolated bronchus

Summary (1) We have studied the effect of epithelium removal (rubbing) and the endopeptidase 24.11 inhibitor, thiorphan, on the contractile response of the guinea-pig isolated bronchi (atropine and indomethacin in the bath) produced by electrical field stimulation, capsaicin or exogenously administered tachykinins (substance P and neurokinin A). (2) The response to field stimulation, thought to involve release of endogenous tachykinins, was potentiated by thiorphan in both epithelium-free and intact bronchi. However, at low frequencies (1–5 Hz), the effect of thiorphan was more evident in intact preparations. (3) The response to capsaicin was enhanced by both epithelium removal and thiorphan administration. (4) The response to exogenous substance P or neurokinin A was potentiated by thiorphan both in epithelium-free and intact bronchi. (5) Capsaicin (1 M) evoked a consistent release of substance P-like immumoreactivity (determined by radioimmunoassay) and tachykinin-like immumoreactivity (determined by a novel immumoenzyme assay), which was enhanced by thiorphan in both epithelium-free and intact bronchi. (6) These findings suggest that a thiorphan-sensitive mechanism, presumably enkephalinase (endopeptidase 24.11), plays a major role in inactivating endogenous tachykinins released from sensory nerves and that this enzymatic activity is still present after removal of the bronchial epithelium. Send offprint requests to C. A. Maggi at the above address     

Dose-response relationships of perphenazine in the treatment of acute psychoses

A group of 26 acute psychotic patients received continuous oral treatment with perphenazine for a period of 5 weeks. Once-weekly blood samples were drawn for measurements of perphenazine levels and, simultaneously, the therapeutic outcome was registered. Another 26 acute psychotic patients received continuous oral treatment with perphenazine for a period of up to 4 weeks. A single blood sample was drawn and the perphenazine concentration was related to the appearance of extrapyramidal side effects. The following conclusions were made: (1) a high risk of provoking extrapyramidal side effects was associated with plasma levels of perphenazine above 3 nmol/l; (2) plasma levels below 2 nmol/l were associated with a poor therapeutic outcome; (3) a therapeutic window between 2 and 3 nmol/l gives maximal therapeutic effect with a low risk of provoking extrapyramidal side effects.     

Investigations of the roles of dihydropyridine and ω-conotoxin-sensitive calcium channels in mediating depolarisation-evoked endogenous dopamine release from striatal slices

Summary The relative roles of L- and N-type voltage-sensitive calcium channels (VSCC) in mediating endogenous dopamine release have been investigated by examining the effects of the dihydropyridine (DHP) agonist BAY K 8644 and the antagonist PN 200-110, as well as the VSCC-blocking peptide -conotoxin GVIA, on depolarisationevoked dopamine release from superfused rat striatal slices. Dopamine release evoked by electrical field stimulation was virtually unaffected by either of the DHP drugs, but release evoked by raising the K⁺ concentration to 25 mmol/l was significantly increased by BAY K 8644 and reduced stereospeciflcally by PN 200-110. Quantitative differences between electrically-evoked and K⁺-evoked dopamine release with respect to their dependence on extracellular calcium concentration were also observed, with electrically-evoked release requiring higher calcium concentrations. The adenylate cyclase activator forskolin itself increased dopamine release, but did not appear to influence the effectiveness of either DHP drug in altering dopamine release. In contrast to the relatively small effects of the DHP drugs, -conotoxin produced a major reduction in electrically-evoked dopamine release as well as a substantial decrease in K⁺-evoked release. Since -conotoxin is thought to block both L- and N-type neuronal VSCC whereas DHP drugs affect only L-type VSCC, these findings suggest that electrically-evoked dopamine release is mediated mainly by calcium influx through N-type VSCC, accounting for the reported lack of effect of many organic calcium antagonists on this process. In contrast, K⁺-evoked dopamine release appears to involve both L- and N-type VSCC, and can occur at lower extracellular calcium concentrations.Abbreviations DHP 1,4-dihydropyridine - HPLC high-performance liquid chromatography - VSCC voltage-sensitive calcium channels Send offprint requests to H. Herdon at the above address     

Gabapentin Blocks L-Type and P/Q-Type Ca2+ Channels Involved in Depolarization-Stimulated Nitric Oxide Synthase Activity in Primary Cultures of Neurons from Mouse Cerebral Cortex

