Effects of nitric oxide synthase inhibition on Basal function and the force-frequency relationship in the normal and failing human heart in vivo.

BACKGROUND: Nitric oxide (NO) exerts autocrine/paracrine effects on cardiac function, including alterations of the inotropic state. In vitro studies suggest that NO modulates the myocardial force-frequency relationship. Basal left ventricular (LV) contractility is depressed and the force-frequency relationship is blunted in human heart failure, and it is speculated that an increase in NO production is involved. METHODS AND RESULTS: We compared the effects of intracoronary NO synthase inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 25 micromol/min) on basal LV function and the response to incremental atrial pacing in patients with dilated cardiomyopathy (n=11; mean age, 51 years) and in control subjects with atypical chest pain and normal cardiac function (n=7; mean age, 54 years). In controls, L-NMMA significantly reduced basal LV dP/dt(max) (from 1826 to 1578 mm Hg/s; P<0.002), but had no effect on heart rate, mean aortic pressure, or right atrial pressure. Pacing-induced increases in LV dP/dt(max) were unaltered by L-NMMA. In patients with dilated cardiomyopathy, L-NMMA had no effect on baseline LV dP/dt(max) (from 1313 to 1337 mm Hg/s; P=NS). The blunted pacing-induced rise in LV dP/dt(max) in these patients was unaltered by L-NMMA. CONCLUSION: Endogenous NO has a small baseline positive inotropic effect in the normal human heart, which is lost in heart failure patients. NO does not significantly influence the force-frequency relationship in either the normal or failing human heart in vivo. Because this study was performed in patients with moderate heart failure, whether the findings apply to subjects with more severe heart failure requires further investigation.

Adenosine A2A receptor antagonists as new agents for the treatment of Parkinson's disease.

There is now good reason to believe that blockade of the adenosine A2A receptor could be of value in the treatment of Parkinson's disease. Peter J. Richardson, Hiroshi Kase and Peter G. Jenner review the actions of this receptor in the striatum, emphasizing its ability to modulate the neuronal activity of striatal GABA-releasing output neurones, and showing that recently developed A2A receptor antagonists are capable of reducing the disabling effects of nigral cell degeneration in primates. They conclude that such antagonists may be useful as novel therapeutic agents for the treatment of Parkinson's disease.

Gene-expression analysis at the single-cell level.

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.

Adenosine A1 and A2A receptors are co-expressed in pyramidal neurons and co-localized in glutamatergic nerve terminals of the rat hippocampus.

Adenosine is a neuromodulator that controls neurotransmitter release through inhibitory A1 and facilitatory A2A receptors. Although both adenosine receptor-mediated inhibition and facilitation of glutamate release have been observed, it is not clear whether both A1 and A2A receptors are located in the same glutamatergic nerve terminal or whether they are located on different populations of these terminals. Thus, we have tested if single pyramidal glutamatergic neurons from the hippocampus simultaneously expressed A1 and A2A receptor mRNA and if A1 and A2A receptors were co-localized in hippocampal glutamatergic nerve terminals. Single cell PCR analysis of visually identified pyramidal neurons revealed the simultaneous presence of A1 and A2A receptor mRNA in four out 16 pyramidal cells possessing glutamatergic markers but not GABAergic or astrocytic markers. Also, A1 and A2A receptor immunoreactivities were co-localized in 26 +/- 4% of nerve terminals labeled with antibodies against vesicular glutamate transporters type 1 or 2, i.e. glutamatergic nerve terminals. This indicates that glutamatergic neurons in the hippocampus co-express A1 and A2A receptors and that these two receptors are co-localized in a subset of glutamatergic nerve terminals.

Analysis of gene expression in single cells.

A cell's structural and functional characteristics are dependent on the specific complement of genes it expresses. The ability to study and compare gene usage at the cellular level will therefore provide valuable insights into cell physiology. Such analyses are complicated by problems associated with sample collection, sample size and the limited sensitivity of expression assays. Advances have been made in approaches to the collection of cellular material and the performance of single-cell gene expression analysis. Recent development in global amplification of mRNA may soon permit expression analyses of single cells to be performed on DNA microarrays.

Effect of acute anaesthesia on synthesis of contractile and non-contractile proteins of heart muscle and mixed proteins of types I and II fibre rich skeletal muscles of rat.

