Pharmacological evidence for the existence of two distinct serotonin receptors in rat brain

The effect of a series of indoleamines on the potassium-evoked release of previously accumulated [³H]serotonin from slices of rat raphe nuclei has been studied. Indoleamine agonists produced a dose-related inhibition of potassium-evoked tritium release which was reversed by methiothepin, metergoline minaserin and methysergide but not cyproheptadine or cinanserin. The relative order of antagonist potency for this effect was different from that obtained for the antagonism of indoleamine-induced inhibition of potassium-evoked tritium release from rat striatal slices previously loaded with [³H]-dopamine. The results show that the serotonin-autoreceptor located on cell bodies and dendrites in the raphe nucleus is different from the postsynaptic serotonin receptor located on dopamine nerve terminals in the striatum.     

Behavioral evidence for two types of cholinergic receptors in the C.N.S

Rats were trained to make a specific behavioral response in a T-maze apparatus conditional upon whether they were injected with 0.4 mg/kg nicotine or saline. Pretreatment with 0.25 mg/kg atropine sulfate had no effect on the rats' ability to discriminate the central cueing effect of nicotine. The administration of 0.25 and 0.50 mg/kg arecoline hydrobromide produced effects similar to saline. The results provide behavioral evidence for the possible existence of specific m-and n-cholinergic receptors in the central nervous system.     

Effects of the 5-HT2 receptor antagonist ritanserin on halothane-induced increase of inositol phosphates in porcine malignant hyperthermia

Recent studies have shown a significant increase of inositol phosphates (IPs) in skeletal muscle during episodes of halothane-induced malignant hyperthermia (MH) in pigs. After treatment with dantrolene and disappearance of MH crisis the IP concentrations returned to basal levels. In order to examine if the increase of IPs during halothane-induced MH may be related to an enhanced IP synthesis in response to activation of 5-HT₂ (5-hydroxytryptamine) receptors, the effects of ritanserin, a selective 5-HT₂ receptor antagonist, on IP levels were investigated. Biopsies of skeletal muscle of the hindlimbs were obtained in random order and IPs were determined in homozygous MH-susceptible (MHS) and MH-non-susceptible (MHN) swine in the following order: (1) basal, (2) after treatment with ritanserin (2.0 mg/kg), (3) after halothane challenge (3 vol% for 20 min). Basal concentrations of all IPs were higher in MHS than in MHN swine. Ritanserin did not cause any significant changes of IP levels compared to the basal concentrations in MHS and MHN pigs. In MHS pigs, ritanserin did not prevent a halothane-induced MH-crisis. After halothane challenge, 1,3,4-IP₃, 1,3,4,6-IP₄ and 1,3,4,5-IP₄ levels were increased in MHS (during MH crisis) vs. basal concentrations, whereas no changes were found in MHN pigs. Since the increases of IP levels in MHS pigs during MH crisis found in the present study were comparable to those without pretreatment with ritanserin, shown by recent studies, it may be concluded that ritanserin does not prevent the increase of IPs during a halothane-induced MH. Thus, the present data indicate that increases of IP levels during halothane-induced MH in swine are due to other mechanisms than 5-HT mediated enhancement of IP synthesis.     

Further evidence for the existence of two receptor sites for bradykinin responsible for the diphasic effect in the rat isolated duodenum.

Low doses of bradykinin (below 10 nM), as well as of K+ (below 10 mM) induced relaxation, whereas higher doses caused contraction of the rat duodenum. The relaxant responses induced by bradykinin and K+ were not affected by ouabain (1 microM), but pre-incubation with 5.9 mM K+ abolished the responses to that ion but not those to bradykinin. The contractile and relaxant components of the response to bradykinin (but not those to K+) increased with the time elapsed after mounting of the preparation, and this was due to stretching by the load of the recording system. Specific and reversible desensitization (tachyphylaxis) was observed with the contractile response (but not the relaxation) induced by bradykinin. Des-Arg9-bradykinin, an analogue specific for B1-receptors, was much less active than bradykinin, and elicited only a contractile response. Among four bradykinin potentiating peptides that were tested, potentiator C enhanced the relaxation only, whereas BPP5a and captopril potentiated only the contraction and BPP9a potentiated both types of response to bradykinin. Our results support the hypothesis that the relaxant and contractile components of the rat duodenum's response to bradykinin are due to actions at different receptor sites, which can be distinguished by their properties (desensitization) and their different apparent affinities for agonists and for potentiating peptides.     

