Subtypes of adenosine receptors in the brainstem mediate opposite blood pressure responses.

Microinjections of selective agonists for adenosine receptor subtypes were made into the caudal NTS of rats. CGS 21680, a selective A2 receptor agonist, elicited pronounced, dose-related decreases in mean arterial blood pressure (ED50 = .021 nmols/rat). Conversely, CPA, a selective A1 receptor agonist, elicited potent dose-related increases in mean arterial blood pressure (ED50 = 0.185 nmols/rat). Additionally, the depressor responses elicited by the A2 agonist and the pressor responses elicited by the A1 agonist were completely and selectively blocked, respectively, by the selective A2 antagonist, CGS 15943A, and the selective A1 antagonist, DPCPX. These data indicate that selective activation of brainstem adenosine receptors in vivo may elicit distinct and opposing response patterns.

The effects of central injections of adenosine analogs on blood pressure and heart rate in the rat.

Rats were implanted with chronic indwelling cannulae into the lateral cerebral ventricle. After recovery from surgery, acute experiments on blood pressure were conducted under methoxyflurane/nitrous oxide anesthesia. Rats were injected intracerebroventricularly with two adenosine analogs, 5'-N-ethylcarboxaminidoadenosine (NECA) and (-)-N-(1-methyl-2-phenylethyl)adenosine(L-phenylisopropyladenosine) (L-PIA), and the effects on blood pressure and heart rate recorded. Both analogs produced dose-related reductions in blood pressure and heart rate with L-PIA producing a more potent depression of heart rate than NECA. These effects on blood pressure and heart rate were antagonized by parenteral injections of caffeine. In separate experiments, the responses of blood pressure and heart rate to microinjection of NECA into the brainstem of rats anaesthetized with methoxyflurane/nitrous oxide were also examined. Microinjection of 2.7 nmol/kg into the fourth ventricle in the region of the area postrema produced a profound and long-lasting depression of blood pressure and heart rate. These results show that central injections of analogs of adenosine can influence the areas of the central nervous system involved in the control of cardiovascular function.

Respiratory effects of cyclic AMP following injection into the nucleus tractus solitarius of rats.

Rats were anesthetized with urethane and a limited occipital craniotomy was performed to expose the caudal medulla in the region of the obex. Injections were made into sites in the brainstem of spontaneously-breathing rats through glass micropipettes. Tidal volume, respiratory frequency, minute volume, blood pressure and heart rate were recorded before and after the administration of 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP), an analog of cyclic AMP. Injections of Br-cAMP into the ventromedial portion of the caudal nucleus tractus solitarius (NTS) produced dose-related decreases in pulmonary ventilation due to effects on both respiratory frequency, as well as minute volume. In larger doses, Br-cAMP produced periodic apnea and irregular breathing. The respiratory depression was accompanied by transient hypotension and bradycardia. The data indicate that cyclic AMP may function as a second messenger in respiratory control regions in the brainstem.

Antagonism of the anti-conflict effects of phenobarbital, but not diazepam, by the A-1 adenosine agonist l-PIA.

The present study examined the effects of the anxiolytics diazepam and phenobarbital, the A-1 adenosine agonist N6-R-phenylisopropyladenosine (l-PIA), and the A-2 adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) on conflict behavior. Water-restricted rats were trained to drink from a tube that was electrified (0.5 mA intensity) on a FI-29s schedule, electrification being signaled by a tone. After 3 weeks of daily 10-min sessions, the animals accepted a stable number of shocks (punished responding) and consumed a consistent volume of water (unpunished responding) per session. Different doses of l-PIA and NECA were then tested separately at weekly intervals. In addition, the effects of diazepam and phenobarbital were determined in animals pretreated with saline, l-PIA, or NECA. Neither l-PIA (15-250 nmole/kg) nor NECA (2.5-20 nmole/kg) produced a significant anti-conflict effect when administered alone. Diazepam (1.25-10 mg/kg) or phenobarbital (10-40 mg/kg) administration to saline-pretreated rats resulted in a dose-dependent increase in punished responding (shocks received) with minimal effects on unpunished responding (water intake). Neither l-PIA nor NECA pretreatment reliably altered the effects of diazepam on conflict behavior. Pretreatment with l-PIA, but not NECA, significantly reduced the anti-conflict effects of phenobarbital on conflict behavior. These data suggest that phenobarbital, but not diazepam, anti-conflict responses may involve interactions with A-1 adenosine receptors.

Interactions between adenosine and nifedipine in the rat cerebral cortex.

