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The endocannabinoid system is overactivated in arterial, pulmonary and portal hypertension. In this paper, we present limited clinical data concerning the role of cannabinoids in human hypertension including polymorphism of endocannabinoid system components. We underline differences between the acute cannabinoid administration and their potential hypotensive effect after chronic application in experimental hypertension. We discuss pleiotropic effects of cannabinoids on the cardiovascular system mediated via numerous neuronal and non-neuronal mechanisms both in normotension and in hypertension. The final results are dependent on the model of hypertension, age, sex, the cannabinoid ligands used or the action via endocannabinoid metabolites. More experimental and clinical studies are needed to clarify the role of endocannabinoids in hypertension, not only in the search for new therapeutic strategies but also in the context of cardiovascular effects of cannabinoids and the steadily increasing legalization of cannabis use for recreational and medical purposes. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

The endocannabinoid system is overactivated in arterial, pulmonary and portal hypertension. In this paper, we present limited clinical data concerning the role of cannabinoids in human hypertension including polymorphism of endocannabinoid system components. We underline differences between the acute cannabinoid administration and their potential hypotensive effect after chronic application in experimental hypertension. We discuss pleiotropic effects of cannabinoids on the cardiovascular system mediated via numerous neuronal and non-neuronal mechanisms both in normotension and in hypertension. The final results are dependent on the model of hypertension, age, sex, the cannabinoid ligands used or the action via endocannabinoid metabolites. More experimental and clinical studies are needed to clarify the role of endocannabinoids in hypertension, not only in the search for new therapeutic strategies but also in the context of cardiovascular effects of cannabinoids and the steadily increasing legalization of cannabis use for recreational and medical purposes. LINKED ARTICLES: This article is part of a themed section on 8

Background and purpose: Intravenous injection of the endocannabinoid anandamide induces complex cardiovascular changes via cannabinoid CB1, CB2 and vanilloid TRPV1 receptors. Recently, evidence has been accumulating that in vitro, but not in vivo, anandamide relaxes blood vessels, via an as yet unidentified, non-CB1 vascular cannabinoid receptor, sensitive to O-1918 (1,3-dimethoxy-5-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene). We here examined whether the anandamide-induced hypotension in urethane-anaesthetized rats was also mediated via a non-CB1 vascular cannabinoid receptor. Experimental approach: Effects of two antagonists (O-1918 and cannabidiol) of the non-CB1 vascular cannabinoid receptor on anandamide-induced changes in mean, systolic and diastolic blood pressure (MBP, SBP, DBP), mesenteric (MBF) and renal (RBF) blood flow and heart rate (HR) in urethane-anaesthetized rats was examined. Key results: In anaesthetized rats, anandamide (1.5–3 µmol·kg−1) and its stable analogue methanandamide (0.5 µmol·kg−1) caused a delayed and prolonged decrease in MBP, SBP, DBP, MBF and RBF by about 10–30% of the respective basal values without changing HR. In pithed rats, anandamide (3 µmol·kg−1) decreased blood pressure by about 15–20% of the basal value without affecting HR, MBF and RBF. All vascular changes were reduced by about 30–70% by cannabidiol and O-1918 (3 µmol·kg−1, each). Conclusions and implications: Non-CB1 cannabinoid vascular receptors, sensitive to O-1918, contribute to the hypotensive effect of anandamide in anaesthetized rats. Activation of these receptors may be therapeutically important as the endocannabinoid system could be activated as a compensatory mechanism in various forms of hypertension.

Background and purpose: We examined whether cannabinoid CB1 and histamine H3 receptors resemble α2-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats. Experimental approach: Effects of the cannabinoid agonist CP-55,940 and the H3 receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with 3H-noradrenaline. Key results: The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB1 or H3 receptors and α2 adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked 3H-noradrenaline release. Conclusions and implications: Urethane, but not pentobarbitone, abolished the H3 receptor-mediated vascular response in pithed rats and attenuated the CB1 receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB1, H3 and α2 receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.

Background and purpose: The endocannabinoid virodhamine is a partial agonist at the cannabinoid CB1 receptor and a full agonist at the CB2 receptor, and relaxes rat mesenteric arteries through endothelial cannabinoid receptors. Its concentration in the periphery exceeds that of the endocannabinoid anandamide. Here, we examined the influence of virodhamine on the human pulmonary artery. Experimental approach: Isolated human pulmonary arteries were obtained during resections for lung carcinoma. Vasorelaxant effects of virodhamine were examined on endothelium-intact vessels precontracted with 5-HT or KCl. Key results: Virodhamine, unlike WIN 55,212-2, relaxed 5-HT-precontracted vessels concentration dependently. The effect of virodhamine was reduced by endothelium denudation, two antagonists of the endothelial cannabinoid receptor, cannabidiol and O-1918, and a high concentration of the CB1 receptor antagonist rimonabant (5 μM), but only slightly attenuated by the NOS inhibitor L-NAME and not affected by a lower concentration of rimonabant (100 nM) or by the CB2 and vanilloid receptor antagonists SR 144528 and capsazepine, respectively. The COX inhibitor indomethacin and the fatty acid amide hydrolase inhibitor URB597 and combined administration of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance Ca2+-activated K+ channels attenuated virodhamine-induced relaxation. The vasorelaxant potency of virodhamine was lower in KCl- than in 5-HT-precontracted preparations. Conclusions and implications: Virodhamine relaxes the human pulmonary artery through the putative endothelial cannabinoid receptor and indirectly through a COX-derived vasorelaxant prostanoid formed from the virodhamine metabolite, arachidonic acid. One or both of these mechanisms may stimulate vasorelaxant Ca2+-activated K+ channels. British Journal of Pharmacology (2008) 155, 1034–1042; doi:10.1038/bjp.2008.371; published online 22 September 2008

Activation by CGP 12177 and cyanopindolol of the human and rat low-affinity state of β1-adrenoceptors increases frequency and contractile force and hastens relaxation in isolated cardiac tissues, and probably relaxes isolated vessels. In order to identify the positive inotropic, positive lusitropic and vasodilator effects of both agonists also in vivo, we have determined their effects on the left ventricular systolic pressure (LVSP), the rate of intraventricular pressure rise (+dP dt−1max) and decline (−dP dt−1max), the diastolic blood pressure (DBP) and the mesenteric blood flow (MBF) in pithed and vagotomized rats. CGP 12177 (0.1–100 nmol kg−1) and cyanopindolol (1–1000 nmol kg−1) dose-dependently enhanced all cardiac parameters. The nonselective β-adrenoceptor antagonist bupranolol 10 μmol kg−1 diminished the CGP 12177 (100 nmol kg−1)-stimulated increases in LVSP from 26.3±8.2 to 13.1±1.8 mmHg (P