STUDY OBJECTIVE: The aim of the study was to determine whether the deleterious effects of anaesthesia on cardiac muscle function were due to disturbance of protein synthesis. Comparative investigations were made on anaerobic and aerobic skeletal muscles, and plasma insulin and growth hormone levels were also measured to see if these were mediating factors. DESIGN: Rats were subjected to acute methoxyfluorane anaesthesia for 10 min. At the end of the study they were killed and plasma growth hormone and insulin were measured. Rates of cardiac and skeletal muscle protein synthesis were also determined with a flooding dose of L[4-3H]phenylalanine. EXPERIMENTAL MATERIAL: Muscle samples were obtained from male Sprague-Dawley rats, weight 191-222 g. MEASUREMENTS AND MAIN RESULTS: Anaesthesia reduced the fractional rate of myocardial mixed protein synthesis and synthesis relative to RNA (p less than 0.05). The anaesthesia induced decrease in the synthesis rates of cardiac contractile proteins (p less than 0.05) was greater than the decrease in the non-contractile protein fractions (p greater than 0.05). Soleus (aerobic, Type I) and plantaris (anaerobic, Type II) muscle rates of protein synthesis were unaltered in response to anaesthesia (p greater than 0.05). Plasma insulin concentrations increased in response to acute anaesthesia (p less than 0.05), but the insulin effect was depressed by the flooding dose of phenylalanine (p less than 0.05). Plasma growth hormone levels were not altered in response to anaesthesia (p greater than 0.05). Thus, the changes in cardiac protein synthesis could not be ascribed to these hormones. CONCLUSIONS: Synthesis of cardiac contractile proteins is selectively sensitive to the effects of acute anaesthesia even in the presence of high plasma insulin concentrations. The fall in cardiac protein synthesis may be a result of the negative inotropic effects of general anaesthesia.

Protein synthesis in pulmonary, cardiac, and skeletal muscle in acute hypertension induced by aortic constriction in the rat.

OBJECTIVE: The aim was to investigate nucleic acid composition and rates of protein synthesis in cardiopulmonary tissues and skeletal muscle in response to hypertension induced by aortic constriction. METHODS: After five days of abdominal aortic constriction, protein, RNA, and DNA contents were measured in the lung, the left and right atria, the left and right ventricles, and gastrocnemius muscle from young male Wistar rats weighing 120-140 g. Rates of protein synthesis were also measured in these tissues with L[4-3H]phenylalanine. RESULTS: Aortic constriction significantly increased the right atrial weight and in contrast reduced the lung weight, compared to pair fed and sham operated controls. The wet weights of all other tissues were unaffected. The concentrations of right atrial proteins, RNA, and DNA were also significantly reduced though total protein, RNA, and DNA contents were unaltered. The left ventricular RNA concentration increased and there were variable alterations in protein and DNA composition. The protein, RNA, and DNA compositions of the other tissues showed patterned responses, which included reductions in lung and skeletal muscle DNA concentrations, reductions in the skeletal muscle RNA/DNA ratio, and a decrease in the lung protein/DNA ratio. In response to aortic constriction there were increases in the left ventricular fractional rate of protein synthesis in mixed, high salt (myofibrillar), and low salt (sarcoplasmic) fractions. Rates of protein synthesis in all other regions of the heart, lung and skeletal muscle were not significantly changed. CONCLUSIONS: We conclude that in abdominal aortic constriction, the left ventricles display early adaptive responses without any concomitant change in mass. Those regions of the rat cardiopulmonary system which are not directly exposed to the acute pressure overloading, ie, right atrium, lungs, and skeletal muscle, also show disturbances.

Distribution of mRNA encoding the inwardly rectifying K+ channel, BIR1 in rat tissues.

The distribution of mRNA encoding the inwardly rectifying K+ channel, BIR1 [1] was investigated in rat tissues, and a comparison made with the expression of related genes rcKATP and GIRK1 using the reverse transcription-polymerase chain reaction (RT-PCR). This showed BIR1 to be expressed in all areas of the brain examined, in the eye but not in any other peripheral tissue. This pattern was distinct from rcKATP and GIRK1. Additional in situ hybridisation studies of the central expression of BIR1 demonstrated high levels of BIR1 mRNA in the hippocampus, dentate gyrus, taenia tecta and cerebellum and at lower levels in the cortex, habenular nucleus, olfactory bulb, primary olfactory cortex, thalamus, pontine nucleus and amygdaloid nucleus.