Nifedipine inhibits the uptake of [(3)H]adenosine into rat cerebral cortical synaptosomes with an IC(50) value of 1.1 ?M. When applied by iontophoresis onto rat cerebral cortical neurons it potentiated the depressant effects of adenosine on spontaneous firing. Some of the calcium-antagonist actions of nifedipine may be mediated by adenosine.

Purine concentrations in the cerebrospinal fluid of unanesthetized rats during and after hypoxia.

A new technique for achieving repeated sampling of fourth ventricular cerebrospinal fluid (CSF) from the cisterna magna of unanesthetized rats is described. The sampling cannula is positioned extracranially, in contrast to previously published techniques which require insertion through the skull. CSF samples, withdrawn from unanesthetized rats before, during and after a 25 min period of inhalation of 5% oxygen in nitrogen, were analyzed for their adenosine and inosine contents by high pressure liquid chromatography. Adenosine and inosine levels increased during the hypoxic episode and were even higher 1 h later. They had declined, but were still above basal levels, 2-3 h after the hypoxic episode. Elevated CSF adenosine concentrations may be responsible for the generation of such persistent effects of hypoxia as post-hypoxic respiratory depression.

Y2 receptors for neuropeptide Y in the nucleus of the solitary tract mediate depressor responses.

In anesthetized, spontaneously breathing rats, microinjections of selective agonists of neuropeptide Y (NPY) receptor subtypes were made into the medial region of the caudal nucleus of the solitary tract (NTS) at the level of the area postrema. This region of the rat NTS exhibits very high densities of NPY binding sites. Microinjections of the long C-terminal NPY fragment, NPY(13-36), a selective agonist at Y2 receptors, into the caudal NTS elicited pronounced, dose-related reductions in blood pressure and respiratory minute volume. Moreover, the specific pattern of cardiorespiratory responses elicited by NPY(13-36) was remarkably similar, over approximately the same dosage range, with the cardiorespiratory response pattern elicited by intact NPY. In contrast to the potent NTS-mediated responses evoked by NPY(13-36), similar microinjections conducted with either NPY(26-36), an inactive C-terminal NPY fragment, or [Leu31,Pro34]NPY, a NPY analog with specific agonist properties at Y1 receptors, into the same caudal NTS sites did not appreciably affect cardiorespiratory parameters even at 10-20-fold higher dosages. The present results with selective agonists for NPY receptor subtypes suggest that the depressor responses and reductions in minute volume elicited by microinjections of intact NPY and NPY(13-36) were mediated by Y2 receptors in the caudal NTS, likely distributed at presynaptic sites in the medial region of the subpostremal NTS.

Respiratory effects of 5'-ethylcarboxamidoadenosine, an analog of adenosine, following microinjections into the nucleus tractus solitarius of rats.

Microinjections of 5'-ethylcarboxamidoadenosine (NECA), an adenosine analog, were made into respiratory-related regions of the nucleus tractus solitarius (NTS) of spontaneously breathing rats and cardiorespiratory parameters were recorded during a 60-min test period. Microinjections of NECA, in the dose range of 0.35-350 picomol per rat, produced significant dose-related reductions in respiratory rate which were accompanied by correlative increases in tidal volume. At the end of the 60-min test period following the administration of NECA, the respiratory rate remained profoundly depressed, whereas blood pressure and heart rate were not significantly affected compared to preinjection control values. The data from this study suggest that adenosine may exert modulatory influences in brainstem respiratory control regions.

Behavioral interaction of adenosine and diazepam in mice.

Mice were implanted with chronic indwelling cannulae in the lateral cerebral ventricle. The behavioral interaction of intraperitoneal (i.p.) injections of diazepam with intracerebroventricular (i.c.v.t.) injections of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA) was examined on spontaneous locomotor activity. Concurrent injections of i.c.v.t. adenosine and i.p. diazepam, at doses which had no significant effect on locomotor activity when given alone, acted synergistically to produce a marked depression of locomotor activity. In contrast, i.p. injections of diazepam did not potentiate the locomotor depressant effects of i.c.v.t. injections of NECA, an uptake resistant analog of adenosine. These findings support the possibility of specific benzodiazepine-adenosine interactions in the central nervous system.

The adenosine analog, 5'-N-ethylcarboxamidoadenosine, exerts mixed agonist action on cardiorespiratory parameters in the intact but not decerebrate rat following microinjections into the nucleus tractus solitarius.