Interactions between intracellular cyclic AMP and agonist-induced inositol phospholipid breakdown in isolated gastric mucosal cells of the rat

Summary The interactions between putative second effector mechanisms for hydrogen ion secretion were studied in isolated gastric cell preparations of the rat containing 60 - 70% parietal cells. Dibutyryl-CAMP and the compounds which increased the level of cAMP (histamine plus rolipram and forskolin plus rolipram) inhibited the carbachol-induced accumulation of [³H]inositol tris-, bis- and monophosphate. There was both a temporal and quantitative correlation between the increase in cAMP and the inhibition of the accumulation of [³H]inositol phosphates. Cimetidine attenuated the inhibitory effect of histamine on the formation of [³H]inositol phosphates. The enhancement of the accumulation of [³H]inositol phosphates by various concentrations of carbachol affected neither the basal nor the histamine-stimulated cAMP levels. In contrast to dibutyryl-cAMP, dibutyryl-cGMP did not modify the carbachol-induced formation of [³H]inositol phosphates. The biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), which activates protein kinase C, inhibited both the basal and carbachol-induced accumulation of [³H]inositol phosphates. We suggest that the inhibition of the formation of inositol trisphosphate by the increase in the intracellular level of cAMP and by the activation of protein kinase C might be intracellular negative feedback systems which prevent the overreaction of the acid-secreting parietal cells under the simultaneous influence of the physiological gastric secretagogues.Fellow of the Alexander von Humboldt Foundation, FRG Send offprint requests to U. Schwabe     

Functional evidence for the existence of adenosine receptors in the human heart

Adenosine added to isolated electrically driven preparations of human ventricular heart muscle antagonized the positive inotropic effect of isoprenaline (mean EC50 19 mumol 1(-1), n = 9). Similar effects were observed with the adenosine receptor agonist (-)-N6-phenylisopropyladenosine (mean EC50 0.5 mumol 1(-1), n = 7). These data provide functional evidence for the existence of adenosine receptors in the human myocardium which may modulate the force of contraction during beta-adrenergic stimulation and thus could be involved in the autoregulation of myocardial contractility.     

Further evidence for the existence of NK2 tachykinin receptor subtypes.

1. We have evaluated the biological activity of a number of neurokinin A (4-10), (NKA (4-10)) analogues in the endothelium-deprived rabbit isolated pulmonary artery (RPA) and hamster isolated trachea (HT), two tissues rich in different NK2 receptor subtypes. 2. MDL 28,564, a pseudopeptide selective for NK2 receptor sites, behaved as a full agonist in the RPA, while in the HT it competitively antagonized NKA or [beta Ala8]-NKA (4-10) contractile effects. 3. The peculiar behaviour of MDL 28,564 in the RPA and HT may be explained neither by a difference in receptor reserve between the two organs (the reserve being three times greater in RPA than in the HT) nor by a different affinity for the two receptor subtypes (identical dissociation constants, pKA or pKB, calculated in the RPA and in the HT). On the other hand, MDL 28,564 displayed a very different intrinsic efficacy for the two receptor subtypes. 4. The novel peptides MEN 10,295 ([Trp7, beta Ala8]-NKA-(4-10)) and MEN 10,296 ([Tyr5, Trp7, beta Ala8]-NKA-(4-10] behaved as weaker agonists than MDL 28,564 in the RPA, but retained appreciable agonist activity also in the HT. 5. The novel peptides: MEN 10,282 ([Tyr5, D-Trp6,8, Trp9, Arg10]-NKA-(4-10], MEN 10,449 ([diI-Try5, D-Trp6,8,9, Arg10]-NKA-(4-10] and the cyclic hexapeptide L 659,877 (cyclo [Leu-Met-Gln-Trp-Phe-Gly]) behaved as competitive antagonists against NKA contractile effects both in the RPA and HT. MEN 10,282 and MEN 10,449 were unable to distinguish between the NK2 receptor subtypes, having almost the same affinity in the two organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of anandamide: evidence for cellular diversity

The endocannabinoid N-arachidonylethanolamine (AEA) is accumulated by many cell types, but the mechanisms are unknown. Data from several laboratories are consistent with the hypothesis that the accumulation of AEA occurs via the action of a transmembrane carrier that binds and transports AEA. However, other data suggest that AEA is sufficiently lipophilic to transverse plasma membranes by passive diffusion and will accumulate if it is catabolized intracellularly. The controversy is muddied by the use of different cellular models and assays, all of which are assumed to be studying the same phenomena. The purpose of the studies reported herein was: first, to compare AEA accumulation and accumulation inhibitors in cerebellar granule neurons with a glioma cell line; and, second, to compare the neuronal accumulation of AEA with a closely related analog, N-palmitoylethanolamine (PEA). We have found that the accumulation of AEA by neurons and C6 glioma exhibits different affinity for AEA and inhibitor profiles. In addition, we find that the accumulation of AEA and PEA by neurons differs in the amount accumulated and in heterologous inhibition. These studies add to the evidence that the neuronal accumulation of AEA uniquely requires more than passive diffusion and fatty acid amide-mediated catabolism of intracellular AEA.     