The role of Coxsackie B viruses in the pathogenesis of myocarditis, dilated cardiomyopathy and inflammatory muscle disease.

Coxsackie B viruses are members of the family Picornaviridae which have been associated by retrospective serology with a range of muscle diseases, particularly myocarditis, dilated cardiomyopathy and epidemic pleurodynia (epidemic myalgia or Bornholm disease). It has been proposed that virus-induced myocarditis disposes to the development of idiopathic dilated cardiomyopathy. However, despite many attempts, isolation of infectious virus or immunofluorescent detection of virus-specific antigens in the affected tissue is rare, although virus may be found in faeces early in infection. This discrepancy awaited the development of nucleic acid probes to resolve the problem of whether virus was present consistently in myocardium or other muscle tissues. We report here the synthesis of Coxsackie B virus-specific complementary DNA (cDNA) probes and their use in molecular hybridizations to quantitative slot-blots of RNA prepared from either endomyocardial or skeletal muscle biopsy specimens. Of 50 patients with histologically proven myocarditis or dilated cardiomyopathy, 28 (56%) had an endomyocardial biopsy specimen positive for the presence of Coxsackie B virus-specific RNA. Twenty-two patients with other cardiac diseases of known aetiology, unrelated to virus infection, were all negative. Multiple biopsies were obtained from 20 patients with myocarditis or dilated cardiomyopathy and 15 of these (75%) had at least one biopsy specimen positive, indicating the focal nature of the disease. In analogous investigations, Coxsackie B virus-specific RNA was detected in four out of seven single skeletal muscle biopsy specimens from patients suffering from juvenile dermatomyositis, and one out of two patients with adult polymyositis. Ten muscle controls, either normal or Duchenne muscular dystrophy, were negative for virus RNA.

Oxidants, antioxidants and alcohol: implications for skeletal and cardiac muscle.

The chronic form of alcoholic skeletal myopathy is characterized by selective atrophy of Type II fibers and affects up to two thirds of all alcohol misusers. Plasma selenium and alpha-tocopherol are reduced in myopathic alcoholics compared to alcoholic patients without myopathy. Plasma carnosinase is also reduced in myopathic alcoholics, implicating a mechanism related to reduced intramuscular carnosine, an imidazole dipeptide with putative antioxidant properties. Together with the observation that alcoholic patients have increased indices of lipid peroxidation, there is evidence suggestive of free radical (i.e., unpaired electrons or reactive oxygen species) mediated damage in the pathogenesis of alcohol-induced muscle disease. Protein synthesis is a multi-step process that encompasses amino acid transport, signal transduction, translation and transcription. Any defect in one or more of the innumerable components of each process will have an impact on protein synthesis, as determined by radiolabelling of constituent proteins. Both acute and chronic alcohol exposure are associated with a reduction in skeletal muscle protein synthesis. Paradoxically, alcohol-feeding studies in rats have shown that the imidazole dipeptide concentrations are increased in myopathic muscles though alpha-tocopherol contents are not significantly altered. In acutely dosed rats, where protein synthesis is reduced, protein carbonyl concentrations (an index of oxidative damage to muscle) also decline slightly or are unaltered, contrary to the expected increase. Alcoholic cardiomyopathy can ensue from heavy consumption of alcohol over a long period of time. The clinical features include poor myocardial contractility with reduced left ventricular ejection volume, raised tissue enzymes, dilation of the left ventricle, raised auto- antibodies and defects in mitochondrial function. Whilst oxidant damage occurs in experimental models, however this issues remains to be confirmed in the clinical setting. In the rat, circulating troponin-T release increases in the presence of ethanol, a mechanism ascribed to free radical mediated damage, as it is prevented with the xanthine oxidase inhibitor and beta-blocker, propranolol. However, whilst propranolol prevents the release of troponin-T, it does not prevent the fall in whole cardiac protein synthesis, suggestive of localized ischemic damage due to ethanol.

Effects of converting enzyme inhibitor on hepatic blood flow in man.

