Differential cotransmission in sympathetic nerves: role of frequency of stimulation and prejunctional autoreceptors.

Recent reports have suggested that sympathetic nerves may store separately and release independently the cotransmitters ATP and norepinephrine (NE). It is conceivable therefore that the quantity of each neurotransmitter that is released from the nerves is not fixed but rather may vary, possibly with the frequency of stimulation. To test this hypothesis we studied the concomitant release at various frequencies and cooperative postjunctional actions of ATP and NE during the first 10 s of electrical field stimulation of the guinea pig vas deferens. We found that at lower frequencies (8 Hz), prejunctional inhibition of the release of NE, which occurs via alpha2-adrenoceptors, modulates the ultimate composition of the cocktail of cotransmitters by limiting the amount of NE that is coreleased with ATP. As the frequency of stimulation increases (above 8 Hz), the autoinhibition of the release of NE is overridden and the amount of NE relative to ATP increases. The smooth muscle of the guinea pig vas deferens reacts to changes in composition of the sympathetic neurochemical messages by increasing the amplitude of its contractions due to the enhancement by NE of the contractile responses triggered by ATP. This evidence suggests that the prejunctional alpha2-adrenoceptor may function as a sensor that "reads" the frequency of action potentials produced during a burst of neuronal activity and converts that information into discrete neurochemical messages with varying proportions of cotransmitters. The mechanism for decoding the informational content of these messages is based on the cooperative postjunctional interactions of the participating cotransmitters.

Regional differences in noradrenaline-induced release of adenosine triphosphate from rat vascular endothelium.

We examined the release of endogenous adenyl purines such as adenosine triphosphate (ATP), ADP, AMP and adenosine from the caudal artery (CA), saphenous artery (SA), renal artery (RA), mesenteric artery (MA), pulmonary artery (PA) and thoracic aorta (TA) of rats, using high-performance liquid chromatography fluorescence detection. Noradrenaline induced the release of adenyl purines from these blood vessels. The total amount of adenyl purines release induced by noradrenaline from the CA was considerably larger than that from the TA. The rank order of the amount of adenyl purines released from the six blood vessels was CA>SA>RA>MA>PA> or =TA. The noradrenaline induced release of adenyl purines from the CA was significantly reduced by the removal of the endothelium. Noradrenaline also induced the release of adenyl purines from cultured endothelial cells of the CA and TA. The total amount of adenyl purines released from the former blood vessel was much larger than that from the latter. These results suggest the existence of vascular endothelial cells that are able to release ATP by an alpha1-adrenoceptor mediated mechanism, and that these cells are not homogeneously distributed in the vasculature.

Source of ATP, ADP, AMP and adenosine released from isolated rat caudal artery exposed to noradrenaline.

Purines of ATP, ADP, AMP and adenosine released from rat caudal artery with and without endothelium and the isolated smooth muscle and endothelial cells were examined, in order to determine the source. Treatment of intact segments of caudal arteries with noradrenaline (10 microM) for 3 min induced a large release of ATP, ADP, AMP and adenosine. However, if the artery segments had been denuded of their endothelial lining, noradrenraline induced only a slight release of purines. Endothelial cells in primary culture prepared from caudal arteries, when exposed to noradrenaline for 3 min released large amounts of purines, whereas vascular smooth muscle cells prepared similarly and passaged endothelial cells did not release purines upon exposure to noradrenaline. These results indicate that, of smooth muscle and endothelial cells of the vascular wall, only intact endothelial cells react to alpha-adrenoceptor stimulation by releasing adenine nucleotides and adenosine.

Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation.

Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.

Differences between the regulation of noradrenaline and ATP release.

