Adenosine potentiates sympathomimetic effects of nicotinic agonists in vivo

We showed previously that circulating adenosine potentiates pressor responses to nicotine in rats, apparently by enhancing the effects of nicotine in sympathetic ganglia. In the present studies, we examined the effects of adenosine (or synthetic adenosine receptor agonists) on a variety of sympathomimetic responses to nicotine (or other nicotinic cholinergic agonists). Indices of sympathetic activity examined were: blood pressure; heart rate; eyelid tension; and vas deferens perfusion pressure. Elevation of arterial plasma adenosine from its basal level (approximately 1.5 microM) to 2 to 3 microM (by i.v. adenosine infusion) had no effect on the basal value of any of these indices, but increased by 2.5 to 4-fold their peak responses to nicotine (40 microgram/kg i.v.). Adenosine also strongly enhanced sympathomimetic responses to inhaled cigarette smoke. The adenosine receptor antagonist caffeine (10 mg/kg) suppressed the ability of adenosine to potentiate these responses to nicotine. Pressor responses to the nicotinic cholinergic receptor agonists dimethylphenylpiperazinium iodide, tetramethylammonium, lobeline and cytisin were also potentiated by adenosine. Low doses (0.25-5 mu/kg) of synthetic adenosine receptor agonists potentiated all four of the tested sympathomimetic responses to nicotine, exhibiting the rank order of potency: N-Cyclopropylcarboxamidoadenosine greater than 2-Chloroadenosine greater than N6-R-Phenylisopropyl adenosine greater than N6-S-Phenylisopropyladenosine. The nonpurinergic vasodilator sodium nitroprusside failed to potentiate responses to nicotine, suggesting that the influence of adenosine on responses to nicotine is not secondary to its direct circulatory effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of ATP-evoked ATP release system in smooth muscles.

Effects of stable ATP analogs such as alpha,beta-methylene ATP (alpha,beta-mATP) and beta,gamma-methylene ATP (beta,gamma-mATP) on ATP release and contractile response were evaluated in the vas deferens and ileal longitudinal muscles of guinea pig. In these smooth muscles, administration of alpha,beta-mATP (10, 30 or 100 microM) produced an ATP release accompanied by a transient contraction, but alpha,beta-methylene ADP (30 or 100 microM) or adenosine (30 microM) failed to elicit both the ATP release and the contraction. However, the peak responses of ATP release and contraction to alpha,beta-mATP (100 microM) in the vas deferens appeared around 2 min and 2.62 sec, respectively, after the injection of the drug. Beta,gamma-mATP (10 or 100 microM) caused an ATP release from the vas deferens. The ATP release as well as the contraction evoked by alpha,beta-mATP or beta,gamma-mATP were effectively inhibited by 300 microM suramin, a P2 purinoceptor antagonist. By contrast, ATP release and contractile response to norepinephrine in the vas deferens and those to bethanechol in the ileum were virtually unaffected by this antagonist. Veratridine and ouabain at (30 or 100 microM) caused markedly acetylcholine release from the ileum and norepinephrine release from the vas deferens, respectively. However, alpha,beta-mATP, even in a high concentration of 100 microM, did not elicit any release of acetylcholine or norepinephrine. These findings suggest that alpha,beta-mATP and probably beta,gamma-mATP evoke ATP release from not neuronal but mainly smooth muscular sites by activating suramin-sensitive P2x receptors, implying that "ATP-evoked ATP release system" exists.     

Behavioral and cardiovascular effects of analogs of adenosine in cynomolgus monkeys