A limited occipital craniotomy was conducted on intact and decerebrate urethane-anesthetized, spontaneously breathing rats to expose the caudal medulla in the region of the obex. Microinjections of 5'-N-ethylcarboxamidoadenosine (NECA), a metabolically stable adenosine analog which exhibits mixed agonist properties for adenosine receptor subtypes, were made into the medial region of the caudal nucleus tractus solitarius (NTS) at the level of the caudal tip of the area postrema, an area of the NTS in which there is known to be a functional co-existence of cardiovascular and respiratory-related neuronal elements. Cardiorespiratory responses were subsequently recorded for a 30-min test period. In the intact rat, microinjections of NECA produced significant dose-related reductions in respiratory rate which were accompanied by dose-dependent increases in tidal volume and these pronounced effects on respiration persisted throughout the test period. On the other hand, microinjections of NECA into this region of the NTS of the intact rat elicited complex, bi-directional cardiovascular responses, producing hypotension (at lower doses) and pressor responses (at higher doses) in addition to bradycardia (at lower doses). In an effort to examine the functional interactions between the NTS and forebrain structures involved in cardiorespiratory control, microinjections of NECA in the identical dose range were made into the same NTS sites of a separate group of urethane-anesthetized, spontaneously breathing rats in which reciprocal connections between forebrain areas and the brainstem had been disrupted by acute supracollicular decerebration. A simulating electrode, placed in the paraventricular nucleus of the hypothalamus (PVH), was used to confirm complete transection during the experiment and to ascertain the integrity of reciprocal connections between the brainstem and rostral brain regions involved in cardiorespiratory control. Although decerebration at the supracollicular level negligibly affected basal cardiorespiratory parameters, microinjections of NECA into the NTS revealed dramatic differences in the cardiovascular response patterns between intact and decerebrate rats. Whereas cardiovascular responses elicited by microinjections into the NTS were significantly affected by supracollicular decerebration, respiratory responses were highly similar for both intact and decerebrate animals. Indeed, repeated measures MANOVA indicated that there were no significant differences in the time-related or dose-related responses in the depression of respiration between decerebrate and intact rats following NECA microinjections.(ABSTRACT TRUNCATED AT 400 WORDS)

Augmented release of serotonin by adenosine A2a receptor activation and desensitization by CGS 21680 in the rat nucleus tractus solitarius.

Rat dorsomedial medullary brain segments containing primarily nucleus tractus solitarius (NTS) were employed for slice superfusion studies of electrically evoked [3H]serotonin ([3H]5-HT) release. Individual slices loaded with [3H]5-HT were stimulated two times, S1 and S2, at 3 Hz, 25 mA, 2 ms pulses for 1 min. Control NTS slices had a S2/S1 ratio of 0.94 (+/- 0.02). Superfusion of tissue slices with 0.1 nM to 100 nM 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680), a selective adenosine A2a receptor agonist, for 5 min prior to the S2 stimulus produced a significant concentration-dependent increase in the S2/S1 fractional release ratio which was maximal (37.2% increase, P < 0.01) at 1.0 nM. However, superfusion of tissue slices with CGS 21680 over the same concentration range for 20 min prior to the S2 stimulus did not significantly alter the S2/S1 ratio from control release ratios. The augmented release of [3H]5-HT mediated by 1.0 nM CGS 21680 with 5 min tissue exposure was abolished by 1.0 nM 9-chloro-2-(2-furanyl)-5, 6-dihydro-[1,2,4]-triazolo[1,5-c]quinazolin-5-imine (CGS 15943) as well as by 100 nM 8-(3-chlorostyryl)caffeine (CSC), both A2a receptor antagonists, but not by 1.0 nM 8-cyclopentyl-1,3,-dipropylxanthine (DPCPX), the A1 receptor antagonist. These results indicate that CGS 21680 augmented the evoked release of [3H]5-HT in the NTS by way of activation of presynaptic adenosine A2a receptors. It was also apparent that this population of adenosine A2a receptors in the NTS desensitized within 20 min since the augmenting action of CGS 21680 on evoked transmitter release was not evident at the longer time interval.

Cardiovascular effects of microinjections of adenosine analogs into the fourth ventricle of rats.

Rats were implanted with chronic indwelling cannulae into the posterior region of the fourth ventricle. After recovery from surgery, acute experiments on blood pressure were conducted under urethane anesthesia. The blood pressure and heart rate responses following administration of two adenosine analogs, NECA and L-PIA were examined. Microinjections of both analogs produced dose-dependent reductions in blood pressure and heart rate. NECA was approximately 20-fold more potent than L-PIA in reducing blood pressure and depressing heart rate. The cardiovascular effects of both analogs were antagonized by parenteral injections of caffeine. These findings show that microinjections of analogs of adenosine into the fourth ventricle can influence areas of the central nervous system involved in cardiovascular control.