The acute effects of the oral angiotension converting enzyme inhibitor captopril on hepatic blood flow and systemic hemodynamics were studied in six patients with essential hypertension. Mean arterial pressure decreased from 141.9 +/- 6.9 mm Hg to 130.2 +/- 6.7 mm Hg (p less than 0.05) one hour after the administration of captopril. There was no significant change in other hemodynamic values, but hepatic blood flow decreased uniformly from 1,127 +/- 115 ml per minute to 841 +/- 93 ml per minute (p less than 0.001).

Characterization of the mechanism of action of tachykinins in rat striatal cholinergic interneurons.

The mechanism by which substance P depolarizes cholinergic interneurons in the rat striatum was studied using whole-cell recording techniques. In all cases the effects of substance P were mimicked by the neurokinin1 receptor agonist [Sar9, Met(O2)11] substance P and were antagonized by the neurokinin1 receptor antagonist SR140333. [Sar9, Met(O2)11] substance P was found to depolarize cholinergic interneurons by the induction of a calcium-activated inward current at -60 mV. This inward current was irreversibly potentiated by photolysis of caged GTPgammaS within neurons implicating the involvement of a G-protein. The [Sar9, Met(O2)11] substance P-induced inward current was inhibited by the phospholipase C inhibitor U-73122, and by the inclusion of the inositol-1,4,5-triphosphate receptor antagonist heparin in the electrode solution. These findings suggest that neurokinin1 receptors depolarize cholinergic interneurons in the rat striatum primarily through a phosphoinositide signalling pathway.

Tachykinins increase [3H]acetylcholine release in mouse striatum through multiple receptor subtypes.

Tachykinins have been suggested to play a significant role in the mammalian striatum, at least in part by the control of acetylcholine release from cholinergic interneurons. In the present study, we have examined the ability of known tachykinin agonists and antagonists to modulate the activity of these interneurons in mouse striatal slices. Using whole-cell patch-clamp recordings, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide each produced a dose-dependent depolarization of visually identified cholinergic interneurons that was retained under conditions designed to interrupt synaptic transmission. The nature of these neurons and the expression of multiple tachykinin receptors was confirmed using single-cell reverse transcriptase-polymerase chain reaction analysis. Using in vitro superfusion techniques, the selective neurokinin-1, neurokinin-2 and neurokinin-3 receptor agonists [sar9,Met(O2)11]substance P, [beta-ala8]neurokinin A(4-10) and senktide, respectively, each produced a dose-dependent increase in acetylcholine release, the selectivity of which was confirmed using the neurokinin-1, neurokinin-2 and neurokinin-3 receptor antagonists SR140333, GR94800 and SR142801 (100 nM). U73122 (10 microM), a phospholipase C inhibitor, blocked [sar9,Met(O2)11]substance P- and senktide-induced acetylcholine release, but had no effect on [beta-ala8]neurokinin A(4-10)-induced release. The protein kinase C inhibitors chelerythrine and Ro-31-8220 (both 1 microM) significantly inhibited responses induced by all three agonists. These findings indicate that tachykinins modulate the activity of mouse striatal cholinergic interneurons. Furthermore, neurokinin-2 receptors are shown to perform a role in mouse that has not been identified previously in other species.

Adenosine receptor-mediated modulation of acetylcholine release from rat striatal synaptosomes.

1. The effects of A1 and A2a adenosine receptor agonists on the veratridine-evoked release of [3H]-acetylcholine ([3H]-ACh) from rat striatal synaptosomes was investigated by use of the A1-selective agonist, R-PIA and the 185 fold selective A2a agonist, CGS 21680. The effects of NECA, which is equipotent at both receptor subtypes, were also studied. 2. The evoked release of [3H]-ACh was significantly enhanced by the A2a agonist CGS 21680 but decreased by the A1 agonist, R-PIA. The effects of NECA were dependent on the concentration used, with high concentrations inhibiting and low concentrations enhancing the evoked release of [3H]-ACh. In the absence of any antagonists, the rank order of potency for these three drugs on increasing [3H]-ACh release was CGS 21680 > NECA > R-PIA. 3. The stimulatory effects of CGS 21680 and low NECA concentrations on evoked [3H]-ACh release were antagonized by the A2a receptor antagonists, CP66,713 (300 nM) and CGS 15943A (50 nM) whilst the inhibitory effects of R-PIA were reversed by the selective A1 antagonist, DPCPX (4 nM). In the presence of DPCPX, NECA greatly enhanced the evoked release of [3H]-ACh at all concentrations studied when, during such A1 receptor blockade, the rank order of potency was NECA >> CGS 21680 > R-PIA. 4. These results demonstrate that both A1 and A2a adenosine receptors modulate the veratridine-evoked release of [3H]-ACh from rat striatal synaptosomes.