The behavioral and cardiovascular effects of six metabolically stable analogs of adenosine were studied in cynomolgus monkeys. Behavioral effects were determined in monkeys trained to respond under a 30-response fixed-ratio (FR) schedule of food presentation; cardiovascular effects were evaluated in a separate group of unanaesthetized monkeys seated at rest. Dose-effect curves for each drug were determined by administering cumulative doses i.v. during sequential components of the experimental session. All six analogs of adenosine produced dose-related decreases in FR responding with the following order of potency: 5'N-ethylcarboxamide adenosine (NECA) greater than 2-chloroadenosine (2-CA) greater than N6-R-phenylisopropyladenosine (R-PIA) greater than N6-cyclopentyladenosine (CPA) greater than N6-cyclohexyladenosine (CHA) greater than N6-S-phenylisopropyladenosine (S-PIA). In cardiovascular studies, all drugs except CPA produced dose-related decreases in mean blood pressure (MBP) with the following order of potency: NECA greater than 2-CA = R-PIA greater than CHA greater than S-PIA. CPA did not alter MBP at doses that decreased both FR responding and heart rate (HR). All drugs except NECA produced dose-related decreases in HR with the following order of potency: CPA greater than 2-CA greater than R-PIA greater than CHA greater than S-PIA. NECA increased HR at doses that decreased MPB and FR responding. Doses of caffeine that did not alter FR responding or cardiovascular function when given alone antagonized the behavioral effects of R-PIA, CHA, CPA and NECA. Caffeine also antagonized the effects of R-PIA, CHA and NECA on MBP and the effects of R-PIA and CPA on HR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purine nucleoside and nucleotide interactions on normal and subsensitive alpha adrenoreceptor responsiveness in guinea-pig vas deferens.

Adenosine and adenosine 5'-monophosphate (AMP) augment contractile responses to norepinephrine (NE) in isolated guinea-pig vas deferens. Dipyridamole slightly enhances, while theophylline antagonizes, adenosine effects on responses to NE. Adenosine triphosphate (ATP) and the nonhydrolyzable analog, adenosine 5'-(beta,gamma-imido)triphosphate (AppNp) depress responses to NE. Adenine and adenosine diphosphate (ADP) are ineffective in influencing alpha adrenoreceptor responsiveness. Repetitive stimulation of isolated vas deferens with maximal concentrations of NE markedly reduce the contractile response to test concentrations of 6 micron NE. Spontaneous resensitization of responses to NE to control levels occurs within 25 to 35 minutes after the end of desensitization treatment. Adenine nucleosides and nucleotides promote a more rapid rate of alpha adrenoreceptor resensitization, with a potency order: AMP greater than adenosine greater than ADP. Adenine and ATP did not influence the rate of alpha adrenoreceptor resensitization. The adenine nucleotides ADP, ATP and the analog AppNp elicit concentration-dependent contractions of guinea-pig vas deferens. Theophylline antagonizes this contractile activity to adenine nucleotides. AMP, adenosine and adenine are devoid of agonistic activity. In the presence of NE, however, AMP and adenosine produce contractile responses of isolated vas deferens strips, and the agonistic activity of ADP, ATP and AppNp is profoundly enhanced. Agonistic actions of purines in the presence of NE are antagonized by phentolamine much more effectively than by theophylline. The results suggest the existence of a purinergic receptor mediating excitatory responses of guinea-pig vas deferens. Furthermore, there appears to be mutual interaction between purinergic and alpha adrenoreceptor mechanisms. That adenyl derivatives are capable of augmenting subsensitive alpha adrenoreceptor responsiveness suggests that adenine nucleosides or nucleotides, released during sympathetic transmission, may be required for maintenance of normal alpha adrenoreceptor sensitivity.     

Pharmacological characterization of purinergic receptors in the rat vas deferens

Using the isolated rat vas deferens, we have confirmed the existence of P1 purinergic receptors whose activation results in an inhibition of the neurogenic twitch of the vas deferens. The observed order of potency for agonists (adenosine ethyl carboxamide greater than 2-chloroadenosine greater than adenosine greater than 5'-AMP greater than 5'-ADP greater than ATP) and antagonism of these effects by theophylline supports a P1-mediated response. Metabolically stable analogs of ATP elicited dose-dependent contractile responses which were quantitatively greater than, but qualitatively comparable to, ATP-induced responses. The order of potency for the eliciting contraction was the following: adenylyl-5-imidodiphosphate = beta-gamma-methylene ATP greater than adenosine tetraphosphate much greater than ATP greater than ADP. Interestingly, these compounds also produced an inhibition of the neurogenic twitch with a similar rank order of potency. This response was not due to the activation of P1 receptors insofar as high concentrations of theophylline failed to attenuate either the inhibition of the neurogenic twitch or the contractile response induced by these agonists. Thus, these data demonstrate the presence of both P1 and P2 purinergic receptors in the rat vas deferens. In addition, the data are consistent with the idea that two distinct classes of P2 receptors exist in this tissue. Furthermore, these data suggest that the rat vas deferens provides a useful tissue for studying compounds which interact with both major subtypes of purinergic receptors.     

