Richard A. Bjur

Characterization of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery

SummaryThe effects of a number of purinoceptor agents on the release of endogenous noradrenaline from the electrically stimulated rat caudal artery were determined. Noradrenaline was quantified by high performance liquid chromatography-electrochemical detection techniques. Both P1-receptor and P2-receptor agonists reduced the release of noradrenaline; the relative order of potency being 2-chloroadenosine > beta, gamma methylene ATP > ATP ≥ adenosine. The adenosine uptake inhibitor S-p-nitrobenzyl-6-thioguanosine potentiated the effects of adenosine but not those of the adenine nucleotides. This suggests that the nucleotides do not need to be converted to adenosine to produce a prejunctional inhibition of the release of noradrenaline. The P1-receptor antagonist 8-(p-sulfophenyl) theophylline reduced the inhibitory effects of both P1- and P2-receptor agonists as did the photolysis of tissues with an intense light source. The findings indicate that prejunctional purinoceptors that mediate an inhibition of the release of noradrenaline from the adrenergic nerves of the caudal artery may not be adequately defined as either P1- or P2-receptors and thus appear to represent a unique receptor. We suggest referring to these receptors as P3-purinoceptors.

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.

In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to α1‐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 α‐adrenoceptor antagonist, but not by idazoxan, an α2‐adrenoceptor antagonist. Clonidine, α 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 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 α1‐adrenoceptor‐mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.

ATP as a Cotransmittera

The notion that one neuron synthesizes, stores, and releases only one transmitter has been seriously questioned recently.’ The possible existence of cotransmitters in postganglionic sympathetic and parasympathetic neurons has been of particular research interest of late, and much credit for focusing attention on this issue goes to G. Burnstock, who published a commentary in Neuroscience in 1976 entitled “Do Some Nerve Cells Release More Than One Transmitter?”’ Although a number of substances have been proposed to act as cotransmitters with the “classical” monoamine or amino acid transmitters,’ the evidence to date is strongest for a nucleotide, most probably ATP. There has been, naturally enough, a general reluctance in accepting the notion that ATP, a compound that plays an essential role in intracellular function, could be released extracellularly. Nevertheless, the evidence is now abundant that adenine nucleotides and nucleosides do appear extracellularly upon stimulation of nerves, and this, together with the knowledge that these compounds exert a variety of pharmacological actions, makes it reasonable to postulate roles as neuromodulators and neurotransmitters. The tissue for which the data is probably the most extensive in support of a cotransmitter role for ATP is the sympathetically innervated vas deferens. In this paper, we will begin by reviewing the evidence from our laboratory and the laboratories of others that norepinephrine ( N E ) and ATP are cotransmitters in the vas deferens. We will then discuss other neuroeffector junctions, especially vascular smooth muscle, from the standpoint of cotransmission. We will also consider the potential sites of release of endogenous ATP upon transmural nerve stimulation of blood vessels.

Release of Endogenous ATP from Rat Caudal Artery

Electrical field stimulation of the rat caudal artery (0.5-ms pulses at 8 Hz for 3 min) results in the release of norepinephrine (quantified by HPLC-electrochemical detection) and adenyl purines including ATP, ADP and AMP (quantified by HPLC-fluorescence detection). The amount of ATP released from the tissue exceeded the amount of norepinephrine. Because postjunctional alpha 1-adrenoceptor stimulation with methoxamine also causes release of ATP, both neuronal and extraneuronal sites may contribute to the overflow of ATP. Results with the alpha 1-adrenoceptor antagonist prazosin lend support to this notion. Prazosin (10(-6) M) completely blocked the release of ATP by methoxamine but only partially reduced the release of ATP by field stimulation.

PARTICIPATION BY PURINES IN THE MODULATION OF NOREPINEPHRINE RELEASE BY METHOXAMINE

The alpha 1-receptor agonist methoxamine reduced, by a prazosin sensitive mechanism, the nerve stimulation evoked release of norepinephrine in the rat caudal artery. The effect of methoxamine was also antagonized by the purinoceptor antagonist 8-(p-sulfophenyl)theophylline suggesting an involvement of endogenous purines in this process. Indeed, methoxamine caused the release of adenine nucleotides and adenosine, an action which was blocked by prazosin. These results suggest that methoxamine releases ATP or a related purine which in turn decreases transmitter release by acting on prejunctional purinoceptors.

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.

TEMPORAL DISSOCIATION OF THE RELEASE OF THE SYMPATHETIC CO‐TRANSMITTERS ATP AND NORADRENALINE

Based partially on an analogy with adrenal chromaffin cells, it is a commonly held view that the sympathetic nerve co-transmitters ATP and noradrenaline (NA) are stored in and released from the same vesicles within the nerve varicosity (Sneddon & Westfall 1984; Stjarne 1989). If ATP and NA originate from the same vesicles then the ratio of released NA to purine should remain constant during stimulation and the time course of release should be similar for both transmitters. We have tested this hypothesis with the in vitro sympathetic nerve smooth muscle preparation of the guinea-pig vas deferens. The approach was to use a superfusion system that rapidly removed the released transmitter from the preparation at a constant rate. The preparations were stimulated for 1 min with transmural nerve stimulation at frequencies of 2 ,4 or 8 Hz. Samples of superfusate were taken at 10 s intervals for analysis of adenine nucleotides and adenosine by HPLC-fluorescence detection and catecholamines by HPLC-electrochemical detection (Sedaa et al. 1990). While what is actually quantified in this type of experiment is the amount of transmitter that overflows from the preparation, the conditions are such that overflow reflects the amount of transmitter that is released from the nerves. Figure 1 shows the time course of overflow of NA and ATP from the vas deferens evoked by transmural stimulation. For both transmitters the magnitude of overflow was increased as the frequency of stimulation increased. It is apparent, however, that the time course of overflow differed markedly for the two transmitters. The release of NA increased with increasing numbers of pulses when stimulated at 2 or 4 Hz. When stimulated at 8 Hz the release of NA reached a peak at 30 s and remained constant for the remainder of the 1 min stimulation period. At the completion of the stimulation the overflow of NA gradually returned toward prestimulation levels. The release of ATP, on the other hand, reached a peak much more quickly, by about 20 s, and then declined dramatically even though the stimulation continued for 1 min. We monitored the overflow not only of ATP but ADP, AMP and adenosine as well. While there were quantitative differences in the overflow of these nucleotides and nucleosides the pattern of release was qualitatively similar for all four. Therefore, the transient nature of the overflow of ATP cannot be accounted for by its metabolism to other purines because, if this were so, one would expect an increasing accumulation with time of the total pool of adenine nucleotides and nucleosides. The primary mechanism for removing NA from the synapse once it is released is re-uptake into sympathetic nerves (Graefe & Bonisch 1988). In order to determine whether neuronal uptake influences the pattern of the overflow of NA we carried out experi-

[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 vas 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 α‐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.

EFFECT OF METHOXAMINE ON NORADRENALINE RELEASE IN THE CAUDAL ARTERY OF HYPERTENSIVE RATS

1. The effect of methoxamine, an α1‐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).