Further characterization of [3H]-CGS 21680 binding sites in the rat striatum and cortex.

1. The putative high affinity binding site for the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethyl-amino-5'-N- ethylcarboxamidoadenosine (CGS 21680) in the rat cerebral cortex was characterized by use of a number of selective A1 and A2 adenosine receptor ligands, and compared to the characteristics of the more abundant striatal A2A receptor. 2. The binding of [3H]-CGS 21680 to cortical membranes was performed at pH 5.5, in order to increase the amount of specific binding. 3. Reduction of the pH from 7.4 to 5.5 increased the apparent affinity of the striatal binding side for both agonists and antagonists. The relative order of potencies of both groups of ligands were the same at both pH values, and were consistent with binding to the A2A receptor. There was no observable change in the Bmax, the values being 415 and 446 fmol mg-1 protein at pH 5.5 and 7.4 respectively. 4. The cortical binding site yielded a Bmax value of 117 fmol mg-1 protein. The relative order of potencies of the adenosine receptor ligands observed at this binding site were not the same as those observed in the striatum, exhibiting a profile with both A1 and A2 characteristics. 5. Further characterization of this cortical binding site in the presence of the A1 selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed a more typical A2A profile. This indicated that under the conditions used there were two components of [3H]-CGS 21680 binding, approximately 20% of the A1 receptor and 80% to the A2A receptor. 6. It is concluded that in the cerebral cortex there is a CGS 21680 binding site showing the characteristic properties of the striatal A2A receptor, and no evidence was obtained for the existence of a novelA2A-like binding site.

Histamine depolarizes cholinergic interneurones in the rat striatum via a H(1)-receptor mediated action.

1. Whole-cell patch clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. Bath application of histamine (EC(50) 6.3 microM) was found to rapidly and reversibly depolarize these neurones through the induction of an inward current at -60 mV. 2. The effects of histamine were mimicked by the H(1) receptor agonist 2-thiazolylethylamine (50 microM) and selectively inhibited by pre-incubation with the H(1) receptor antagonist triprolidine (1 microM). 3. Ion substitution experiments under voltage clamp conditions revealed that the histamine activated current was comprised of two components. One component was sensitive to the concentration of extracellular Na(+), whilst the other component was inhibited by intracellular Cs(+) or extracellular Ba(2+). 4. In situ hybridization experiments revealed that the majority of cholinergic interneurones in the rat striatum express the histamine H(1) receptor but few neurones express H(2) receptors. These findings were confirmed using single cell RT - PCR. 5. It is concluded that histamine depolarizes cholinergic interneurones in the rat striatum via a H(1)-receptor mediated mechanism.

Adenosine receptors in post-mortem human brain.

1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.

Effects of pH on responses to adenosine, CGS 21680, carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat.

1. The receptors mediating the vasodilator responses to adenosine in the isolated mesenteric arterial bed of the rat were identified by use of selective agonists and antagonists and the involvement of the endothelium was examined. 2. Adenosine-mediated dilatation of the mesentery was potentiated by the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 100 microM), but in contrast, removal of the endothelium substantially reduced the responses to adenosine. 3. The order of potency of adenosine receptor agonists was: 5'-N-ethylcarboxamidoadenosine (NECA) > 2-p-(-2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) > 2-chloro-N6-cyclopentyl-adenosine (CCPA) > or = adenosine, suggesting the presence of A2A receptors. 4. Adenosine-mediated dilatation was inhibited by the non-selective adenosine receptor antagonist, 8-phenyltheophylline (3 microM) and by the A2A receptor antagonist 8-(3-chlorostyryl)caffeine (500 nM), but was unaffected by the A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM). 5. Reducing the pH of the perfusate to 6.8 potentiated the actions of both CGS 21680 and adenosine, but the vasodilator effects of carbachol were the same at both pH values. The adenosine response at the lower pH as at pH 7.4, was unaffected by DPCPX. The actions of the nitrovasodilator, sodium nitroprusside, were also potentiated at pH 6.8 relative to those at the higher pH value but smaller responses were obtained at the lower pH value with forskolin, a stimulator of adenylyl cyclase, than at pH 7.4. 6. It is concluded that the adenosine receptor mediating dilatation of the rat mesenteric arterial bed is of the A2A subtype, that the response, under the conditions used, is apparently partly dependent on the endothelium (but not due to the release of nitric oxide), and that the response to activation of this receptor is potentiated by a reduction in pH which is similar to that seen in ischaemic conditions.