Ligand selectivity of dog coronary adenosine receptor resembles that of adenylate cyclase stimulatory (Ra) receptors

Adenosine receptors on the surface of coronary myocytes initiate coronary relaxation, but events subsequent to receptor occupancy are uncertain. We assayed the coronary vasoactivity of receptor-selective adenosine analogs in open chest dogs to test the hypothesis that the coronary adenosine receptor is an adenylate cyclase stimulatory (Ra) receptor. The potency order was: ethyl adenosine-5'-uronamide greater than cyclopropyl adenosine-5'-uronamide greater than 2-chloroadenosine greater than N6-[R(-)-1-phenyl-2-propyl] adenosine (R-PIA) greater than N6-cyclohexyladenosine greater than adenosine greater than S-PIA. Such a hierarchy of activity resembles that of the Ra receptors of thyroid and aortic myocyte adenylate cyclases and of tracheal smooth muscle relaxation. The potency order R-PIA greater than adenosine and the stereoselective coronary vasoactivity of R-PIA suggest that the coronary receptor may be a hybrid receptor which contains the "N6 site" typical of inhibitory (Ri) receptors in addition to the "5'-uronamide site' which expresses Ra activity.     

Identification of A1 and A2 adenosine receptors in the rat spinal cord

The adenosine receptors in membranes prepared from rat ventral and dorsal lumbar spinal cord were characterized by comparing the binding characteristics of [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA), an agonist with nearly equal affinities at the A1 and A2 adenosine receptor subtypes, with those of [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine ([3H]R-PIA), an A1-selective agonist. Saturation isotherms of the ventral and dorsal spinal cord yielded Kd values 1.9 to 2.3 nM for [3H]R-PIA and 18.1 to 19.5 nM for [3H]NECA. The Bmax for [3H]NeCA was approximately twice the Bmax for [3H]R-PIA in ventral and dorsal halves (267 vs. 128 fmol/mg of protein and 402 vs. 206 fmol/mg of protein, respectively). Displacement of specific [3H]NECA binding by the A2-selective agonist, 2-(phenylamino)adenosine, the relatively nonselective antagonist, theophylline and six A1-selective agonists, R-PIA, S-PIA, N6-(cyclohexyl)adenosine, N6-(cyclopentyl)adenosine, N6-(m-aminophenyl)adenosine and N6-(m-iodophenyl)adenosine, revealed two [3H]NECA binding components with the characteristics of A1 and A2 receptors. All curves best fit a two-site model when analyzed by the computer program LIGAND. R-PIA, N6-(cyclohexyl)adenosine and N6-(cyclopentyl)adenosine were the most potent displacers at the first site (Ki = 0.6-1.4 nM). All A1-selective agonists were poor displacers of [3H]NECA at the second site (Ki = 0.6-18.6 microM). The A2-selective agonist, 2-(phenylamino)adenosine, was as potent as R-PIA in displacing [3H]NECA from this site with a Ki value 0.57 microM. Finally, the A1 and A2 adenosine receptor-mediated inhibition and stimulation of adenylate cyclase were demonstrated directly in synaptic membranes prepared from the spinal cord.     

Discriminative-stimulus effects of adenosine analogs: mediation by adenosine A2 receptors