Cardiorespiratory function is altered by picomole injections of 5'-N-ethylcarboxamidoadenosine into the nucleus tractus solitarius of rats.

A limited occipital craniotomy was conducted on urethane-anesthetized, spontaneously breathing rats to expose the caudal medulla in the region of the obex. Microinjections of 5'-N-ethylcarboxamidoadenosine (NECA), an adenosine analog, were made into the medial region of the caudal nucleus tractus solitarius (NTS) at the level of the caudal tip of the area postrema, an area of the NTS in which there is known to be a functional co-existence of cardiovascular and respiratory-related neuronal elements. Cardiorespiratory responses were subsequently recorded for a 60 min test period. Microinjections of NECA, in the dose range of 0.35-350 pmol per rat, produced significant dose-related reductions in respiratory rate which were accompanied by dose-dependent increases in tidal volume and these pronounced effects on respiration persisted throughout the test period. In contrast, the effects of NECA microinjections on cardiovascular parameters in this region of the NTS were bidirectional and elicited considerably more complex responses during the test period. During the initial period (2-5 min) following injection, NECA elicited significant hypotension (at lower doses) and pressor responses (at higher doses) in addition to significant bradycardia (at lower doses) whereas by the end of the 60 min test period, almost all doses of NECA had resulted in hypertension and tachycardia. Multivariate analysis of variance (MANOVA) and correlation statistics indicated that the effects of NECA on blood pressure during the initial 2-5 min were dose-dependent and unlikely related to depression of respiratory frequency. A further examination of the data by MANOVA indicated that the pharmacological effects of NECA during the 60 min test period exhibited a highly significant and specific dose-dependent and time-related response pattern for the respiratory, but not the cardiovascular, parameters. Taken together, these manifold response patterns suggest that the respiratory effects of NECA may be mediated by different intrinsic mechanisms in the NTS than are the cardiovascular effects of NECA. At the end of the 60 min test period following the administration of NECA, the respiratory rate remained profoundly depressed. In view of previous studies showing that microinjections of cyclic AMP analogs, forskolin, isoproterenol and adenosine into the same NTS sites elicit a similar depression of respiration, the results with NECA in the present study further support the notion that cyclic AMP may serve as a second messenger in NTS respiratory control regions and these respiratory depressant effects may be mediated by a single adenosine receptor subtype.(ABSTRACT TRUNCATED AT 400 WORDS)

Central effects of adenosine analogs on locomotor activity in mice and antagonism of caffeine.

Mice implanted with chronic indwelling cannulas were injected in the lateral cerebral ventricle with a series of adenosine analogs and the effects on spontaneous locomotor activity were recorded. All analogs produced dose-related decreases in locomotor activity. The relative order of potency for locomotor depression was: NECA much greater than L-PIA greater than CADO greater than D-PIA. Caffeine at the lowest dose produced a significant decrease in locomotor activity. At higher doses caffeine had no effect on locomotor activity but it did antagonize the depressant effects of NECA, a finding consistent with the notion that the central stimulant action of methylxanthines is due to their antagonism of central adenosine receptors.

Cardiorespiratory effects of inositol hexakisphosphate following microinjections into the nucleus tractus solitarii.

Microinjections of inositol hexakisphosphate (IP6), a metabolite of inositol recently found to occur in high concentrations in the brainstem, were made into the caudal portion of the nucleus tractus solitarii (NTS) of spontaneously breathing rats and cardiorespiratory parameters recorded for a 30 min test period. Microinjections of IP6, in the dose range of 100-500 pmol/rat, produced significant dose-related reductions in mean arterial blood pressure and respiratory rate. The onset for hypotensive action and respiratory depression following microinjections of IP6 was very rapid and a transient apnea could be elicited at the higher doses. Moreover, the sodium and calcium salts of IP6 were relatively equipotent in depressing cardiorespiratory parameters, with the exception of heart rate wherein the sodium salt elicited a much more pronounced bradycardia. These results confirm and extend the findings of a previous study suggesting that IP6 and closely related metabolites may act on extracellular receptors. Taken together, these data provide further support to the notion that inositol lipid signalling pathways may generate both intracellular and extracellular signals in the brain.

A possible role of endogenous adenosine in the sedative action of meprobamate.