1. We have studied the effects of adrenergic receptor agonists and antagonists and various calcium channel antagonists on the overflow of adenine nucleotides (ATP, ADP, AMP), adenosine (ADO) and noradrenaline (NA) from superfused guinea-pig vasa deferentia evoked by electrical field stimulation (EFS). 2. Samples of superfusate were taken at 10 s intervals for analysis of purines (HPLC with fluorescence detection) and of NA (HPLC with electrochemical detection). During 1 min of EFS the overflow of ATP peaked at about 20 s and then abruptly decreased even though stimulation continued. The overflow of NA reached a peak at about 40 s and remained at a constant level for the duration of the stimulation. 3. Pretreatment with the alpha 2-receptor antagonists idazoxan and yohimbine produced a substantial increase in the overflow of NA and a lesser increase in the overflow of ATP, indicating that endogenously released NA has a greater influence on its own release than on that of ATP. Interestingly, certain alpha 2-agonists. e.g. xylazine and clonidine, produce a greater reduction in ATP release than NA. Together the results suggest that the release of ATP and NA may be regulated by different subsets of prejunctional alpha 2-receptors. 4. The N-type calcium channel antagonist omega-conotoxin reduced the EFS-evoked release of NA to a greater extent than ATP while the P-type calcium channel antagonist omega-agatoxin did the reverse. These results indicate that NA release may be more dependent on calcium influx through N-type channels whereas ATP release is coupled to calcium entry through P-type channels. 5. These differences in the pharmacological regulation of ATP and NA release lend credence to the idea that these two co-transmitters originate from different release sites in adrenergic nerves.

Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea-pig vas deferens.

1. Experiments were carried out to quantify the stimulation-evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve-smooth muscle preparation of the guinea-pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC-fluorescence detection and catecholamines by HPLC-electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS-evoked overflow of NA and the purines from the guniea-pig vas deferens preparation was examined after treatment with the neuronal amine-uptake inhibitors desipramine and cocaine, the alpha 1-adrenoceptor agonist methoxamine, the alpha 1-adrenoceptor antagonist prazosin, the alpha 2-adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline-depleting drug reserpine and the adrenergic neuron-blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non-neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon-ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation-dependent metabolism of ATP.

The effects of age on the release of adenine nucleosides and nucleotides from rat caudal artery.

1. The spontaneous and alpha-adrenoceptor-induced release of ATP, ADP, AMP and adenosine were determined from arterial segments and from isolated endothelial cells from caudal arteries of young (5-week-old), adult (30-week-old) and old (100- to 110-week-old) Wistar rats. 2. The spontaneous (non-evoked) release of the sum total of the four purines was significantly greater from artery segments of young rats than from adult and old rats. 3. The release of the adenine nucleotides and adenosine induced by methoxamine (10 microM), an alpha 1-adrenoceptor agonist, was greater from artery segments from young rats than from old rats. 4. The spontaneous release of the sum total of the four purines was significantly greater from endothelial cells prepared from caudal arteries of young rats than of old rats. 5. The noradrenaline (10 microM)-induced release of the sum total of the four purines was significantly greater from endothelial cells prepared from caudal arteries of young rats than of old rats. 6. The levels of adenine nucleotides and adenosine, determined in plasma from anaesthetized rats, were significantly higher in young rats compared with adult and old rats. 7. These findings suggest that the release of ATP from the vascular endothelial cells is reduced with advancing age.

Differences in purinoceptor modulation of norepinephrine release between caudal arteries of normotensive and hypertensive rats.

The effects of 2-chloroadenosine, a purinoceptor agonist, on the overflow of endogenous norepinephrine (NE) was examined in the electrically field-stimulated (EFS) caudal artery obtained from Wistar-Kyoto (WKY) rats and age-matched spontaneously hypertensive rats (SHRs). 2-Chloroadenosine at 10(-6) and 10(-5) M reduced the EFS-evoked release of NE from arteries of WKY rats but was without effect on the release of NE from arteries of SHRs. Methoxamine (10(-5) M), an alpha adrenoceptor agonist, caused the release of ATP, ADP, AMP and adenosine from caudal arteries of Wistar rats, WKY rats and SHRs, and decreased the release of EFS-evoked release of endogenous NE in arteries from normotensive rats, but not from arteries of SHRs. Thus, both exogenously applied and endogenously released purines are ineffective in reducing the evoked release of NE in this model of hypertension. The enhanced release of NE from sympathetic nerves of SHRs may be due in part to the loss of prejunctional modulation by purines.

Effect of methoxamine on noradrenaline release in the caudal artery of hypertensive rats.

1. The effect of methoxamine, an alpha1-adrenoceptor agonist, on the overflow of endogenous noradrenaline (NA) was examined in the electrically field stimulated (EFS) caudal artery obtained from Wistar rats, Wistar-Kyoto rats (WKY) and age-matched spontaneously hypertensive rats (SHR). 2. Methoxamine inhibited the EFS-evoked release of endogenous NA in the arteries from Wistar rats and WKY, but not in the arteries of SHR. 2-chloroadenosine, a purinoceptor agonist, also inhibited the NA release in the arteries from normotensive rats but not in the arteries of SHR. 3. The inhibitory effect of methoxamine was blocked by adenosine deaminase and potentiated by adenosine uptake inhibitor, dipyridamole. 4. Methoxamine caused the release of adenine nucleotides and adenosine from the caudal arteries of WKY and SHR. 5. These suggest that the inhibitory effect of methoxamine on NA release is mediated by endogenous adenyl purines and that the failure of methoxamine to inhibit NA release in the caudal artery of SHR is due to a dysfunction of the prejunctional purinoceptors on sympathetic nerve terminals.