Squirrel monkeys were trained to discriminate either the nonselective adenosine analog 5'-N-ethylcarboxamide adenosine (NECA) or the A1-selective analog N6-cyclopentyladenosine (CPA) from saline. After i.v. injections of 0.03 mumol/kg of NECA or 1.0 mumol/kg of CPA, 10 consecutive responses on one lever produced food, whereas after i.v. injections of saline, 10 consecutive responses on the alternate lever produced food. The discriminative-stimulus effects of the adenosine analogs NECA, CPA, 2-chloroadenosine and the R- and S-isomers of N6-phenylisopropyladenosine (PIA), of the adenosine antagonists caffeine and 8-cyclopentyltheophylline (CPT), and of selected drugs from other classes (haloperidol, pentobarbital, diazepam and morphine) were determined by administering cumulative doses i.v. before sequential components of the experimental session. All adenosine analogs engendered dose-related increases in the percentage of responses on the NECA- or CPA-associated levers reaching a maximum of greater than or equal to 90%. The rank order of potency was similar in both NECA-trained and CPA-trained monkeys: NECA greater than 2-chloroadenosine greater than or equal to CPA greater than or equal to R-PIA greater than S-PIA. None of the other drugs had discriminative-stimulus effects comparable to those of the adenosine analogs. In additional studies, the nonselective adenosine antagonist caffeine or the A1-selective antagonist CPT were administered before cumulative doses of NECA or CPA. Both adenosine antagonists blocked the discriminative-stimulus effects of NECA and CPA, with CPT being about one order of magnitude more potent than caffeine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of K+-channel blockers on A1-adenosine receptor-mediated negative inotropy and chronotropy of guinea-pig isolated left and right atria

Adenosine has previously been shown to stimulate K(+)-efflux and to block L-type calcium channels in atrial myocytes. The aim of the present study was to evaluate the contribution of K(+)-channels in the development of the negative inotropic and chronotropic responses to adenosine agonists in guinea-pig left and right atria, respectively. Tetraethylammonium (TEA) potentiated the negative inotropic and chronotropic responses to R-(-)-N6-(2-phenyl-isopropyl)-adenosine (R-PIA), seen as leftward shifts of the concentration-response curves. Glibenclamide had no effect on the negative inotropic response to R-PIA but increased the rate of onset of the negative chronotropic response in right atria. 4-Aminopyridine (4-AP, 10 mM), potentiated the left atrial inotropic responses to R-PIA, seen as a leftward shift of the concentration-response curve, but slowed the speed of onset of the response to a single concentration (10(-6) M) of R-PIA. This reduction in speed of onset of the response can explain the differences in effects of 4-AP on concentration-response curves reported here and previously. In the right atria, 4-AP (10 mM) inhibited the negative chronotropic responses to R-PIA, seen as a rightward shift of the concentration-response curve and reduction of the maximum response. 4-AP also slowed the onset of the right atrial rate response to R-PIA. These results support the theory that K(+)-efflux plays only a minor role in the negative inotropic responses of guinea-pig left atria to R-PIA. This apparently controls the speed of onset of the response. The negative chronotropic response of guinea-pig right atria to R-PIA appears to be much more dependent upon K(+)-efflux than for the negative inotropic response of the left atria.     

Two dissociable phases in the contractile response of the guinea pig isolated vas deferens to adenosine triphosphate

The effects of the ATP affinity label periodate-oxidized ATP (ATP-2',3'-dialdehyde; P-ATP) on contractile responses of the guinea pig vas deferens to ATP was characterized and compared to the effects of the specific P2x-purinoceptor photoaffinity label antagonist, arylazido aminopropionyl ATP (ANAPP3). After incubation of vas deferens with 10(-2) M P-ATP for 5 min, the second phase of biphasic contractions to ATP was inhibited selectively. The inhibitory effect of P-ATP was specific for ATP, and resulted from affinity labeling in that it was long-lasting, was not reversed by washing, was related in magnitude to exposure period and was attenuated by ATP present during the incubation. In contrast to P-ATP, ANAPP3 inhibited selectively the first phase of ATP-induced responses. P-ATP and ANAPP3 together inhibited both phases of response. P-ATP inhibited selectively the second phase of responses to 5'-substituted ATP analogs which develop a prolonged second phase [e.g., adenosine 5'-O-(3-thiotriphosphate) and adenosine tetraphosphate] or an abbreviated second phase (e.g., beta,gamma-methylene ATP, beta,gamma-imido ATP and alpha,beta-methylene ATP). The greater the duration of the second phase, the more pronounced was the inhibitory effect. Two distinct and dissociable mechanisms mediate the biphasic response to ATP: the initial phase involves ANAPP3-sensitive P2x-purinoceptors, whereas the second is blocked by P-ATP and appears to involve cleavage of the phosphate chain. An additional effect observed as a result of P-ATP-treatment was potentiation of the first phase of contraction to ATP. This novel and irreversible effect may arise from the inhibition of degradative ecto-phosphohydrolase activity.     

