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.

[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.

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.

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

The 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 greater than beta, gamma methylene ATP greater than ATP greater than or equal to adenosine. The adenosine uptake inhibitor S-p-nitro-benzyl-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.

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.

Effect of blood sampling and shipment to slaughter on plasma catecholamine concentrations in bulls.

The effect of venipuncture on blood serum catecholamine concentrations was studied in five yearling Red Angus bulls. Treatments consisted of sampling jugular vein blood via indwelling cannulas in a squeeze chute every 10 min for 1 h; sampling jugular vein blood from an indwelling catheter before, during and after venipuncture simulation; sampling jugular vein blood by venipuncture in a chute after shipment of 320 km to a slaughter plant; and sampling pooled blood from the neck during exsanguination at slaughter. Plasma epinephrine (E) and norepinephrine (NE) concentrations were determined by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). Prevenipuncture NE and E in blood sampled from an indwelling catheter extended through a solid wall were 1.0 and .5 pmol/ml plasma, respectively. Plasma NE and E during venipuncture (1.6 and 1.1 pmol/ml, respectively) and immediately after cannulation in a squeeze chute (1.8 and 1.2 pmol/ml, respectively) were higher than prevenipuncture concentrations. Plasma NE and E, 1.9 and 1.2 pmol/ml plasma after shipment of 320 km, increased during exsanguination to 135.8 and 81.3 pmol/ml plasma, respectively. The NE and E in blood samples collected by venipuncture in a chute or box stall do not represent resting concentrations of these catecholamines.

The activity of tyrosine hydroxylase in intact adrenergic neurons of the mouse vas deferens.

A procedure is described for measuring the activity of tyrosine hydroxylase in the intact adrenergic neurons of the mouse vas deferens. In this procedure, the L-dopa-1-14C which is formed from L-tyrosine-1-14C by the action of tyrosine hydroxylase is selectively and quantitatively decarboxylated by endogenous aromatic-L-amino acid decarboxylase. Although considerable tyrosine hydroxylase activity can be demonstrated with the intact mouse vas deferens in the absence of exogenous tetrahydropterin cofactor, the addition of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine results in increased activity. Exogenous norepinephrine inhibits the activity of tyrosine hydroxylase in the intact mouse vas deferens and this inhibitory effect is competitively antagonized by 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. When the vesicular catecholamine uptake mechanism is blocked by treatment of mice with 5 mg/kg of reserpine 24 hours prior to sacrifice, the activity of tyrosine hydroxylase in intact vas deferens is reduced and the inhibitory effect of exogenous norepinephrine is enhanced. Inhibition of monoamine oxidase with 0.15 mM pargyline does not affect the activity of tyrosine hydroxylase in the intact mouse vas deferens when the vesicular catecholamine uptake mechanism is intact but has a pronounced inhibitory effect following reserpine treatment. These observations lend further support to the conclusion that the activity of tyrosine hydroxylase in the intact adrenergic neuron is inversely related to the catecholamine concentration within an extravesicular pool. They also suggest that the catecholamines tend to accumulate within this extravesicular pool and thus become accessible to the action of monoamine oxidase when the vesicular uptake mechanism is inactivated or when the vesicular stores are filled to capacity.

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.

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.

Effects of alpha,beta-methylene ATP on the prejunctional purinoceptors of the sympathetic nerves of the rat caudal artery.

The effects of alpha,beta-methylene ATP, an agent known to stimulate and then to desensitize P2-purinoceptors, on the release of endogenous norepinephrine from the electrically stimulated rat caudal artery were determined. Norepinephrine was quantified by high-performance liquid chromatography-electrochemical detection techniques. alpha,beta-Methylene ATP over the concentration range of 1 to 100 microM did not affect the release of norepinephrine evoked by stimulation for 3 min at 1 Hz. In contrast, 2-chloroadenosine, a P1 receptor agonist, and beta,gamma-methylene ATP, a P2 receptor agonist, produced a concentration-related inhibition of the release of norepinephrine presumably by activating prejunctional purinoceptors. The failure of alpha,beta-methylene ATP to inhibit transmitter release was apparently not related to the length of pretreatment with this agent because pretreatments of 0.5 to 15 min yielded similar results. These findings indicate that the ability of alpha,beta-methylene ATP to decrease excitatory junction potentials and vasoconstriction of the caudal artery, as reported by others, is not due to a decrease in the release of transmitter. In spite of not possessing agonistic properties, alpha,beta-methylene ATP does interact with prejunctional purinoceptors as judged by the finding that the inhibitory effects of 2-chloroadenosine and beta,gamma-methylene ATP, but not those of the alpha-2 agonist clonidine, were antagonized by alpha,beta-methylene ATP. alpha,beta-Methylene ATP thus appears to be an antagonist at prejunctional purinoceptors (classified by us previously as P3-purinoceptors). alpha,beta-Methylene ATP also appears to act as an antagonist against endogenously released adenine nucleosides and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.