Functional evidence for the existence of a capsaicin-sensitive innervation in the rat urinary bladder

Capsaicin (0.03-3 microM) induces contractions of the rat isolated bladder which are unaffected by either atropine (3 microM) or tetrodotoxin (0.5 microM). In the presence of capsaicin (0.1 microM) an enhancement of field stimulation-induced contractions was observed. Capsaicin-desensitization did not modify the height of these. The neurogenic nature of the capsaicin-induced contractions was proved by the observation that 'chronic' (48 h) denervation prevented, while 'acute' (2 h) denervation did not modify the effect of capsaicin. Denervated bladders maintained their responsiveness to acetylcholine but not to field stimulation. Isolated bladders from rat pups (1-2 days old) did not respond to capsaicin while strong contractile responses to acetylcholine or field stimulation were obtained in these preparations. In bladders from two week old animals, capsaicin produced similar contractions to those observed in preparations from adult animals. The bladders from rats receiving a high dose of capsaicin (50 mg kg-1 s.c.) at birth were heavier than those of their age-matched, vehicle-treated controls. Isolated bladders from 2 month old animals pretreated with capsaicin at birth were unresponsive to capsaicin while responsiveness to acetylcholine, substance P or field stimulation was unaffected compared with that of vehicle-treated controls. These experiments provide evidence that a capsaicin-sensitive innervation exists in the rat urinary bladder which undergoes a postnatal development at end organ level.     

Control of alveolar surfactant in rats at rest and during prolonged hyperpnoea: pharmacological evidence for two tissue pools of surfactant.

1. Propranolol, atropine and indomethacin (i.p.) affect neither the amount (PLalv), nor the specific activity (PLalvsp.act.) of alveolar surfactant-type phospholipids lavaged from the lungs of unanaesthetized rats, either at rest or made hyperpnoeic for 24 h with 5%CO2/13%O2/82%N2. 2. Whereas salbutamol (280 micrograms kg-1 body weight, i.p.) consistently increased PLalv and PLalvsp.act., pilocarpine (1.5, 3, 10 and 50 mg kg-1, i.p.) and labetalol (1 and 5 mg kg-1, i.p.) had no effect. The dose of pilocarpine reported by others to release surfactant (150 mg kg-1) induced marked salivation, diarrhoea, chromodacryorrhoea and a three-fold increase in tidal volume. 3. In the isolated perfused lung of the rat, salbutamol (1.5 microM) consistently increased PLalvsp.act, whereas pilocarpine (0.1 and 1 microM) had no effect on these variables. 4. In the isolated perfused lung, the maximum amount of surfactant that could be released by salbutamol (0.5 mM) was smaller than that which could be released in response to an increase in tidal volume (peak inflation pressure 28 cmH2O). 5. When the concentration of salbutamol in the isolated perfused lung was adjusted to produce the same increase in PLalv as did a single simulated deep breath, the PLalvsp.act was significantly increased by salbutamol, but not by the simulated deep breath. 6. We conclude, that neither the autonomic nervous system nor the prostaglandin system is essential for the release of surfactant at rest or during hyperpnoea. Furthermore, we suggest that two pools of surfactant, with different release mechanisms, exist in lung tissue.     

Further evidence for the existence of alpha2-mediated adrenergic vasoconstriction in human vessels

Summary To confirm the presence of alpha₂-mediated vasoconstriction in human vasculature, the effect of selective alpha₁- and alpha₂-agonists (methoxamine and B-HT 933) and antagonists (indoramin and Yohimbine) was studied in fourteen patients with mild, uncomplicated, essential hypertension. Drugs were infused, into the brachial artery at systemically ineffective rates, and concomitant changes in forearm blood flow were measured by strain gauge venous plethysmography.During control conditions, cumulative infusions either of methoxamine or B-HT 933 caused dose-related vasoconstriction, while both indoramin and yohimbine doubled forearm blood flow.Subsequently, the alpha₁-adrenoceptor mediated vasoconstriction produced by methoxamine was shown to be completely blocked by indoramin pretreatment, and to be left unchanged by yohimbine. The alpha₂-vascular stimulation by B-HT 933 was antagonized by previous yohimbine but not by indoramin pretreatment, thus fulfilling the pharmacological requirements for identification of distinct alpha-adrenoceptor mediated excitation-contraction pathways.The data provide further evidence of the existence of alpha₂-mediated vasoconstriction in human forearm vessels.     

