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.

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.

Presynaptic displacement of serotonin by alpha-methyl-5-hydroxytryptamine in the nucleus tractus solitarius of the rat.

Rat brain slices containing the nucleus tractus solitarius loaded with [3H]5-hydroxytryptamine ([3H]5-HT) for superfusion were stimulated at (3 Hz, 25 mA, 1 min) resulting in fractional release ratios S2/S1 of 0.89 for [3H]5-HT in the presence of the serotonin uptake inhibitor 1.0 microM 6-nitroquipazine (6-NQ). alpha-Methylserotonin (alpha-Me-5-HT; 1.0 microM), a non-selective 5-HT1 and 5-HT2 receptor agonist, significantly reduced the S2/S1 ratio of [3H]5-HT without affecting the basal release ratios B2/B1 1.00 +/- 0.04. Without 6-NQ in the perfusion medium 1.0 microM alpha-Me-5-HT sharply increased the basal release B2/B1 to 2.21 (P < 0.01). In low Ca2+ medium the S2/S1 ratio was reduced to 0.06 and alpha-Me-5-HT promoted a B2/B1 release of 2.17 (P < 0.01). The 5-HT3 antagonist LY-278,584 did not block alpha-Me-5-HT induced basal release of [3H]5-HT. Both pindolol and LY-53,857 blocked the autoinhibitory effects of alpha-Me-5-HT, but only LY-53,857 and 6-NQ blocked the basal release induced by alpha-Me-5-HT. These results suggest that alpha-Me-5-HT reduces neurotransmitter release through the serotonin autoreceptor and displaces serotonin through a non-exocytotic release mechanism.

Acute severe carbon monoxide exposure in the rat: effects of hyperglycemia and hypoglycemia on mortality, recovery, and neurologic deficit.

Human and animal studies suggest a poorer outcome in the presence of abnormal blood glucose concentration during cerebral hypoxia-ischemia. It is unknown whether this is also the case in acute severe carbon monoxide poisoning. Using Levine-prepared rats, three groups were established and exposed to CO to answer this question: (1) hyperglycemics resulting from the administration of a 50% glucose solution, (2) hypoglycemics resulting from the administration of normal saline, and (3) untreated controls. The rats inhaled 2400 ppm CO for 90 min in the absence of anesthesia. Blood glucose was raised to a mean value of 402 mg/dL just prior to CO exposure in group 1. This resulted in an increased mortality rate (i.e., 54%), and during 4 h of room air recovery an impaired ability to regain body temperature, an increased plasma lactate dehydrogenase activity, and an increased neurologic deficit as compared with group 3. Hypoglycemia, which developed during CO exposure in group 2 (mean minimum glucose after 90 min, 44 mg/dL), resulted in an increased mortality rate (i.e., 46%), and during 4 h of room air recovery an impaired ability to regain body temperature and an increased neurologic deficit as compared with group 3. Blood glucose concentration in the rats in groups 2 and 3 that died during or shortly after CO exposure was significantly depressed relative to the survivors of those groups. Plasma insulin activity was elevated during CO exposure in group 1 as compared with group 3, but fell during recovery; insulin remained low throughout CO exposure and recovery in group 2. The results demonstrate the deleterious effects of both a very high and a very low blood glucose concentration during acute CO exposure.

Elevated blood glucose is associated with poor outcome in the carbon-monoxide-poisoned rat.

Levine-prepared, unanesthetized rats exposed to 2400 and 2700 ppm carbon monoxide (CO) for 90 min were used to examine the effect of acute CO poisoning on plasma glucose and insulin concentrations, and neurologic dysfunction. Body temperature and mean arterial blood pressure fell progressively during CO exposure. Glucose rose during initial CO exposure, then declined: glucose increased again after 2 h of room air recovery. Neurologic deficit, behaviorally-assessed after 4 h of recovery, was strongly correlated (r = 0.71, P less than 0.001) with glucose increase during the first 2 h of recovery. Recovery hyperglycemia was, in turn, correlated (r = 0.69, P less than 0.001) with the fall in glucose during the second half of CO exposure. Neurologic deficit was also correlated, but less strongly so, with hypoglycemia during CO exposure. Failure to rapidly regain body temperature during recovery was correlated with the post-CO rise in glucose concentration and with increased neurologic deficit. Plasma insulin activity was depressed immediately following CO exposure, and increased during recovery. CO-induced hypothermia was greater at 2700 than at 2400 ppm CO, as were post-CO recovery hyperglycemia, neurologic deficit and mortality, while body temperature recovery was less complete. The results provide evidence of an association between neurologic deficit and general morbidity following acute CO poisoning and the magnitude of post-CO hyperglycemia.

Carboxyhemoglobin in the rat: improvements in the spectrophotometric measurement and comparison to other studies.

An improved and simplified spectrophotometric method for the determination of carboxyhemoglobin (COHb) is described, which employs an equation to correct for the dissociation error during analysis. Two microliters, or less, of blood is diluted with an ammonium hydroxide solution directly in the measuring cuvette. A layer of light mineral oil overlying the diluent was found to increase measured COHb saturation of blood equilibrated with 100% CO. Sodium dithionite treatment of the oil further increased this value in one case. The measured COHb was shown to be affected directly by factors that alter hemoglobin concentration in the diluent (i.e. blood volume, hematocrit). Blood samples kept cold and under oil may be stored safely for as long as 10 days. Measurement of COHb by this method in rats exposed to 525, 900, 1800 and 2400 ppm CO produces higher values than those obtained with the 1965 spectrophotometric method of Commins and Lawther. Variations on the method of Commins and Lawther, as well as COHb values available in the literature for animals exposed to CO, are reviewed briefly.