The behavioral interaction of intraperitoneal (i.p.) injections of meprobamate with intracerebroventricular (i.c.v.) injections of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA) was examined on spontaneous locomotor activity in mice. The locomotor depressant effect of meprobamate, an adenosine uptake inhibitor, was potentiated by adenosine, but not by NECA, an uptake-resistant adenosine analogue. These findings suggest that heightened endogenous adenosine levels could mediate some of the central actions of meprobamate.

The effects of 5'-N-ethylcarboxamidoadenosine on evoked release of [3H]serotonin in the rat nucleus tractus solitarius.

5'-N-Ethylcarboxamidoadenosine (NECA) a non-selective adenosine A1 and A2a receptor agonist was employed in stimulated (3 Hz, 25 mA, 1 min) superfused nucleus tractus solitarius (NTS) brain slices loaded with [3H]5-hydroxytryptamine ([3H]5-HT), and ligand binding with [3H]NECA on NTS membranes. Superfusion of NTS slices with 1.0 and 3.0 nM NECA for 5 min prior to S2 stimulation produced an augmented release of [3H]5-HT (35.7%) above the control S2/S1 ratio. When the duration of NECA perfusion was increased to 20 min prior to S2, the S2/S1 ratio was depressed 21% at 1.0 nM for [3H]5-HT release. The augmented release of [3H]5-HT by NECA at 5 min was blocked by the adenosine A2a antagonist 8-(3-chlorostyryl)caffeine (CSC; 100 nM), and was reduced but not blocked by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 nM). Saturation binding assays with [3H]NECA on NTS membranes showed two binding sites, a high affinity site with a KD 2.18 nM and low affinity site with a KD of 44.9 nM. With the selective adenosine A2a antagonist CSC the high affinity site was blocked while 10 nM DPCPX, the A1 antagonist, reduced binding of the low affinity site, but did not abolish it. NECA binds to two different adenosine receptor sites in NTS membranes with the A2a receptor being the high affinity site. The same A2a site is associated with the augmented neurotransmitter release of [3H]5-HT with 5 min tissue exposure since it is blocked by CSC. Longer tissue exposure to NECA resulted in desensitization and finally inhibition of release possibly associated with adenosine A1 receptors.

Behavioral interaction of adenosine and trifluoperazine in mice.

Mice were implanted with chronic indwelling cannulae in the lateral cerebral ventricle. A dose-response curve was established for the effect of i.p. injections of trifluoperazine (TFP) on spontaneous locomotor activity. In addition, the behavioral interaction of i.p. injections of TFP with intracerebroventricular (i.c.v.) injections of adenosine (ADO) was examined. TFP depressed locomotor activity in a dose-dependent manner. A dose of ADO, which had no effect on locomotor activity when given alone, enhanced the depressant effects of TFP at all doses. As a control for the specificity of this behavioral interaction, mice also were given i.p. injections of TFP in combination with i.c.v. injections of 5'-N-ethylcarboxamidoadenosine (NECA), an uptake-resistant adenosine analogue. TFP and NECA did not interact to produce a significantly more pronounced locomotor depression. These results substantiate the notion that the sedative actions of TFP involve the inhibition of adenosine uptake and thus potentiation of extracellular adenosine levels.

Characterization of muscarinic receptors in the rat nucleus tractus solitarius.

Crude membrane preparations from the dorsomedial medulla and whole heart of adult rats were used to characterize muscarinic receptors (mChRs) in the nucleus tractus solitarius (NTS). Isotherm binding assays with [3H]quinuclidinyl benzilate ([3H]QNB) indicated a uniform population of muscarinic cholinergic receptors in both tissues with a Kd of 60.3 pM and a Bmax of 401 fmol/mg protein for NTS membranes while heart membranes had a Kd of 56.6 pM and a Bmax of 193 fmol/mg protein. Competitive binding assays with selective muscarinic antagonists on NTS membranes using 80 pM [3H]QNB showed the following relative Ki values consistent with M2 mChRs: atropine, 1.9 nM; methoctramine, 7.7 nM; 4-diphenylacetoxy-N-methylpiperidine methobromide, 9.4 nM; AF-DX 116, 87.6 nM; and pirenzepine, 657 nM. Similarly, two agonists, carbachol and oxotremorine, showed Ki values of 1.82 microM and 0.379 microM, respectively. All of the competitive binding assays exhibited Hill coefficients close to 1.0 and InPlot analysis showed a single binding site for each agonist and antagonist examined.