In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.

Inhibitory and facilitatory effects of purines on transmitter release from sympathetic nerves.

The effects of several purinoceptor agonists and antagonists on norepinephrine overflow were examined in the electrically field stimulated vas deferens and saphenous artery of the rabbit. Both the adenine nucleotides ATP and beta,gamma-methylene ATP and the adenine nucleosides adenosine and 2-chloroadenosine reduced the electrical field stimulation-evoked release of norepinephrine from the in vitro vas deferens. These responses are antagonized by both 8-(p-sulfophenyl)-theophylline and alpha,beta-methylene ATP, indicating that this effect is mediated by P3-receptors. Interestingly, the nucleotide and nucleoside agonists facilitated, rather than inhibited, the electrical field stimulation-evoked release of norepinephrine from the in vitro perfused saphenous artery. This facilitation was antagonized by 8-(p-sulfophenyl)-theophylline but not by alpha,beta-methylene ATP. These results indicate that there may be facilitatory prejunctional purinoceptors that exhibit structure-activity relationships similar to, but perhaps not identical to, those exhibited by inhibitory prejunctional purinoceptors.

[Ca2+]i-sensitive, IP3-independent Ca2+ influx in smooth muscle of rat vas deferens revealed by procaine.

1. The actions of procaine (10 mM) on noradrenaline-induced effects on 45Ca-influx, 45Ca-efflux, 45Ca-content, total inositol phosphates, inositol-1,4,5-trisphosphate, and contractile status of the rat was deferens were examined. 2. Noradrenaline alone had no effect on 45Ca-influx or 45Ca-content, but released Ca2+ from intracellular stores as indicated by an increased 45Ca-efflux and increased total inositol phosphates, specifically inositol-1,4,5-trisphosphate, leading to contraction of the rat vas deferens. 3. Noradrenaline, in the presence of 10 mM procaine, increased 45Ca-influx and 45Ca-content. Procaine blocked the noradrenaline-induced 45Ca-efflux, the increase in total inositol phosphates, the increase in inositol-1,4,5-trisphosphate, and contraction. 4. The noradrenaline-induced increase in 45Ca influx which was observed in the presence of procaine was abolished by phentolamine and nifedipine but was not altered significantly by propranolol suggesting that, in the presence of procaine, noradrenaline activates dihydropyridine-sensitive calcium channels through alpha-adrenoceptors. 5. These findings indicate that, in the rat vas deferens, noradrenaline induces contraction by releasing intracellularly stored Ca2+. The effects of procaine appear to be due to its ability to block the release of Ca2+ from intracellular stores. Furthermore, the simultaneous increase in 45Ca influx and inhibition of inositol-1,4,5-trisphosphate formation in tissues treated with procaine plus noradrenaline indicates that Ca2+ influx is independent of inositol-1,4,5-trisphosphate formation.

Time-dependent increase in Ca2+ influx in rabbit abdominal aorta: role of Na-Ca exchange.

Exposure of the rabbit abdominal aorta to the combination of high K+ and norepinephrine resulted in a time-dependent increase in the rate of 45Ca influx and 45Ca and 22Na content over that observed after stimulation with either K+ or norepinephrine alone. The increase in 45Ca influx, but not the increase in 22Na content, was extracellular Ca2+ (Cao2+) dependent. This time-dependent increase in 45Ca influx was prevented by incubating the tissue in Na(+)-free medium. Nifedipine inhibited both the initial depolarization-induced 45Ca influx and time-dependent increase in 45Ca influx and 22Na content. The effect of nifedipine on time-dependent fluxes was prevented by ouabain. Phorbol dibutyrate mimicked the effects of norepinephrine on 22Na retention and 45Ca fluxes. The effects of phorbol dibutyrate and norepinephrine were not additive. It is concluded that, in rabbit abdominal aorta, norepinephrine plus K+ causes 22Na retention (possibly through inhibition of the sodium pump) and a Cao(2+)- and intracellular Na+ (Nai+)-dependent increase in 45Ca influx. This latter effect is possibly the result of increased Nai(+)-Cao2+ exchange.