1. We wanted to search for the mechanism(s) responsible for the brief pressor response induced by anandamide in urethane-anaesthetized rats. 2. The anandamide-induced pressor effect was not modified by the antagonists of cannabinoid CB(1) and vanilloid TRPV(1) receptors, SR 141716A (3 micromol kg(-1)) and capsazepine (1 micromol kg(-1)), respectively, by bilateral vagotomy and by pithing. Replacement of urethane by pentobarbitone virtually abolished the pressor effect of anandamide, both in pithed and vagotomized and in 'intact' rats (i.e. not treated in this manner). 3. The pressor effect of anandamide was reduced by the nonselective TRPV family inhibitor ruthenium red (3 micromol kg(-1)) and by the blocker of L-type calcium channels nifedipine (1 micromol kg(-1)), both in pithed urethane-anaesthetized rats and in 'intact' urethane-anaesthetized rats. The nonselective beta-adrenoceptor antagonist propranolol (0.1 or 0.3 micromol kg(-1)) and the nonselective NMDA receptor antagonist MK-801 (1 micromol kg(-1)) diminished the anandamide-induced vasopressor response in 'intact' but not in pithed rats. The inhibitory effect of propranolol in 'intact' rats was mimicked by the beta(2)-adrenoceptor antagonist ICI 118551 (1 micromol kg(-1)), but not by the beta(1)-adrenoceptor antagonist CGP 20712 (1 micromol kg(-1)). 4. The present study revealed that two mechanisms may be responsible for the anandamide-induced pressor response in urethane-anaesthetized rats. The first involves the central nervous system (probably the medulla oblongata) and is sensitive to propranolol and MK-801. The second, which is located peripherally (most probably in blood vessels), is sensitive to nifedipine, ruthenium red and pentobarbitone and, hence, probably represents a Ca(2+)-dependent mode of action.

Our study was undertaken to investigate whether bacterial endotoxin/lipopolysaccharide (LPS) affects the neurogenic vasopressor response in rats in vivo by presynaptic mechanisms and, if so, to characterize the type of presynaptic receptor(s) operating in the initial phase of septic shock. In pithed and vagotomized rats treated with pancuronium, electrical stimulation (ES) (1 Hz, 1 ms, 50 V for 10 s) of the preganglionic sympathetic nerve fibers or intravenous bolus injection of noradrenaline (NA) (1–3 nmol kg−1) increased the diastolic blood pressure (DBP) by about 30 mmHg. Administration of LPS (0.4 and 4 mg kg−1) under continuous infusion of vasopressin inhibited the neurogenic vasopressor response by 25 and 50%, respectively. LPS did not affect the increase in DBP induced by exogenous NA. The LPS-induced inhibition of the neurogenic vasopressor response was counteracted by the cannabinoid CB1 receptor antagonist SR 141716A (0.1 μmol kg−1), but not by the CB2 receptor antagonist SR 144528 (3 μmol kg−1), the vanilloid VR1 receptor antagonist capsazepine (1 μmol kg−1) or the histamine H3 receptor antagonist clobenpropit (0.1 μmol kg−1). The four antagonists by themselves did not affect the increase in DBP induced by ES or by injection of NA in rats not exposed to LPS. We conclude that in the initial phase of septic shock, the activation of presynaptic CB1 receptors by endogenously formed cannabinoids contributes to the inhibition of the neurogenic vasopressor response. British Journal of Pharmacology (2004) 142, 701–708. doi:10.1038/sj.bjp.0705839

We studied whether cannabinoid CB1 receptor gene disruption (to yield CB1−/− mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [3H]-noradrenaline (NA) (‘noradrenaline release') and from cerebral cortex slices preincubated with [3H]-choline (‘acetylcholine release'). NA release was higher by 37% in vas deferens from CB1−/− mice than in vas deferens from CB1+/+ mice. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and the CB1 receptor inverse agonist/antagonist SR 141716, increased NA release in vas deferens from CB1+/+ mice without affecting it in vas deferens from CB1−/− mice. Atrial NA release did not differ between CB1+/+ and CB1−/− mice nor did WIN 55,212-2 affect NA release in either strain. Cortical acetylcholine (Ach) release did not differ between CB1+/+ and CB1−/− mice. WIN 55,212-2 inhibited, but SR 141716 did not affect, Ach release in the cortex from CB1+/+ mice. Both drugs did not alter Ach release in the cortex from CB1−/− mice. Tritium content did not differ between CB1+/+ and CB1−/− mice in any preparation. In conclusion, the increase in NA release associated with CB1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB1 receptors of CB1+/+ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB1 receptors, whereas the involvement of non-CB1–non-CB2 receptors can be excluded. Keywords: Cerebral cortex, vas deferens, atrium, acetylcholine release, noradrenaline release, cannabinoid CB1 receptors, CB1 receptor-deficient mouse, presynaptic receptors, SR 141716 Introduction Cannabinoid CB1 receptors serve as presynaptic inhibitory receptors on a variety of central and peripheral neurones (for review, see Schlicker & Kathmann, 2001; Howlett et al., 2002). These receptors are activated by endocannabinoids like anandamide, 2-arachidonoylglycerol, noladin ether and virodhamine (Hanus et al., 2001; Porter & Felder, 2001; Porter et al., 2002). Many types of presynaptic CB1 receptors appear to be subject to an endogenous tone as suggested by the fact that CB1 receptor inverse agonists/antagonists like SR 141716 facilitate the release of the respective neurotransmitter (for review, see Pertwee, 1999; Schlicker & Kathmann, 2001). It is of interest in this context whether lack of CB1 receptors will lead to the same result. We have indeed found recently (Kathmann et al., 2001b) that in hippocampal slices, in which SR 141716 increases acetylcholine (Ach) release (Kathmann et al., 2001a), the release of this transmitter was also increased by CB1 receptor deficiency (CB1 receptor knockout mouse (CB1−/−) generated by Zimmer et al. (1999) from C57BL/6J mice). This alteration in transmitter release is very specific inasmuch as hippocampal noradrenaline (NA) release and striatal Ach release (both of which are not subject to modulation via CB1 receptors; Schlicker et al., 1997; Kathmann et al., 2001a) did not differ between both strains (Kathmann et al., 2001b). The aim of the present study was to examine whether parallel effects of SR 141716 and of CB1 receptor deficiency also occur in other isolated tissues of the mouse. For this purpose, we determined the influence of CB1 receptor gene disruption on NA release in the vas deferens and in the atrium. NA release in the vas deferens is inhibited via presynaptic CB1 receptors subject to an endogenous tone (Rinaldi-Carmona et al., 1994; Pertwee et al., 1996), whereas atrial NA release is not affected by presynaptic CB1 receptors at all (Trendelenburg et al., 2000). Our study was extended to cerebral cortex slices in which Ach release was determined. This experimental model differs from the latter two and from the models considered in our previous study (Kathmann et al., 2001b) in that the release of the transmitter is inhibited via presynaptic CB1 receptors, which are, however, not subject to an endogenous tone, that is, Ach release is not facilitated by SR 141716 (Kathmann et al., 2001a).