Naloxone potentiation of cardiovascular responses to sympathomimetic amines in the rat

The work was aimed at analyzing the ability of naloxone to potentiate 1) the arterial pressure responses to sympathomimetic amines administered i.v. in normotensive anesthetized, pithed, chemically sympathectomized or acutely adrenalectomized rats and 2) the chronotropic responses to norepinephrine in the isolated rat atria. In anesthetized rats, naloxone (2.5-10 mg/kg i.v.) potentiated the pressor responses to epinephrine (2 micrograms/kg). Naloxone (5 mg/kg) significantly potentiated the pressor responses to norepinephrine (1-4 micrograms/kg), phenylephrine (10-50 micrograms/kg) and the reflex pressor responses to a 60-sec carotid occlusion. On the contrary, naloxone did not potentiate the arterial pressure responses to methoxamine (100 micrograms/kg), angiotensin (0.5-2 micrograms/kg) and isoproterenol (1 micrograms/kg). Pithing or acute adrenalectomy did not alter the naloxone-induced potentiation of the pressor responses to norepinephrine (0.125-0.5 micrograms/kg). 6-Hydroxydopamine pretreatment abolished completely the naloxone-induced potentiation of the pressor responses to norepinephrine (0.25-1 micrograms/kg). In isolated rat atria, naloxone (1.4 and 2.8 x 10(-5) M) potentiated the chronotropic responses to norepinephrine (1.5-6 x 10(-8) M). It is suggested that naloxone potentiates cardiovascular responses to sympathomimetic amines by interacting with presynaptic adrenergic mechanisms which could additionally contribute to its pressor effects in acute hypotensive conditions.     

Adenosine receptor antagonism attenuates angiotensin II effects on adrenergic neurotransmission in the rabbit isolated vas deferens

Angiotensin II augments adrenergic neurotransmission in the rabbit isolated vas deferens and suppresses purinergic neurotransmission. This study tests the hypothesis that angiotensin II augments adrenergic neurotransmission by depressing the neuronal release of ATP, resulting in suppressed formation of the inhibitory neuromodulator, adenosine or a related purine. Exogenous ATP added to the vasa deferentia increased adenosine formation and depressed adrenergic neurotransmission thus providing indirect support for the hypothesis. The adenosine receptor antagonist, 8-(sulfophenyl)theophylline (10 and 100 microM) depressed responses to exogenous adenosine and ATP but did not alter contractile responses to nerve stimulation or exogenously administered norepinephrine thus indicating that endogenous adenosine had no basal influence upon neurotransmission. However, the 8-(sulfophenyl)theophylline reduced angiotensin II effects on both adrenergic neurogenic contractions and evoked norepinephrine release. Additionally, the augmentation of adrenergic neurogenic contractions by angiotensin II was enhanced in the presence of ATP. These results are consistent with an ATP involvement in angiotensin effects on adrenergic neurotransmission and contrary to the initial hypothesis, suggest that purines enhance adrenergic neurotransmission in the presence of angiotensin II.     

Relaxant effects of adenosine analogs on guinea pig trachea in vitro: xanthine-sensitive and xanthine-insensitive mechanisms

