Jian-Xin Bao

Frequency- and train length-dependent variation in the roles of postjunctional α1- and α2-adrenoceptors for the field stimulation-induced neurogenic contraction of rat tail artery

SummaryThe present paper examines the roles of postjunctional α1- and α2-adrenoceptors for the noradrenaline (NA)-induced neurogenic contractile response to field stimulation mainly with 1–100 pulses at 2 or 20 Hz, in the tail artery of adult normotensive rats. Pharmacological tools were employed to isolate and characterize the α1- and α2-adrenoceptor-mediated components of this response. The degree to which the drugs influenced NA release or reuptake was assessed by their effects on the electrochemically determined, stimulation-induced rise in the NA concentration at the innervated outer surface of the media. This response was unaffected by α,β-methylene ATP (10 μM) or suramin (500 μM), added to desensitize or block P2-purinoceptors, respectively prazosin (0.1 μM) or SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxyl]-3-methyl-1H-2, 3, 4, 5-tetrohydro-3-benzazepine, 0.1 μM), used to block postjunctional α1- and α2-adrenoceptors respectively, nifedipine (10 μM), blocker of Ca2+ influx through L-type channels, and ryanodine (10 μM), which blocks mobilization of Ca2+ from intracellular stores; it was moderately enhanced by yohimbine (0.1 μM), blocker of pre- and postjunctional α2-adrenoceptors, and strongly enhanced by cocaine (3 μM) or desipramine (1 μM), blockers of NA reuptake. Judging from their inhibitory effects on the contractile responses to the α1- and α2-adrenoceptor agonists, phenylephrine andxylazine, prazosin (0.1 μM)and SK & F 104078 (0.1 μM) could be used to selectively block α1- and α2-adrenoceptors respectively, while yohimbine (0.1 μM) was less selective, strongly depressing α2- and slightly depressing α1-adrenoceptor-mediated responses. The α1-adrenoceptor-mediated component of the contractile response to short trains at 20 Hz was fast in onset, brief in duration and abolished by ryanodine; that mediated by α2-adrenoceptors was more delayed, prolonged and insensitive to ryanodine. Both components were dose-dependently depressed by nifedipine (0.1–10 μM). The small contractile responses to single pulses, or up to 50 pulses at 2 Hz, or short train (< 4 pulses) at 20 Hz, were more markedly depressed by 0.1 μM yohimbine or SK & F 104078 than by 0.1 μM prazosin and, hence, mediated mainly by α2-adrenoceptors. The reverse was true of the much larger response to longer trains at 20 Hz, which thus probably was mediated mainly by α1-adrenoceptors. Cocaine or desipramine, as well as α,β-methylene ATP or suramin, amplified both components of the NA induced contractile response especially that mediated via a1-adrenoceptors and caused by single pulses or short trains.The main conclusions are (i) that the small NA-induced contractile responses of this artery to single pulses, or pulses at low frequency, or in short trains at high frequency, are mediated mainly via α2-, and the larger responses to longer trains at high frequency increasingly via α1-adrenoceptors, (ii) that the α1- and α2-adrenoceptor-mediated components interact cooperatively, probably at least in part by utilizing two different pathways to increase the intracellular Ca2+, (iii) that neuronal reuptake of NA strongly restricts both components of the NA-induced contraction, especially the α1-adrenoceptor-mediated response to single pulses or short trains, and (iv) that both components of the NA-induced contraction, especially that mediated by α1-adrenoceptors, may be depressed by ATP released by field stimulation and acting via P2x-purinoceptors on smooth muscle. Based on these results a novel working hypothesis is proposed, in which it is assumed that the geometry of NA-mediated neuromuscular transmission in this vessel varies with the frequency and number of impulses in a stimulus train.

Dual contractile effects of ATP released by field stimulation revealed by effects of α,β‐methylene ATP and suramin in rat tail artery