1. Atypical beta-adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats. 2. CGP 12177, an agonist of the atypical beta-adrenoceptor, increased heart rate dose-dependently. Its dose-response curve was shifted to the right by S-(-)-bupranolol 10 micro mol kg(-1) by a factor of 8.4, but not affected by the same dose of R-(+)-bupranolol. 3. Desmethylbupranolol and compounds BK-21, BK-22, BK-23 and BK-25 also increased heart rate dose-dependently. The beta(1)-adrenoceptor antagonist CGP 20712 given in combination with the beta(2)-adrenoceptor antagonist ICI 118,551 (0.1 micro mol kg(-1) each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r=0.91, P

The cannabinoid CB1 receptor inverse agonist/antagonist SR 141716 increases acetylcholine release in rodent hippocampus and improves memory in some experimental paradigms. Since drugs like SR 141716 may represent a novel class of cognition-enhancing drugs, we wanted to check whether the function of the CB1 receptor is preserved during ageing. Hippocampal and striatal slices from 2- to 3- and 24- to 28-month-old C57BL/6J mice were preincubated with [3H]-choline or [3H]-noradrenaline ([3H]-NA) and superfused. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and SR 141716 facilitated, the electrically (3 Hz) evoked tritium overflow in hippocampal slices (preincubated with [3H]-choline) from young and aged mice to the same extent. The evoked overflow per se was less by 33% in slices from aged animals. WIN 55,212-2 and SR 141716 did not affect, but the muscarinic receptor agonist oxotremorine inhibited, the evoked (3 Hz) overflow in striatal slices (preincubated with [3H]-choline) from young and aged mice to the same extent. The evoked overflow per se tended to be less in slices from aged animals. The evoked (0.3 Hz) overflow in hippocampal slices (preincubated with [3H]-NA) was not affected by WIN 55,212-2 and SR 141716, but was inhibited by histamine (via H3 receptors) in slices from young mice and, to a somewhat less extent, in slices from aged mice. The evoked overflow per se did not differ between age groups. In conclusion, the function of the CB1 receptor involved in the tonic inhibition of hippocampal acetylcholine release is preserved in aged mice. Keywords: Ageing, acetylcholine release, noradrenaline release, hippocampus, striatum, cannabinoid CB1 receptor, muscarinic receptors, histamine H3 receptor, C57BL/6J mouse, SR 141716 Introduction Hashish/marijuana and agonists at cannabinoid CB1 receptors impair learning and memory in humans and animals (for review, see Ameri, 1999; Sullivan, 2000). Two lines of evidence suggest that the endocannabinoid system is involved in cognitive processes. First, the CB1 receptor inverse agonist/antagonist SR 141716 facilitates memory tasks in some behavioural paradigms (Terranova et al., 1996; Lichtman, 2000). Second, in each of the three CB1 receptor knockout mice, marked alterations of learning were observed (Reibaud et al., 1999; Marsicano et al., 2002; Varvel & Lichtman, 2002). CB1 receptors are frequently located presynaptically on nerve endings, where their activation results in inhibition of release of the respective neurotransmitter (for review, see Schlicker & Kathmann, 2001; Howlett et al., 2002). CB1 receptor activation also causes inhibition of acetylcholine release in the hippocampus of rodents, whereas SR 141716 or CB1 receptor deficiency increases the release of this transmitter (for review, see Schlicker & Kathmann, 2001). Thus, the possibility has to be considered that the septohippocampal cholinergic system, the morphological and functional integrity of which is important for learning and memory (Dutar et al., 1995), is implicated in the detrimental effects of cannabinoids on cognitive processes. Even if this should not hold true, the fact that CB1 receptor inverse agonists/antagonists (e.g. SR 141716), like the reversible cholinesterase inhibitors (e.g. donepezil; for review, see Fodero & Small, 2002), increase synaptic acetylcholine levels might mean that CB1 receptor inverse agonists/antagonists might represent a new class of cognition-enhancing agents for use in humans. In this context, it is of interest that CB1 receptor-mediated inhibition of acetylcholine release and its facilitation by SR 141716 have recently also been shown in superfused slices of the human brain (Steffens et al., 2002). Since cognitive decline in humans most frequently occurs in aged individuals, the question has to be addressed whether the molecular substrates for potential cognition-enhancing drugs retain their function during the ageing process. With respect to CB1 receptors, this aspect has so far been studied only rarely. An age-induced reduction of cannabinoid receptor binding and mRNA levels has been shown in the striatum and other extrapyramidal brain regions (Mailleux & Vanderhaeghen, 1992; Romero et al., 1998), but is less marked in other brain regions including the cerebral cortex or the hippocampus (Berrendero et al., 1998). In the study of Romero et al. (1998), a functional parameter, the (CB1 receptor-mediated) stimulation of [35S]-guanylyl-5′-O-(γ-thio)-triphosphate binding by WIN 55,212-2, was also studied in extrapyramidal areas and was found to be decreased in some brain regions. The aim of the present study was to check whether the function of the CB1 receptors involved in the inhibition of hippocampal acetylcholine release is preserved in aged mice of the C57BL/6 strain (frequently used for studies of the ageing brain; for review, see Jucker & Ingram, 1997). We examined the effects of cannabinoid receptor ligands on the electrically evoked tritium overflow from superfused hippocampal slices preincubated with [3H]-choline. For the sake of comparison, the electrically evoked tritium overflow from striatal slices preincubated with [3H]-choline and from hippocampal slices preincubated with [3H]-noradrenaline and its modulation via presynaptic receptors were studied as well.