Adenosine analogs were tested for their ability to relax carbachol-contracted trachea in vitro. The rank order of potency was: 5'-N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine (2-CIADO) greater than 5'-chloroadenosine = N6-R-1-phenyl-2-propyladenosine (R-PIA) greater than N6-cyclohexyladenosine greater than 2-phenylaminoadenosine (CV1808) greater than 5'-methylthioadenosine (MTA). The rank order of potency for NECA, 2-CIADO and R-PIA is characteristic of an A2 subtype of adenosine receptor. 8-Para-sulfophenyltheophylline (8-p-ST) and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), were used to antagonize tracheal relaxation elicited by adenosine analogs. 8-p-ST antagonized the 2-CIADO, N6-cyclohexyladenosine, R-PIA and 5'-chloroadenosine responses, but had little or no effect on the CV1808 and MTA responses. 8-p-ST antagonized responses to NECA at concentrations of NECA up to approximately 30 microM, but had no effect on responses to higher concentrations of NECA. The differences in antagonist potency of 8-p-ST and the clear biphasic response of NECA are indicative of at least two mechanisms of adenosine analog action leading to tracheal relaxation. One mechanism is mediated through a xanthine-sensitive site, at which NECA acted in a potent manner, whereas the other mechanism or mechanisms are insensitive to blockade by xanthines and account for the effects of action of MTA and CV1808, as well as for NECA at high concentrations. The low potency of the A1-selective antagonist DPCPX indicates that the xanthine-sensitive site is an A2 type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenosine A1 receptor activation mediates suppression of (-) bicuculline methiodide-induced seizures in rat prepiriform cortex

The protective effects of a series of stable adenosine analogs against generalized seizures initiated by focal injection of bicuculline methiodide into the rat prepiriform cortex (PPC) were studied by microinjection of these compounds into this brain area. The adenosine agonists, 5'-N-(ethyl)carboxamido-adenosine (NECA), cyclohexyladenosine, cyclopentyadenosine, 2-chloroadenosine and R- and S-phenylisopropyladenosine (R- and S-PIA), protected animals against seizures in a dose-dependent, and extremely potent manner. NECA, the most potent compound evaluated, completely prevented seizures at doses greater than or equal to 6.8 pmol. In contrast, heroic doses of the A2 selective ligand, 2-phenylaminoadenosine, afforded no protection against seizures. The rank order of potency of these compounds in suppressing seizures is as follows: NECA greater than cyclohexyladenosine greater than cyclopentyladenosine greater than or equal to R-PIA greater than 2-chloroadenosine greater than S-PIA much greater than 2-phenylaminoadenosine. These data suggest that the antiseizure activity of these compounds in the PPC results from activation of A1 adenosine receptors. Quantitative autoradiographic analysis of the distribution of tritiated adenosine agonists 30 min after microinjection in the PPC reveals that [3H]NECA diffuses to a significantly greater extent than R-[3H]PIA, which may contribute to the relatively greater potency of the former compound in suppressing bicuculline methiodide-induced seizures. These results suggest that adenosine A1 receptors may participate in the normal inhibitory regulation of the PPC, a forebrain area which may play a significant role in the pathobiology of epilepsy.     

A1- and A2-selective adenosine antagonists: in vivo characterization of cardiovascular effects

Caffeine, a nonselective adenosine receptor antagonist, 7-methyl-1,3-dipropylxanthine, a purported A2 selective antagonist and a 1,3-dipropyl-8-phenylxanthine amine congener (XAC), an A1 selective antagonist, were evaluated for their in vivo selectivity at A1 vs. A2 adenosine receptors. Blockade of the negative chronotropic effect of bolus i.v. injections of 2-chloroadenosine, R-phenylisopropyladenosine and N-ethylcarboxamidoadenosine was utilized as an index of antagonism at A1 receptors; blockade of the hypotensive effect of the same series of adenosine agonists was used as an index of activity at A2 receptors. In addition, blockade of the potentiating effect of adenosine on the hypertensive and chronotropic effects of nicotine was studied to assess further the role of A1 and A2 adenosine receptors in this response. The potent antagonist XAC displayed considerable A1 selectivity as demonstrated by blockade of adenosine receptor-mediated bradycardia at doses 5- to 10-fold lower than those antagonizing adenosine receptor-mediated hypotension. XAC also selectively blocked potentiation by adenosine of the positive chronotropic effect of nicotine, at doses which had minimal effects on the enhancement of the hypertensive effect of nicotine. The caffeine homolog 7-methyl-1,3-dipropylxanthine exhibited A2 selectivity as demonstrated by prevention of adenosine receptor-mediated hypotension at doses which only minimally attenuated the bradycardiac effect of adenosine analogs. Caffeine displayed no selectivity for A1 vs. A2 adenosine receptors. The results indicate that selective analogs such as XAC and F-methyl-1,3-dipropylxanthine will be useful probes for investigation of receptors involved in the physiological functions of adenosine.     