Pharmacological analysis of the interaction between Bay K 8644 and 5-HT in rabbit aorta.

Bay K 8644 potentiated and augmented 5-hydroxytryptamine (5-HT)-induced contractions in the rabbit, isolated aorta preparation, as manifested in leftward shift and increase in the asymptote of 5-HT E/[A] (effect vs concentration) curves. The operational model of agonism (Black & Leff, 1983) was used to analyse this interaction and the concomitant effects of irreversible receptor alkylation by phenoxybenzamine. The competitive effects of spiperone in the presence and absence of Bay K 8644 were also examined. From these analyses it is concluded that Bay K 8644 elicits its potentiating effects by increasing the efficacy of 5-HT at the 5-HT2 receptor with no alteration in affinity. This is consistent with the known effect of Bay K 8644 of causing an increase in the functional concentration of plasmalemmal calcium channels coupled to the 5-HT2 receptors in this preparation. The positively co-operative shape of the 5-HT E/[A] curves obtained in the aorta and the quantitative nature of their potentiation by Bay K 8644 indicated that the coupling of 5-HT2 receptor occupancy to intracellular calcium concentration is linear and that the co-operativity resides in the subsequent relation between intracellular calcium and pharmacological effect. Bay K 8644 may serve as a probe for differentiating between the types of calcium channels that transduce 5-HT receptor-mediated effects in different systems. Such information would be useful in the classification of agonist interactions with 5-HT receptors.

Inhibition by KF17837 of adenosine A2A receptor-mediated modulation of striatal GABA and ACh release.

1. The effect of the A2A adenosine receptor agonist, 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS 21680) on the potassium evoked release of [3H]-gamma-aminobutyric acid ([3H]-GABA) from nerve terminals derived from the caudate-putamen and the globus pallidus of the rat was compared. In both preparations CGS 21680 (1 nM) inhibited the [3H]-GABA release evoked by 15 mM KCl but had no effect on that evoked by 30 mM KCl. 2. The ability of CGS 21680 (1 nM) to inhibit the release of [3H]-GABA from striatal nerve terminals was unaffected by the presence of the GABA receptor antagonists, bicuculline (10 microM), phaclofen (100 microM) and 2-hydroxysaclofen (100 microM). Similarly the opioid receptor antagonist, naloxone (10 microM), the adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 40 nM), and the cholinoceptor antagonists, mecamylamine (10 microM) and atropine (100 nM) had no effect on this inhibition. 3. The ability of CGS 21680 (0.1 nM) to stimulate the release of [3H]-acetylcholine ([3H]-ACh) from striatal nerve terminals was unaffected by the presence of bicuculline (10 microM), 2-hydroxysaclofen (100 microM), phaclofen (100 microM), naloxone (10 microM) and DPCPX (4 nM). 4. The novel A2A receptor antagonist, (E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF 17837), blocked the CGS 21680 (1 nM)-induced inhibition of [3H]-GABA efflux with an EC50 of approximately 30 nM and also antagonized the CGS 21680 (0.1 nM)-induced stimulation of [3H]-ACh release with an EC50 of approximately 0.3 nM. 5. It is concluded that the A2A adenosine receptor is present on both GABAergic and cholinergic nerve terminals of the rat striatum and that in both the caudate-putamen and the globus pallidus this receptor inhibits [3H]-GABA release. No evidence was seen for a difference in the ligand binding sites of this receptor in the two groups of nerve terminals.