We determined the effects of nociceptin/orphanin FQ and the NOP receptor ligands acetyl-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (Ac-RYYRIK-NH2) and naloxone benzoylhydrazone on transmitter release in vitro. The electrically evoked tritium overflow from guinea-pig and mouse striatal slices and guinea-pig retinal discs preincubated with [3H]-dopamine was inhibited by nociceptin/orphanin FQ (pEC50 7.9, 7.6 and 8.6; Emax 30, 50 and 55%). Ac-RYYRIK-NH2 0.032 μM and naloxone benzoylhydrazone 5 μM antagonized the effect of nociceptin/orphanin FQ in striatal slices of the guinea-pig (apparent pA2 9.1 and 6.8) and the mouse (apparent pA2 9.2 and 7.5) and strongly attenuated the effect of nociceptin/orphanin FQ 0.1 μM in guinea-pig retinal discs. Ac-RYYRIK-NH2 0.032 μM did not affect the evoked overflow by itself whereas naloxone benzoylhydrazone 5 μM inhibited it in each tissue. The electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]-noradrenaline was inhibited by nociceptin/orphanin FQ (pEC50 7.9, Emax 85%), Ac-RYYRIK-NH2 (pEC50 8.3, Emax 47%) but not affected by naloxone benzoylhydrazone 5 μM. Ac-RYYRIK-NH2 and naloxone benzoylhydrazone showed apparent pA2 values of 8.6 and 6.9. In conclusion, the inhibitory effect of nociceptin/orphanin FQ on dopamine release in the striatum and retina and on noradrenaline release in the cerebral cortex is mediated via NOP receptors. Ac-RYYRIK-NH2 behaves as an extremely potent NOP receptor antagonist in the striatum and retina and as a partial agonist in the cortex. Keywords: NOP receptor, nociceptin, orphanin FQ, dopamine release, noradrenaline release, striatum, guinea-pig retina, mouse cerebral cortex, Ac-RYYRIK-NH2, naloxone benzoylhydrazone Introduction A fourth type of opioid receptor, which has a homology in the amino acid sequence to the three classical opioid receptors of about 50%, was first identified in 1994 (for review, see Mogil & Pasternak, 2001). This receptor, termed NOP according to the nomenclature of IUPHAR (International Union of Pharmacology; Cox et al., 2000), is activated by a heptadecapeptide, termed nociceptin/orphanin FQ(N/OFQ) (for review, see Mogil & Pasternak, 2001). The NOP and the classical opioid receptors resemble each other with respect to the transduction mechanisms, i.e. inhibition of adenylyl cyclase (Hawes et al., 2000) and of voltage-dependent Ca2+ channels and activation of voltage-dependent K+ channels (Moran et al., 2000). However, the NOP receptor markedly differs from the classical opioid receptors in its pharmacological properties (e.g. lack of antagonistic effect of the opioid receptor blocking agent naloxone) (Calo et al., 2000) and its distribution in the brain (Mollereau & Mouledous, 2000). The most striking difference, however, occurs with respect to the influence on nociception. Thus, N/OFQ elicits hyperalgesia rather than analgesia when administered at a supraspinal site (for review, see Mogil & Pasternak, 2001). The action of N/OFQ is, however, not restricted to nociception since the heptadecapeptide also affects feeding (Polidori et al., 2000), learning and memory (Noda et al., 2000) and anxiety (Jenck et al., 1997) as well as numerous vegetative functions (Mogil & Pasternak, 2001). One aspect in which the classical opioid and the NOP receptors resemble each other is that they occur presynaptically on a variety of peripheral and central neurones where they cause inhibition of the release of the respective neurotransmitter (Moran et al., 2000; Schlicker & Morari, 2000). N/OFQ e.g. affects the release of dopamine and this has been shown by direct measurement of transmitter release or electrophysiological techniques for the (i) nigrostriatal (Konya et al., 1998; Schlicker et al., 1998a; Shieh & Pan, 2001), (ii) mesolimbic (Murphy et al., 1996; Murphy & Maidment, 1999; Lutfy et al., 2001; Shieh & Pan, 2001; Zheng et al., 2002), (iii) tuberoinfundibular (Wagner et al., 1998; Shieh & Pan, 2001) and (iv) incertohypothalamic system (Shieh & Pan, 2001). The involvement of NOP receptors in the effect of N/OFQ has so far been proven only in the studies by Shieh & Pan (2001; use of an antisense oligodeoxynucleotide) and by Zheng et al. (2002; use of the weakly potent NOP receptor antagonist [Phe1Ψ(CH2-NH)Gly2]-N/OFQ(1-13)NH2). The need for such experiments is highlighted by a study on conscious rats in which intrastriatal administration of N/OFQ increased dopamine release in a manner sensitive to naloxone thereby arguing against the involvement of NOP receptors (Konya et al., 1998). We therefore examined in two in vitro models of the striatum, i.e. superfused guinea-pig and mouse striatal slices, in which N/OFQ inhibits dopamine release in the presence of naloxone (Schlicker et al., 1998a), whether this effect is counteracted by NOP receptor antagonists. We used the selective and highly potent NOP receptor antagonist acetyl-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (Ac-RYYRIK-NH2) (Dooley et al., 1997; Berger et al., 1999) and a non-selective and less potent NOP receptor antagonist, naloxone benzoylhydrazone, used by our own group (Schlicker et al., 1998b; Malinowska et al., 2000a) and by other investigators (Bucher, 1998; Siniscalchi et al., 1999; Ho et al., 2000; Bigoni et al., 2002). Furthermore, we studied whether N/OFQ affects dopamine release also in guinea-pig retinal discs since the influence of N/OFQ on the retinal dopamine system has so far not been examined. Finally, we compared the potencies of Ac-RYYRIK-NH2 and naloxone benzoylhydrazone at the NOP receptor causing inhibition of noradrenaline release in the mouse brain cortex, i.e. at an NOP receptor previously identified and characterized by our group (Schlicker et al., 1998b; Bauer et al., 1999; Werthwein et al., 1999).