Purpose: The effect of gabapentin [1-(aminomethyl)cyclohexane acetic acid] on Ca²⁺ channels involving the activation of nitric oxide synthase (NOS) was investigated in primary neuronal culture of mouse cerebral cortex. Methods:The expression of ₂ subunits of Ca²⁺ channels was investigated by RT-PCR using specific primer sets. The K⁺-evoked NOS activity was estimated by guanosine 3'5' cyclic monophosphate (cGMP) formation. Results:mRNA for ₂ subunits of Ca²⁺ channels is found in these cells. Gabapentin blocked the K⁺-evoked NOS activity estimated from cGMP formation in a concentration dependent manner. The increase in NOS activity by the K⁺-stimulation was almost completely reversed by the combination of nifedipine, an L-type Ca²⁺ channel blocker, and -conotoxin GVIA, an N-type Ca²⁺ channel blocker, was failed to reverse the increase in NOS activity by the K⁺-stimulation, indicating that the activation of NOS by the depolarizing stimulation might be not mediated by N-type Ca²⁺ channel. Under the presence of nifedipine or -agatoxin IVA, gabapentin inhibited the increase in NOS activity concentration-dependently. Conclusions: These results suggest that gabapentin inhibits depolarization-induced NOS activation in murine cortical neuronal culture via blockade of both P/Q-type and L-type Ca²⁺ channels.     

Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ‘fear’-motivated behaviour

Summary An elevated X-maze with alternating open and enclosed arms was investigated as a model for the study of fear-induced behaviour. As predicted, the anxiolytics diazepam and amylobarbitone increased, and the putative anxiogenics ACTH and picrotoxin decreased the proportion of open arm entries. The ₁-adrenoceptor agonists phenylephrine and ST587, and the ₂-adrenoceptor antagonists idazoxan, piperoxane, RS-21361 and yohimbine decreased relative open-arm entries, thus resembling the putative anxiogenics. On the other hand, azepexole, clonidine and guanabenz, agonists at ₂-adrenoceptors, and the ₁-adrenoceptor antagonists prazosin and thymoxamine, enhanced the proportion of open arm entries at low doses, suggesting anxiolytic-like properties. A paradoxical fall in open arm entries occurred with these agents at higher doses. These results provide further evidence for the involvement of noradrenergic systems in fear-motivated behaviour.     

Effects of pertussis toxin on opioid regulation of catecholamine release from rat and guinea pig brain slices

Summary Opioid agonists selective for µ-, -, and -receptors are all capable of regulating the stimulated release of noradrenaline from three terminal fields (cortex, hippocampus, and cerebellum) of the noradrenergic projections from locus coeruleus in the guinea pig brain. Intracerebroventricular injections of pertussis toxin abolished the ability of a µ-selective agonist and of a -selective agonist to inhibit stimulated noradrenaline release, but left unaffected the concentration-related inhibition of NE release by a agonist. Thus, µ- and -receptors have been shown to be coupled to their effector systems in these noradrenergic neurons via guanyl nucleotide binding proteins (G proteins) which are sensitive to pertussis toxin, while -receptors in the same neurons appear to be coupled through a different mechanism which is significantly less sensitive to pertussis toxin. In contrast to opioid receptor regulation of noradrenaline release in guinea pig hippocampus, µ-, but not - or -agonists are capable of regulation of stimulated noradrenaline release from rat hippocampus and cortex, and -, but not µ- or -agonists are capable of inhibiting the stimulated release of dopamine from rat striatum and cortex. Pertussis toxin injections significantly attenuated µ-agonist inhibition of noradrenaline release, but had no effect on the ability of a -selective agonist to regulated dopamine release, confirming the insensitivity of the -receptor-effector coupling system to pertussis toxin.This work was supported by a grant from the National Institute on Drug Abuse. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. Animals used in this study were acquired and cared for in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 85-23 Revised 1985) Send offprint requests to L. L. Werling at the above address     

Inhibition of Nuclear Transcription Factor-κB by Specific IκB Kinase Peptide Inhibitor

Pharmaceutical Research -     

2,2′,3′,4,4′,5,5′-Heptachlorobiphenyl (PCB 180) — on its toxicokinetics,biotransformation and porphyrinogenic action in female rats

The toxicokinetics and biotransformation of 2,2,3,4,4,5,5-heptachlorobiphenyl, as well as its influence on the activity of microsomal and cytosolic enzymes and on the porphyrin pathway in the liver were studied in female rats following oral treatment with 7 mg/kg every other day for 3 months. One day after cessation of treatment the concentration of the compound in liver, spleen, CNS and blood was 100–500 times and in the trachea it was only 5 times less than in the adipose tissue. The daily excretion with the feces and urine amounted to 35 and 1.5 g, respectively. In both excreta, heptachlorobiphenylol was identified as a metabolite. The biotransformation rate was estimated to be about 5%. Investigations of the liver revealed increases in the relative liver weight, total cytochrome P-450 content, O-deethylation of 7-ethoxycoumarin and in the activity of glutathione S-transferases. Disturbances of the hepatic porphyrin pathway were not detected. Only at the end of a post-dosing period of 12 months did the hepatic uroporphyrinogen decarboxylase show diminished activity. Only one of these animals with diminished enzyme activity showed drastically elevated porphyrins. In these animals, the fecal and urinary porphyrins did not differ from controls. At no time did heptachlorobiphenyl influence the urinary excretion of delta-aminolevulinic acid and porphobilinogen. The results indicate 1) that this congener shows expected toxicokinetics with the exception of being accumulated in the trachea and 2) that this congener induces disturbances of the hepatic porphyrin pathway several months after cessation of treatment.     