Adenosine A1 and A2 receptors mediate presynaptic inhibition and postsynaptic excitation in guinea pig submucosal neurons

Intracellular recordings were made from guinea pig submucosal neurons in vitro. Adenosine, 2-[p-(carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS21680), 2-chloroadenosine (CADO), 5'-N-ethylcarboxamidoadenosine (NECA), R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclohexyladenosine (CHA) and 1-deaza-2-chloro-N6-cyclopentyladenosine (DCCPA) were applied by adding them to the superfusion solution. Adenosine (30 nM to 30 microM) depolarized S-type neurons neurons and this was mimicked by analogs with potency order: CGS21680 = NECA greater than R-PIA greater than CADO greater than adenosine greater than CHA much greater than DCCPA. 8-Cyclopentyltheophylline (CPT) blocked the depolarizing action of CADO or R-PIA; this antagonism was surmountable and the dissociation equilibrium constant (KD) estimated by the Schild method was 295 nM. Synaptic potentials were evoked by focal stimulation of nerve strands running between submucosal ganglia. The nicotinic excitatory postsynaptic potential was reduced by adenosine and analogs with potency order CHA = R-PIA greater than DCCPA = NECA = CADO greater than adenosine much greater than CGS21680; CPT competitively antagonized this effect of CADO or CHA with a KD of 13 nM. The noradrenergic inhibitory postsynaptic potential was also reduced; the potency order was R-PIA greater than CHA = CADO = NECA = DCCPA greater than adenosine much greater than CGS21680; the KD of CPT as an antagonist was 7 nM. It is concluded that adenosine directly depolarizes submucosal neurons by acting at an A2 receptor, and that it inhibits the release of acetylcholine (from intramural nerves) and noradrenaline (from sympathetic nerves) by acting at a presynaptic A1 receptor.     

Opioid receptor selectivity of beta-endorphin in vitro and in vivo: mu, delta and epsilon receptors

The relative contributions of mu and delta opioid receptors in the response to Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn - Ala-Ileu-Ileu-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (B-endorphin) were assessed as reductions in B-endorphin potency in the presence of mu and delta receptor selective antagonists in the guinea pig ileum, mouse vas deferens, rat vas deferens and in analgesic and gastrointestinal transit time tests in mice. We used the nonselective antagonist naloxone, the mu antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) and the delta antagonist N,N,diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864) in each test system at concentrations that effectively antagonized the respective mu and delta agonists, Tyr-Pro-N-MePhe-D-Pro-NH2 and Tyr-D-Pen-Gly-Phe-D-Pen. In the guinea pig ileum, the inhibitory effects of 1 microM B-endorphin were blocked by 1 microM CTP and 1 microM naloxone, but not by 1 microM ICI 174,864. In the mouse vas deferens, B-endorphin (0.2 microM) was antagonized by 1 microM CTP, 1 microM ICI 174,864 and by 1 microM naloxone. In contrast, in the rat vas deferens, B-endorphin (0.01-1 microM) produced potent inhibitory actions that were blocked by 1 microM naloxone, but not by 1 microM-CTP or by 1 microM ICI 174,864. The mu agonist, Tyr-Pro-N-MePhe-D-Pro-NH2 (0.1-10 microM), like B-endorphin, also had inhibitory actions in the rat vas deferens, but its effects were blocked by 1 microM CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sympathetic denervation does not alter the density or properties of alpha-1 adrenergic receptors in rat vas deferens