We determined the affinities of eight novel histamine H(3)-receptor ligands (ethers and carbamates) for H(3)-receptor binding sites and their agonistic/antagonistic effects in two functional H(3)-receptor models. The compounds differ from histamine in that the ethylamine chain is replaced by a propyloxy chain; in the three ethers mentioned below (FUB 335, 373 and 407), R is n-pentyl, 3-methylbutyl and 3,3-dimethylbutyl, respectively. The compounds monophasically inhibited [(3)H]-N(alpha)-methylhistamine binding to mouse cerebral cortex membranes (pK(i) 7.51 - 9.53). The concentration-response curve of histamine for its inhibitory effect on the electrically evoked [(3)H]-noradrenaline overflow from mouse cortex slices was shifted to the right by these compounds (apparent pA(2) 6.61 - 8.00). Only FUB 373 and 407 inhibited the evoked overflow by themselves (intrinsic activities 0.3 and 0.4); these effects were counteracted by the H(3)-receptor antagonist clobenpropit. [(35)S]-GTPgammaS binding to mouse cortex membranes was stimulated by the H(3)-receptor agonist (R)-alpha-methylhistamine in a manner sensitive to clobenpropit. Among the novel compounds only FUB 373 and 407 stimulated [(35)S]-GTPgammaS binding (intrinsic activities 0.6 and 0.4). In conclusion, the novel compounds are partial H(3)-receptor agonists (FUB 373 and 407) or H(3)-receptor antagonists; comparison with FUB 335 shows that the transition from antagonist to agonist is caused by a slight structural change. A protonated N atom in the side chain is not necessary for agonism at H(3) receptors, proposing a receptor-ligand interaction different from that of classical agonists.

We examined whether acetylcholine release in the hippocampus and striatum and noradrenaline release in the hippocampus is altered in CB(1) receptor-deficient mice. The electrically evoked tritium overflow from hippocampal slices preincubated with [(3)H]-choline was increased by about 100% in CB(1)(-/-) compared to CB(1)(+/+) mice whereas the electrically evoked tritium overflow from striatal slices preincubated with [(3)H]-choline and from hippocampal slices preincubated with [(3)H]-noradrenaline did not differ. The cannabinoid receptor agonist, WIN 55,212-2, inhibited, and the CB(1) receptor antagonist, SR 141716, facilitated, the evoked tritium overflow from hippocampal slices (preincubated with [(3)H]-choline) from CB(1)(+/+) as opposed to CB(1)(-/-) mice. Both drugs did not affect the evoked tritium overflow from striatal slices (preincubated with [(3)H]-choline) and from hippocampal slices (preincubated with [(3)H]-noradrenaline) from CB(1)(+/+) and CB(1)(-/-) mice. The selective increase in acetylcholine release in CB(1)(-/-) mice may indicate that the presynaptic CB(1) receptors on the cholinergic neurones of the mouse hippocampus are tonically activated and/or constitutively active in vivo.

In segments of rat vena cava preincubated with [(3)H]-noradrenaline and superfused with physiological salt solution, the influence of agmatine on the electrically evoked [(3)H]-noradrenaline release, the EP(3) prostaglandin receptor-mediated and the alpha(2D)-adrenoceptor-mediated inhibition of evoked [(3)H]-noradrenaline release was investigated. Agmatine (0.1-10 microM) by itself was without effect on evoked [(3)H]-noradrenaline release. In the presence of 10 microM agmatine, the prostaglandin E(2)(PGE(2))-induced EP(3)-receptor-mediated inhibition of [(3)H]-noradrenaline release was not modified, whereas the alpha(2D)-adrenoceptor-mediated inhibition of [(3)H]-noradrenaline release induced by noradrenaline, moxonidine or clonidine was more pronounced than in the absence of agmatine. However, 1 mM agmatine antagonized the moxonidine-induced inhibition of [(3)H]-noradrenaline release. Agmatine concentration-dependently inhibited the binding of [(3)H]-clonidine and [(3)H]-rauwolscine to rat brain cortex membranes (K(i) values 6 microM and 12 microM, respectively). In addition, 30 and 100 microM agmatine increased the rate of association and decreased the rate of dissociation of [(3)H]-clonidine resulting in an increased affinity of the radioligand for the alpha(2D)-adrenoceptors. [(14)C]-agmatine labelled specific binding sites on rat brain cortex membranes. In competition experiments. [(14)C]-agmatine was inhibited from binding to its specific recognition sites by unlabelled agmatine, but not by rauwolscine and moxonidine. In conclusion, the present data indicate that agmatine both acts as an antagonist at the ligand recognition site of the alpha(2D)-adrenoceptor and enhances the effects of alpha(2)-adrenoceptor agonists probably by binding to an allosteric binding site of the alpha(2D)-adrenoceptor which seems to be labelled by [(14)C]-agmatine.

Mouse brain slices preincubated with [3H]-noradrenaline or [3H]-serotonin were superfused with medium containing naloxone 10 μM; we studied whether nociceptin (the endogenous ligand at ORL1 receptors) affects monoamine release. Furthermore, the affinities of ORL1 ligands were determined using [3H]-nociceptin binding. The electrically (0.3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]-noradrenaline was inhibited by nociceptin and [Tyr14]-nociceptin (maximally by 80%; pEC50 7.52 and 8.28) but not affected by [des-Phe1]-nociceptin (pEC50