Simultaneous determination of nerve-induced adenine nucleotides and nucleosides released from rabbit pulmonary artery.

Electrical field stimulation elicits the release of catecholamines, adenine nucleotides, and adenosine from the rabbit pulmonary artery in a frequency dependent manner. To enhance our ability to investigate the release of endogenous adenine nucleotides and adenosine from this and other biological preparations, a new analytical procedure has been developed. This procedure involves the use of an internal standard, 9-beta-D-arabinofuranosyladenine (IS), the derivatization of ATP, ADP, AMP, adenosine (Ado), and IS with chloroacetaldehyde, the isocratic high-performance liquid chromatographic separation of these ethenopurine derivatives on an Ultron N-phenyl HPLC column, and their detection and quantitation by fluorescence spectroscopy. This procedure has enhanced sensitivity and reliability over existing procedures due to the stability of the chromatographic baseline and the use of an internal standard. When this analytical procedure was utilized to measure the adenine nucleotides and Ado that are released from the rabbit pulmonary artery in response to electrical field stimulation, it was observed that the release of endogenous ATP, ADP, AMP, and Ado exceeded that of endogenous norepinephrine. A molar ratio (6-amino purines:catecholamines) of approximately 2000:1 was obtained at a stimulation frequency of 16 Hz. This observation suggests an important extracellular role for adenine nucleotides and nucleosides in the physiology of vascular tissues.

Norepinephrine increases Na-Ca exchange in rabbit abdominal aorta.

Na-Ca exchange was measured as intracellular Na+ (Na+i)-dependent 45Ca uptake in rabbit abdominal aortic rings. The amount of Na+i-dependent 45Ca uptake was proportional to both the concentration of Na+ in the Na(+)-loading solution and the concentration of Ca2+ in the assay medium. Na+i-dependent 45Ca uptake was inhibited by incorporation of Na+ in the assay medium and by amiloride analogues. Norepinephrine significantly enhanced the rate of 45Ca uptake in Na(+)-loaded tissue but had no effect on Na+i-independent 45Ca uptake. The effect of norepinephrine was prevented by phentolamine but not by propranolol. The stimulatory effect of norepinephrine was absent when the concentration of extracellular Ca2+(Ca2+o) was 0.3 mM or lower but became significant at 0.6 mM and higher. Na-Ca exchange was also increased by phorbol 12,13-dibutyrate but not by its inactive analogue (4 alpha-phorbol 12,13-didecanoate). 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, blocked the stimulatory effect of norepinephrine on Na-Ca exchange. It is suggested that alpha-adrenoceptor stimulation increases Na-Ca exchange in rabbit abdominal aorta in a Na+i- and Ca2+o-dependent fashion. This effect is possibly mediated through the activation of protein kinase C.

Nucleotide modulation of norepinephrine release from sympathetic nerves in the rat vas deferens.

The effects of a number of purinoceptor agonists and antagonists on norepinephrine (NE) overflow were examined in the electrically field-stimulated rat vas deferens. The P1 receptor agonists adenosine and 2-chloroadenosine and the P2 receptor agonists ATP and beta, gamma-methylene ATP all reduced the overflow of NE, which was quantified by high-performance liquid chromatography-electrochemical detection techniques. The P1 receptor antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) and the P2 receptor desensitizing agent alpha, beta-methylene ATP blocked the inhibitory effects of both P1 and P2 receptor agonists. The pyrimidine nucleotide UTP also inhibited NE overflow and this effect was antagonized by 8-SPT. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine potentiated and adenosine deaminase blocked the inhibitory effect of adenosine on NE overflow but neither had any effect on the ability of the adenine nucleotides to inhibit NE overflow. These results indicate that adenine nucleotides can act per se, without conversion to adenosine, on a prejunctional receptor to inhibit the release of NE. Because the effects of the adenine nucleotides are antagonized by 8-SPT, it appears that they act at the same receptor as the adenine nucleosides. UTP apparently acts at this receptor as well. These findings suggest that prejunctional purinoceptors on the sympathetic nerves of the rat vas deferens differ from P1 or P2 receptors as usually defined and thus may represent a unique class of receptor (P3) as has been suggested for the prejunctional receptors of the rat caudal artery.