Alpha-1 adrenergic receptors in surgically denervated rat vas deferens were studied using radioligand binding assays of [125I] BE 2254 ([125I]BE) and contraction measurements. Scatchard analysis of saturation isotherms of specific [125I]BE binding showed no change in the affinity or density of binding sites 4, 7 or 14 days after denervation of rat vas deferens. The potency of norepinephrine in inhibiting specific [125I]BE binding was also unchanged 7 days after denervation of vas deferens. The potency of phenylephrine in causing contraction in vitro did not change 4, 7 or 14 days after denervation of vas deferens; however, there was a significant increase in the maximum contractile response to phenylephrine at all time points. After partial inactivation of alpha-1 adrenergic receptors in vitro with phenoxybenzamine, there was an equivalent reduction in the number of [125I]BE binding sites in the control and 14-day denervated vas deferens. The equilibrium dissociation constants calculated from contractile measurements for norepinephrine were the same in the control and denervated tissues. However, there was a 2.2-fold increase in contractile sensitivity to norepinephrine 14 days after denervation and a 3.6-fold increase in contractile sensitivity to methacholine 7 days after denervation. These results show that surgical denervation of the rat vas deferens results in an increase in contractile sensitivity to norepinephrine and methacholine and an increase in maximum contraction. However, there is no change in alpha-1 adrenergic receptor density or properties at any time after denervation. Thus, alterations in alpha-1 adrenergic receptors do not contribute to contractile supersensitivity of denervated rat vas deferens.     

Cardiac adenylate cyclase activity, positive chronotropic and inotropic effects of forskolin analogs with either low, medium or high binding site affinity

A series of in vitro studies were conducted examining the adenylate cyclase stimulation, positive chronotropic and inotropic effects of forskolin and nine analogs which exhibited a range of [3H]forskolin binding site affinities (K1) from 0.020 to 3.174 microM. A significant (P less than .001) linear correlation (r = 0.94) was found between binding site affinity and adenylate cyclase stimulation (EC50) for forskolin and the nine structural analogs. Adenylate cyclase activity was also significantly correlated with the positive chronotropic and inotropic effects of these substances on isolated guinea pig atria. Compounds with K1 values between 0.020 and 1.136 microM produced concentration-dependent increases in heart rate and contractile force in isolated spontaneous and electrically paced guinea pig atria, respectively. In contrast, an analog with a K1 of 3.174 microM caused significant (P less than .05) negative chronotropic and inotropic effects at concentrations above 10 microM. The optimal separation between positive inotropic and chronotropic activity was found with compounds displaying potent [3H]forskolin binding site affinity but moderate adenylate cyclase stimulation, i.e., K1 and EC50 values of approximately 0.05 to 0.10 and 3 microM, respectively. The results of this study show that the forskolin analog, P87-7692 [7-desacetyl-7-(O-propionyl)-hydroxyl amino-carbonyl-forskolin], has marked activity with a wide separation between positive inotropic (248 +/- 41%) and chronotropic effects (43 +/- 13%) at 6.2 microM and may serve as a prototype for a forskolin-based cardiotonic.     

Adenosine promotes histamine H1-mediated negative chronotropic and inotropic effects on human atrial myocardium

Because histamine and adenosine are coreleased from the ischemic heart, we investigated the effects of their interaction on human myocardium. Surgical specimens of human right atrium (i.e., pectinate muscles) responded to histamine with increases in spontaneous rate and contractile force. Adenosine, and the A1-selective adenosine agonist N6-cyclopentyladenosine (CPA), reduced the spontaneous rate and suppressed the positive chronotropic and inotropic effects of histamine. CPA was more potent than adenosine in slowing the spontaneous rate and in suppressing histamine's positive chronotropic effect, suggesting that the responses to CPA and adenosine are A1-mediated. CPA was also more potent than adenosine in attenuating histamine's positive inotropic effect on human ventricular papillary muscle. The adenosine-induced suppression of histamine's effects on pectinate muscles was mimicked by carbachol, which like adenosine is known to attenuate H2-mediated histamine-induced adenylate cyclase activation. The H1-selective histamine antagonist pyrilamine potentiated histamine's chronotropic and inotropic responses, and inhibited the attenuation of these responses by adenosine or carbachol. In contrast, pyrilamine failed to modify the adenosine-induced attenuation of the cardiac stimulatory effects of dimaprit, an H2-selective histamine agonist. Our data suggest that adenosine-induced suppression of histamine's positive chronotropic and inotropic effects on human myocardium results both from an A1-mediated attenuation of H2-stimulatory effects and from the uncovering of H1 negative chronotropic and inotropic effects. Thus, the results of the histamine-adenosine interaction may exceed the "retaliatory" purpose of adenosine release and uncover H1-mediated myocardial depression.