1. We have previously shown (Malinowska & Schlicker, 1996) that the atypical beta-adrenoceptor involved in the positive chronotropic effect of the so-called non-conventional partial beta-adrenoceptor agonists CGP 12177 and cyanopindolol in the pithed rat possesses properties markedly different from those observed for beta3-adrenoceptors in the literature. In the present study, we have directly compared the pharmacological properties of the atypical cardiostimulant beta-adrenoceptor and of the beta3-adrenoceptor mediating the thermogenic response in the brown adipose tissue in pithed and vagotomized rats. 2. Heart rate was dose-dependently increased by CGP 12177 and cyanopindolol by maximally 150 and 100 beats min(-1), yielding pED50 values of 8.0 and 7.3, respectively (pED50, -log10 of the dose in mol kg(-1) body weight i.v. causing the half-maximum effect), but not affected by the selective beta3-adrenoceptor agonist CL 316243 (pED50 > 6.0). 3. CGP 12177, cyanopindolol and CL 316243 increased temperature in the brown adipose tissue by maximally 1 degree C (pED50 values 7.4, 6.3 and 8.6, respectively). 4. The beta1-adrenoceptor antagonist CGP 20712 10 micromol kg(-1), attenuated the cardiostimulatory effect of CGP 12177 and, at a still higher dose (30 micromol kg(-1)), also antagonized its thermogenic effect. The -log10 values of the doses causing a two fold shift of the dose-response curves (DRCs) of CGP 12177 to the right were 6.1 and 5.2, respectively, and were much lower than the corresponding value for the antagonism of CGP 20712 against the beta1-adrenoceptor-mediated positive chronotropic effect which was 8.6. 5. The cardiostimulant and the thermogenic effect of CGP 12177 were not affected by the beta2-adrenoceptor antagonist ICI 118551 10 micromol kg(-1). 6. The beta3-adrenoceptor antagonist SR 59230A (which, by itself, caused a beta1-adrenoceptor-mediated increase in heart rate and, for this reason, was studied after administration of a low dose of CGP 20712) attenuated the cardiostimulant and the thermogenic effect of CGP 12177 to a similar extent. The -log10 values of the doses causing two fold rightward shifts of the DRCs of CGP 12177 were 5.9 and 5.7, respectively. 7. The non-selective beta-adrenoceptor antagonist bupranolol diminished the cardiostimulant and thermogenic response to a very similar extent. The -log10 values causing two fold rightward shifts of the DRCs of CGP 12177 were 5.6 and 5.7, respectively, and were much lower than the corresponding values for the antagonism of bupranolol against the beta1-adrenoceptor-mediated positive chronotropic effect and the beta2-adrenoceptor-mediated decrease in diastolic blood pressure which were 7.6 and 8.3, respectively. 8. The rank order of agonistic potencies for the cardiostimulant effect (CGP 12177 > cyanopindolol > CL 316243) differs from that for the thermogenic response in the brown adipose tissue (CL 316243 > CGP 12177 > cyanopindolol); furthermore, there is a difference with respect to the rank orders of antagonistic potencies for cardiostimulation (CGP 20712 > or = SR 59230A > or = bupranolol > ICI 118551) and thermogenesis (SR 59230A = bupranolol > CGP 20712 > ICI 118551). 9. In conclusion, our study provides further evidence that the atypical cardiostimulant beta-adrenoceptors (causing an increase in heart rate) and beta3-adrenoceptors are pharmacologically different.

1. We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring-containing alkyl residue [corrected]. 2. The specific binding of [3H]-N alpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the ten compounds at pKi values ranging from 7.56 to 8.68. 3. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]-noradrenaline was antagonized by the ten compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.07 to 9.20. 4. The electrically induced contraction in guinea-pig ileum strips (which was abolished by atropine) was inhibited by the H3 receptor agonists R-(-)-alpha-methylhistamine (pEC50 7.76), N alpha-methylhistamine (7.90) and imetit (8.18). The concentration-response curve of R-(-)-alpha-methylhistamine was shifted to the right by thioperamide (apparent pA2 8.79) and by the ten novel compounds (range of pA2 values 6.64-8.81). 5. The affinities and potencies were compared by linear regression analysis. This analysis was extended to thioperamide, the standard H3 receptor antagonist, which is also capable of differentiating between H3A and H3B sites. Comparison of the apparent pA2 values in the two functional H3 receptor models yielded a regression coefficient of 0.77 (P0.02). When the pA2 of the drugs in the mouse brain cortex were compared to the pXj for H3 sites (ten novel compounds) and for H3A sites (thioperamide), a significant correlation (r = 0.87; P0.001) was obtained. There was, however, no significant correlation when the pKi of thioperamide for H3B sites was used instead (r = 0.52). In a similar manner, comparison of the pA2 in the guinea-pig ileum with the pKi in the binding assay yielded a significant correlation(r = 0.70, P0.05) only when the pKi of thioperamide for H3A sites was used but not when its pKi forH3B sites was considered (r = 0.17, NS).6 On the basis of these results, structure-activity relationships for the novel H3 receptor antagonists,and the nature of the H3 receptors in the guinea-pig ileum and mouse brain, are considered.

Intravenous injection of the endocannabinoid anandamide induces complex cardiovascular changes via cannabinoid CB(1), CB(2) and vanilloid TRPV1 receptors. Recently, evidence has been accumulating that in vitro, but not in vivo, anandamide relaxes blood vessels, via an as yet unidentified, non-CB(1) vascular cannabinoid receptor, sensitive to O-1918 (1,3-dimethoxy-5-2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-benzene). We here examined whether the anandamide-induced hypotension in urethane-anaesthetized rats was also mediated via a non-CB(1) vascular cannabinoid receptor.Effects of two antagonists (O-1918 and cannabidiol) of the non-CB(1) vascular cannabinoid receptor on anandamide-induced changes in mean, systolic and diastolic blood pressure (MBP, SBP, DBP), mesenteric (MBF) and renal (RBF) blood flow and heart rate (HR) in urethane-anaesthetized rats was examined.In anaesthetized rats, anandamide (1.5-3 micromol.kg(-1)) and its stable analogue methanandamide (0.5 micromol.kg(-1)) caused a delayed and prolonged decrease in MBP, SBP, DBP, MBF and RBF by about 10-30% of the respective basal values without changing HR. In pithed rats, anandamide (3 micromol.kg(-1)) decreased blood pressure by about 15-20% of the basal value without affecting HR, MBF and RBF. All vascular changes were reduced by about 30-70% by cannabidiol and O-1918 (3 micromol.kg(-1), each).Non-CB(1) cannabinoid vascular receptors, sensitive to O-1918, contribute to the hypotensive effect of anandamide in anaesthetized rats. Activation of these receptors may be therapeutically important as the endocannabinoid system could be activated as a compensatory mechanism in various forms of hypertension.