Biochemical evidence of adrenergic interaction with cholinergic function in the central nervous system of the rat

The effect of centrally administered norepinephrine (NE) into the lateral ventricle on choline acetylase activity (ChAc), endogenous levels of NE, dopamine (DA) and serotonin (5HT) was studied in different regions of the rat brain. In normal rats, the highest activity of choline acetylase was found in the cerebral cortex, followed by the brain stem, the diencephalon, the hypothalamus and least in the cerebellum. Chronic administration of NE significantly increased the choline acetylase activity in all regions studied, whereas both the acute and the in vitro studies showed no significant change except in the brain stem. Serotonin level was increased in the cerebellum, but decreased in the diencephalon and the brain stem. There was no significant alteration in the level of NE in all areas studied except in the hypothalamus where there was an increase in the mean concentration. Tissue level of DA showed a significant increase in the cerebral cortex and the hypothalamus. Behaviourally, there was a significant increase in food intake on the first day of treatment with no significant change in water intake. The data suggests that NE may be involved in the regulation of acetylcholine synthesis. The significance of interaction between different monoamines and acetylcholine may be important in the study of drug tolerance phenomena.This work was supported by USPHS grant MH-12383; Dr. Ho is a recipient of a PMA Foundation grant award in morphology-pharmacology.     

Behavioral analysis of diazepam-induced memory deficits: evidence for sedation-like effects

Rats were trained on a nonmatching-to-sample task with delays of 2, 5, and 10 min. Subsequently, performance was assessed in three groups of rats following treatment with saline or diazepam (2.0 mg/kg) administered acutely or tested chronically in six administrations. Relative to treatment with saline, diazepam produced a deficit in discrimination performance, which was greater in the acutely treated rats than in those treated chronically. The deficit was not dependent on the length of the delays. Diazepam-treated animals differed from controls in erring on trials in which they failed to investigate both test objects, failed to investigate the test object for a long enough period of time, and displaced the test object on the preferred side of the apparatus. The hypothesis that these effects represented a sedation-like reduction in behavioral variability was also supported by evidence of a diazepam-induced decrease in gross bodily activity, increase in inactivity, and increase in latencies to respond to objects. No support was found for the involvement of diazepam-induced changes in habituation, extinction, or reward effects.     

Further evidence for the existence of opiate binding sites on neurosecretory LHRH mediobasal hypothalamic terminals

Opiate binding sites as well as LHRH and SRIF content were evaluated in mediobasal hypothalamus (MBH) from normal male rats and animals subjected to anterolateral deafferentation of the hypothalamus. A 87 and 44% depletion of LHRH and SRIF content respectively and a 50% decrease in the number of specific opiate binding sites was observed in the deafferented MBH. Dopa-decarboxylase activity remained unchanged. These data indicate that presynaptic opiate binding sites are present on LHRH and SRIF mediobasal hypothalamic nerve endings.     

Further evidence for the existence of a homogenous beta-endorphin-sensitive receptor population in the rat tail artery

Isolated rat tail arteries were perfused and vasoconstriction was evoked by electrical field stimulation (2 pulses at 1 Hz every 2 min). The vasoconstriction was depressed by DAGO (IC50 = 611 nM) and beta-endorphin (IC50 = 37 nM). Structuraly analogues and shorter fragments of beta-endorphin were also tested. beta-Endorphin and beta-endorphin-(1-26) were about equipotent whereas the beta-endorphin fragments 1-17, 1-16 and 6-31 were inactive. The potencies of beta-endorphin, beta-endorphin-(1-26), -(1-17) and -(1-16) were not changed in the presence of peptidase inhibitors. Structural analogues such as [D-Ala2]beta-endorphin or [Leu5]beta-endorphin had a somewhat lower potency than beta-endorphin itself. Naloxone 30 nM antagonized the effects of DAGO and beta-endorphin to a similar extent with dissociation constants 3.8 and 3.7 nM, respectively for the antagonist against the agonists. The results support the existence in the rat tail artery of a homogenous population of beta-endorphin-sensitive receptors which may belong to the epsilon-type.