Failure of “antigastrin” (SC-15 396) to inhibit gastric acid and pepsin secretion in anaesthetized gastric fistula cats

Summary 1. The effect of antigastrin (SC-15 396) on gastric acid and pepsin secretion produced by the gastrin-analogue tetrapeptide amide Try. Met. Asp. Phe-NH₂ and by electrical stimulation of the vagus was investigated in anaesthetized gastric fistula cats.2. Antigastrin failed to inhibit both acid and pepsin response stimulated by either the tetrapeptide or vagus excitation.3. It was concluded that the ineffectiveness of antigastrin in cats is due to a species difference between rats and dogs on the one hand and cats on the other, and that antigastrin is not a specific gastrin antagonist.Supported by the Deutsche Forschungsgemeinschaft and by the Alfred Teufel-Stiftung.     

Inhibition by interleukin-1\ of noradrenaline release in rat spleen: involvement of lymphocytes,NO and opioid receptors

Effects of indomethacin, N-nitro-L-arginine (NNA) and naloxone, and of pretreatment with cyclophosphamide (CY), on the interleukin (IL)-I\ induced inhibition of exocytotic noradrenaline release were investigated in the isolated, vascularly perfused spleen of the rat. Neurotransmitter release was evoked by perivascular electrical stimulation (4 Hz) and the overflow of endogenous noradrenaline was determined by HPLC with electrochemical detection.Perfusion of the spleen with Tyrode's solution containing IL-1\ (100 pg/ml) for 90 min caused an inhibition of the stimulation-evoked noradrenaline overflow which persisted for at least 20 min after washout of the IL. The evoked overflow was reduced in the presence of NNA 30 mol/l, but remained unaffected by indomethacin 3 mol/l, naloxone 0.1 mol/l or treatment of the rats with CY (250 mg/kg). The opioid agonist etorphine 10 mol/1 inhibited the evoked overflow of noradrenaline and this effect was prevented by naloxone 0.1 mol/1. The inhibition of evoked overflow by IL-1\ was not affected by indomethacin but was reduced or even prevented in the presence of NNA or naloxone, or after lymphocyte depletion of spleens by CY.The results are compatible with the idea that in the rat spleen exocytotic noradrenaline release is accompanied by a concomitant secretion of a nitric oxide (NO)-like compound which, in turn, reinforces noradrenaline release, and that the release can be inhibited via prejunctional opioid receptors. The IL-1\ induced inhibition of evoked release appears to be a complex process which involves as one of many steps a decrease of the facilitatory NO-like compound and the release of endogenous opioids probably from spleen lymphocytes.     

Inhibitory effect of tyramine-induced release of catecholamines on renin secretion

Summary The effect of the indirect sympathomimetic agent tyramine on the isoprenaline-induced increase in plasma renin concentration was investigated in conscious rats. Tyramine caused a dose-dependent decrease in the isoprenaline-induced elevation of plasma renin concentration. Pretreatment of the rats with reserpine abolished this effect of tyramine, indicating that tyramine released catecholamines which acted on the inhibitory adrenoceptors. Pretreatment with phenoxybenzamine, an -adrenoceptor antagonist, also abolished the inhibitory effect of tyramine on renin release, indicating that -adrenoceptors mediated the observed inhibition of renin release.In rats with chronically denervated kidneys tyramine did not inhibit renin release. It is concluded that catecholamines which are released from renal sympathetic nerve endings can suppress renin release by activating -adrenoceptors.Supported by DFG Me 541/1     

Role of ω-conotoxin-sensitive calcium channels in inositolphosphate production and noradrenaline release due to potassium depolarization or stimulation with carbachol