1. The purpose of the present study was to relate the effects of the novel drug, anpirtoline, on 5-hydroxytryptamine (5-HT) receptor subtypes to its antinociceptive and antidepressant-like actions in rodents. 2. Binding assays with rat brain membranes have shown that anpirtoline bound with a much higher affinity to 5-HT1B receptor (Ki = 28 nM) than to 5-HT1A (Ki = 150 nM) and 5-HT2 (Ki = 1.49 microM) receptors. 3. Like 5-HT, anpirtoline concentration-dependently inhibited forskolin-stimulated adenylate cyclase activity in homogenates from the rat substantia nigra. Both effects were not additive, and could be prevented by 5-HT1B receptor antagonists such as propranolol and penbutolol. 4. In superfused rat and pig brain cortex slices preincubated with [3H]-5-HT, the electrically evoked tritium overflow was inhibited by anpirtoline and 5-HT. Whereas 5-HT was equipotent in both tissues (EC50 = 69 nM), anpirtoline was markedly less potent in pig brain cortex slices (EC50 = 1190 nM) than in rat brain cortex slices (EC50 = 55 nM). The concentration-response curve for anpirtoline was shifted to the right by metitepine in both preparations. 5. In the social behaviour deficit test, anpirtoline and trifluoromethylphenyl-piperazine were effective in reversing the isolation-induced impairments in mice, an effect shown only by compounds with agonist properties at the 5-HT1B receptor. 6. In the electrostimulated pain test using mice, anpirtoline dose-dependently increased the pain threshold with an ED50 of 0.52 mg kg-1, i.p. The antinociceptive activity of anpirtoline was abolished by pretreatment with cyproheptadine or propranolol.7. In the forced swimming test in rats, anpirtoline induced a dose-related increase in swimming activity. With an ED50 value of 4.6mgkg-1, i.p., anpirtoline was 4 times more potent than the two standard compounds imipramine and desipramine. The decrease of immobility time or the increase of active periods in this model of behavioural despair is suggested to be characteristic of antidepressant drugs.8. Anpirtoline exhibits both antinociceptive and antidepressant-like activities in animals. It is probable that anpirtoline elicits these pharmacological effects via its agonist effect on 5-HT1B and 5-HT1A receptors.

1 Segments of the rat vena cava preincubated with [3H]-noradrenaline were superfused with [3H]-noradrenaline-free solution containing desipramine and corticosterone and the effects of γ-aminobutyric acid (GABA) receptor ligands and of histamine on tritium overflow evoked by transmural electrical stimulation were studied. 2 GABA inhibited, and histamine failed to affect, the electrically (0.66 Hz) evoked tritium overflow both in the absence and presence of rauwolscine (which was present in the superfusion medium in the subsequent experiments). The effect of GABA was less pronounced at a stimulation frequency of 2 Hz. 3 The inhibitory effect of GABA (pIC35 5.83) on the electrically (0.66 Hz) evoked overflow was mimicked by the GABAB receptor agonist, R-(−)-baclofen (6.07) and less potently by S-(+)-baclofen (3.30) and the GABAA receptor agonist, muscimol (3.70). The concentration-response curve of GABA was shifted to the right by the GABAB receptor antagonist, CGP 35348 (P-(3-aminopropyl)-P-diethoxymethyl-phosphinic acid; apparent pA2 4.76), but not affected by the GABAA receptor antagonist, (−)-bicuculline methiodide 100 μmol1−1. Given alone, (−)-bicuculline methiodide slightly increased, and CGP 35348 did not affect, the evoked overflow. 4 The effect of GABA in veins from rats treated for 14 days with RS-baclofen (10 mg kg−1, i.p. once daily) did not differ from that in veins from rats which received the vehicle instead. The effect of GABA also did not differ in veins from rats treated once either with RS-baclofen or with its vehicle. 5 The results suggest that the postganglionic sympathetic nerve fibres in the rat vena cava are endowed with presynaptic GABAB receptors, but not with receptors for histamine. (−)-Bicuculline methiodide increases noradrenaline release by a mechanism not related to GABAA, GABAB, α2-receptors or noradrenaline uptake.

1. The effects of gamma-aminobutyric acid (GABA) and related drugs on the vasopressor response induced by electrical stimulation (single pulse of 30 V and 1 ms) of the preganglionic sympathetic nerve fibres or by injection of noradrenaline 0.3 nmol kg-1 were studied in the pithed rat. 2. The electrically-induced increase in diastolic blood pressure was inhibited by GABA and the GABAB-receptor agonist R-(--)-baclofen but was not affected by its S-(+)-enantiomer and by the GABAA-receptor agonists muscimol and 3-aminopropane sulphonic acid. 3. The dose-response curve of R-(--)-baclofen for its inhibitory effect on the electrically-induced vasopressor response was shifted to the right by the GABAB-receptor antagonist 2-hydroxysaclofen, but was not affected by the GABAA-receptor antagonist bicuculline. 2-Hydroxysaclofen and bicuculline by themselves did not affect the electrically-induced vasopressor response. 4. The increase in diastolic blood pressure induced by exogenous noradrenaline was not affected by the GABA-related drugs, which also had no (or very slight) effects on the basal diastolic blood pressure. 5. It is concluded that GABA inhibits catecholamine release in the resistance vessels of the rat via GABAB-receptors, probably located presynaptically on the postganglionic sympathetic nerve fibres.

1. Our study was undertaken to investigate whether bacterial endotoxin/lipopolysaccharide (LPS) affects the neurogenic vasopressor response in rats in vivo by presynaptic mechanisms and, if so, to characterize the type of presynaptic receptor(s) operating in the initial phase of septic shock. 2. In pithed and vagotomized rats treated with pancuronium, electrical stimulation (ES) (1 Hz, 1 ms, 50 V for 10 s) of the preganglionic sympathetic nerve fibers or intravenous bolus injection of noradrenaline (NA) (1-3 nmol x kg(-1)) increased the diastolic blood pressure (DBP) by about 30 mmHg. Administration of LPS (0.4 and 4 mg x kg(-1)) under continuous infusion of vasopressin inhibited the neurogenic vasopressor response by 25 and 50%, respectively. LPS did not affect the increase in DBP induced by exogenous NA. 3. The LPS-induced inhibition of the neurogenic vasopressor response was counteracted by the cannabinoid CB(1) receptor antagonist SR 141716A (0.1 micromol x kg(-1)), but not by the CB(2) receptor antagonist SR 144528 (3 micromol x kg(-1)), the vanilloid VR1 receptor antagonist capsazepine (1 micromol x kg(-1)) or the histamine H(3) receptor antagonist clobenpropit (0.1 micromol x kg(-1)). The four antagonists by themselves did not affect the increase in DBP induced by ES or by injection of NA in rats not exposed to LPS. 4. We conclude that in the initial phase of septic shock, the activation of presynaptic CB(1) receptors by endogenously formed cannabinoids contributes to the inhibition of the neurogenic vasopressor response.