Summary -Conotoxin GVIA (CTX) has been used to assess the role of voltage-sensitive Ca²⁺ channels involved in inositolphosphate (InsP) production and in noradrenaline (NA) release from washed brain homogenates. Stimulation was performed by depolarization with high [K⁺] and by the M-cholinergic agonist carbachol. In chicken brain CTX (1 mol/l and less) nearly completely inhibited both InsP production and NA release due to high [K⁺]. The peptide depressed InsP production moderately but NA release largely when evoked with carbachol. In rat brain inhibition by CTX of InsP production or NA release was weak or even absent independent of the mode of stimulation. Independent of species or test system, the dihydropyridine derivative nitrendipine (1 gmol/l and above) was inactive.Because of its preferential CTX sensitivity chicken brain is particularly suitable to study the role of voltage-sensitive Ca²⁺ channels in presynaptic events. Here InsP production, like NA release, depends on entry of extracellular Ca²⁺ nearly completely when evoked by depolarization. When evoked by way of cholinergic M-receptor stimulation, InsP production is triggered by a second pathway in addition to the CTX-sensitive Ca²⁺ entry.Abbreviations InsP inositolphosphate(s) - CTX omega-conotoxin GVIA - NA noradrenaline - TTX tetrodotoxin - NTP nitrendipine - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SDS sodium dodecylsulfate - KRH Krebs-Ringer solution buffered with Hepes - BSA bovine serum albumin Send offprint requests to E. Habermann at the above address     

Differences between full and partial α-adrenoceptor agonists in eliciting phasic and tonic types of responses in the longitudinal smooth muscle of the rat portal vein

Summary The aim of the present investigation was to study, taking into account both quantitative and qualitative differences, the influence of full and partial -adrenoceptor agonists on spontaneous myogenic activity in the rat portal vein.We found that the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, phenylephrine, St 587, Sgd 101/75, B-HT 920 and UK-14,304 could increase the amplitude of the phasic myogenic contractions in the rat portal vein with apparent differences in EC₅₀ and E_max values. In addition to an increase in phasic myogenic activity, the -adrenoceptor agonists cirazoline, adrenaline, noradrenaline, and phenylephrine were also able (in higher concentrations) to increase the basal tone of the rat portal vein preparation, again with apparent differences in EC₅₀ and E_max values. Changing the extracellular Ca²⁺ concentration from 0.9 mmol/l to 2.5 mmol/1 had no influence on the phasic character and the concentration range in which St 587 and UK-14,304 increased spontaneous myogenic activity, although changes in amplitude and frequency of the spontaneous myogenic contractions were less pronounced at a higher extracellular Ca²⁺ concentration (2.5 mmol/1). By the use of Schild analysis with the competitive a-adrenoceptor antagonists prazosin (pA₂ = 8.74) and 5-methyl-urapidil (pA₂ = 8.37), it was established that the contractile responses to St 587 were mediated by the same ₁-adrenoceptor subtype as the phasic and tonic type of contraction elicited by phenylephrine as described in a previous study. The concentration-response curve of UK-14,304 was significantly shifted to the right by low concentrations of prazosin (3 nmol/1–30 nmol/1), indicating stimulation of ₁-adrenoceptors by UK-14,304 in the rat portal vein. The -adrenoceptor antagonists phenoxybenzamine and chloroethylclonidine irreversibly blocked the contractile responses to St 587. Based on the method of receptor alkylation with phenoxybenzamine an affinity constant was calculated for St 587 (pK_a = 5.91). Phenoxybenzamine was approximately 1000-fold more potent in inactivating ₁-adrenoceptors than chloroethylclonidine.In conclusion there appeared to be a divergence in the excitation-contraction coupling of ₁-adrenoceptors in the rat portal vein, which is reflected by two types of contraction (phasic versus tonic). The extent to which both the phasic and tonic types of contraction are stimulated by agonists depends on the affinity and intrinsic efficacy for each of the receptor-coupled effector pathways. Thus, partial and full agonism can only meaningfully be discussed if confined to one particular effector pathway. Send offprint requests to H. R. Schwietert at the above address     

Effects of indomethacin on changes in renal blood flow induced by adenosine and its analogues in conscious dogs

Summary The effects of indomethacin on changes in renal blood flow induced by adenosine, NECA (adenosine-5-N-ethyl-carboxamide) and 2,3-dinitro-NECA were investigated in 6 chronically instrumented conscious dogs. Adenosine (187.5, 375 and 750 nmol/kg, i.v.) induced a dose-dependent initial decrease, followed by a reactive increase in renal blood flow. NECA (1.5 nmol/kg, i.v.) also induced an initial decrease, which was, however, followed by a prolonged reactive increase in renal blood fow. 2,3-dinitro-NECA (50 nmol/kg, orally) induced only an increase in renal blood flow. Indomethacin (27.9 mol/kg, i.v.) caused no relevant change of the initial decrease and a significant attenuation of the reactive increase in renal blood flow induced by adenosine. NECA-induced changes in blood flow were affected by indomethacin in the same direction but to a greater extent than were adenosine-induced changes in blood flow. Indomethacin reversed the increase to a decrease in renal blood flow induced by 2,3-dinitro-NECA. Thus, prostaglandins seem to be involved in mediating the response of renal blood flow to adenosine, NECA and 2,3-dinitro-NECA.Part of this study was presented at the fall meeting of the German Pharmacological Society, September 1982 in Vienna, Austria