1. We studied whether cannabinoid CB1 receptor gene disruption (to yield CB1-/- mice) affects the electrically evoked tritium overflow from vas deferens and atrial pieces preincubated with [3H]-noradrenaline (NA) ('noradrenaline release') and from cerebral cortex slices preincubated with [3H]-choline ('acetylcholine release'). 2. NA release was higher by 37% in vas deferens from CB1-/- mice than in vas deferens from CB1+/+ mice. The cannabinoid receptor agonist WIN 55,212-2 inhibited, and the CB1 receptor inverse agonist/antagonist SR 141716, increased NA release in vas deferens from CB1+/+ mice without affecting it in vas deferens from CB1-/- mice. 3. Atrial NA release did not differ between CB1+/+ and CB1-/- mice nor did WIN 55,212-2 affect NA release in either strain. 4. Cortical acetylcholine (Ach) release did not differ between CB1+/+ and CB1-/- mice. WIN 55,212-2 inhibited, but SR 141716 did not affect, Ach release in the cortex from CB1+/+ mice. Both drugs did not alter Ach release in the cortex from CB1-/- mice. 5. Tritium content did not differ between CB1+/+ and CB1-/- mice in any preparation. 6. In conclusion, the increase in NA release associated with CB1 receptor deficiency in the vas deferens, which cannot be ascribed to an alteration of tritium content of the preparations, suggests an endogenous tone at the CB1 receptors of CB1+/+ mice in this tissue. Furthermore, the effect of WIN 55,212-2 on NA release in the vas deferens and on cortical Ach release involves CB1 receptors, whereas the involvement of non-CB1-non-CB2 receptors can be excluded.

1. The cannabinoid CB(1) receptor inverse agonist/antagonist SR 141716 increases acetylcholine release in rodent hippocampus and improves memory in some experimental paradigms. Since drugs like SR 141716 may represent a novel class of cognition-enhancing drugs, we wanted to check whether the function of the CB(1) receptor is preserved during ageing. 2. Hippocampal and striatal slices from 2- to 3- and 24- to 28-month-old C57BL/6J mice were preincubated with [(3)H]-choline or [(3)H]-noradrenaline ([(3)H]-NA) and superfused. 3. The cannabinoid receptor agonist WIN 55212-2 inhibited, and SR 141716 facilitated, the electrically (3 Hz) evoked tritium overflow in hippocampal slices (preincubated with [(3)H]-choline) from young and aged mice to the same extent. The evoked overflow per se was less by 33% in slices from aged animals. 4. WIN 55212-2 and SR 141716 did not affect, but the muscarinic receptor agonist oxotremorine inhibited, the evoked (3 Hz) overflow in striatal slices (preincubated with [(3)H]-choline) from young and aged mice to the same extent. The evoked overflow per se tended to be less in slices from aged animals. 5. The evoked (0.3 Hz) overflow in hippocampal slices (preincubated with [(3)H]-NA) was not affected by WIN 55212-2 and SR 141716, but was inhibited by histamine (via H(3) receptors) in slices from young mice and, to a somewhat less extent, in slices from aged mice. The evoked overflow per se did not differ between age groups. 6. In conclusion, the function of the CB(1) receptor involved in the tonic inhibition of hippocampal acetylcholine release is preserved in aged mice.

1. In segments of the rat vena cava preincubated with [3H]-noradrenaline and superfused with physiological salt solution (containing desipramine and corticosterone), we studied the effects of prostaglandins of the D, E and F series, of a prostacyclin analogue and a thromboxane-mimetic and of subtype-selective prostaglandin E-receptor (EP-receptor) ligands on the electrically (0.66 Hz)-evoked tritium overflow. 2. The electrically-evoked tritium overflow was inhibited by prostaglandin E2 (maximum inhibition by about 80%; pIC40 7.49). The effect of prostaglandin E2 was not affected by rauwolscine, which, by itself, increased the evoked overflow; the alpha 2-adrenoceptor antagonist was added to the superfusion medium in all subsequent experiments. Indomethacin failed to affect either the evoked tritium overflow or its inhibition by prostaglandin E2. 3. The inhibitory effect of prostaglandin E2 on the electrically-evoked tritium overflow was not altered by the EP1-receptor antagonist. AH 6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) at a concentration at least 30 fold higher than its pA2 value at EP1-receptors. The following compounds mimicked the effect of prostaglandin E2 showing the following rank order of potencies: misoprostol (EP2-/EP3-receptor agonist) congruent to sulprostone (EP1-/EP3-receptor agonist) congruent to prostaglandin E1 = prostaglandin E2iloprost (EP1-/IP-receptor agonist) = prostaglandin F2 alpha. The evoked overflow was not affected by high concentrations of prostaglandin D2 or the thromboxane-mimetic U46619 (9,11-dideoxy-11 alpha, 9 alpha-epoxy-methano-prostaglandin F2 alpha). 4. The present results suggest that the postganglionic sympathetic nerve fibres innervating the rat vena cava are endowed with presynaptic EP3-receptors.They are not tonically activated by endogenously formed products of cyclo-oxygenase and do not interact with the presynaptic M2-adrenoceptors.

1 A microsomal fraction was prepared from human umbilical arteries by differential centrifugation. The preparation was capable of an oxalate-stimulated Ca2+ uptake at a mean rate of 0.74 nmol Ca2+ mg-1 protein min-1 which could be inhibited by a Ca2+ ionophore, A 23 187, and by Tween 80. 2 The rate of Ca2+ uptake in the fraction obtained by density gradient fractionation paralleled 5'-nucleotidase activity suggesting that vesicles of predominantly sarcolemmal origin were responsible for the microsomal Ca2+ uptake. 3 Cyclic adenosine 3',5'-monophosphate-dependent protein kinase enhanced membrane phosphorylation but did not affect Ca2+ uptake. Preincubation with alkaline phosphatase reduced membrane phosphorylation to a greater extent than Ca2+ uptake. These data are not in favour of a close correlation between Ca2+ uptake and phosphorylation. 4 None of 15 vasodilator drugs (bencyclane, carbocromen, diazoxide, dilazep, hydralazine, indapamide, isosorbide dinitrate, methyl-isobutyl-xanthine, minoxidil, naftidrofuryl, nitroglycerine, prenylamine, sodium nitroprusside, tetracaine, and verapamil) had any effect on Ca2+ uptake at 10(-5) M. This suggests that vasodilator drugs do not act by a direct influence on the Ca2+ pumps of vascular smooth muscle cells.