1 The field stimulation‐induced release of endogenous ATP and noradrenaline (NA) and contractile response in rat isolated tail artery were examined. The release of ATP was studied by extracellular electrophysiological recording and that of NA by a novel voltammetrical technique. The effects of the P2‐purinceptor antagonist, suramin, on these parameters were compared with those of α,β‐methylene ATP, a P2X‐purinoceptor desensitizing agent. 2 Neither α,β‐methylene ATP (10 μm) nor suramin (100−500 μm) had significant effects on the extracellularly recorded nerve terminal action potential but both abolished the ATP‐induced excitatory junction current caused by stimulation at 0.1 Hz. Neither agent affected significantly the voltammetrically measured release of NA induced by 10 or 100 pulses at 20 Hz. 3 Combined blockade of both postjunctional α1‐ and α2‐adrenoceptors by prazosin and yohimbine (both 0.1 μm) profoundly depressed the contractile response to 10 pulses at 20 Hz. The small and fast residual contraction in the presence of these agents was abolished by α,β‐methylene ATP (10 μm) and inhibited by suramin in a concentration‐dependent manner (10–500 μm; IC50 75 μm) and was hence probably caused by ATP or a related nucleotide. 4 When added first, α,β‐methylene ATP (10 μm) or suramin (100–500 μm) delayed the onset and enhanced the amplitude of the neurogenic contraction. This enhanced response was abolished by further addition of prazosin and yohimbine (both 0.1 μm). 5 The K+ channel blocker, tetraethylammonium (10 mm), dramatically enhanced the contractile response to 100 pulses at 1 Hz and caused it to become diphasic. Addition of α,β‐methylene ATP (10 μm) or suramin (100–500 μm) abolished the large initial twitch component of this contraction and depressed the tonic phase. 6 Like α,β‐methylene ATP, suramin (500 μm) had no effect on the contraction caused by exogenous NA (10 nm − 100 μm) or KCl (60 mm); both agents almost abolished the contraction caused by ATP (100 μm). 7 In conclusion, (i) the contractile response of rat tail artery to electrical field stimulation is mediated by both ATP and NA, and is thus an expression of ATP‐NA co‐transmission, (ii) the released ATP exerts two opposite effects via ‘P2X‐like’ purinoceptors, triggering the initial rapid phase of the neurogenic contraction and restricting the NA‐mediated component of the contraction; and (iii) the source and possible physiological role of the ATP which causes the inhibitory effect are unknown at present.

Fast and Local Electrochemical Monitoring of Noradrenaline Release from Sympathetic Terminals in Isolated Rat Tail Artery

Abstract: Noradrenaline release from sympathetic nerve terminals was evoked by electrical nerve stimulation of an isolated segment of rat tail artery. This release was recorded by a carbon fiber electrode combined with differential pulse amperometry. The active part of the electrode (one carbon fiber 8 μm in diameter and 50 μm in length) was placed in close contact with the arterial surface. The oxidation current appearing at +120 mV and corresponding to the local noradrenaline concentration at the electrode surface was recorded every 0.5 s. No oxidation current was detected under resting conditions, but electrical stimulation evoked an immediate increase in this current. This response was suppressed when tetrodotoxin was added to the perfusion medium and was enhanced when noradrenaline reuptake was inhibited by cocaine. The amplitude of the response was increased with increasing stimulation frequencies (2–25 Hz) and train lengths (1–16 pulses). Finally, the time resolution of the method (0.5 s) was good enough to show that noradrenaline release precedes the postsynaptic response, i.e., the electrically evoked contraction of the artery.

Electrochemical and electrophysiological analysis of the effects of SK&F 104078 on prejunctlonal α2-adrenoceptors

SK&F 104078 is proposed to be a selective postjunctional alpha 2-adrenoceptor antagonist. We examined its effects on the electrically evoked release of endogenous noradrenaline (NA) from the rat tail artery and of ATP from the mouse vas deferens, as determined electrochemically or by extracellular recording, respectively. The alpha 2-adrenoceptor antagonist yohimbine (0.1 and 1 microM) increased NA release at both 2 and 20 Hz; the effect of yohimbine was further enhanced by the NA uptake blocker cocaine. SK&F 104078 (0.01-1 microM) did not change NA release at 2 or 20 Hz in the absence or presence of cocaine. NA release at 2 Hz was dose dependently depressed by the alpha 2-adrenoceptor agonist xylazine, an effect reversed by yohimbine (1 microM) but unaffected by SK&F 104078 (0.1 and 1 microM). Similarly, the inhibitory effect of xylazine on ATP release at 0.1 Hz was not influenced by SK&F 104078 (1 microM) but partially reversed by yohimbine (1 microM). The results show (i) that prejunctional alpha 2-adrenoceptors mediate autoinhibition of the release of endogenous NA and ATP from sympathetic nerves in the rat tail artery and in the mouse vas deferens, respectively, and (ii) that SK&F 104078 does not block these receptors in the concentration range 0.01-1 microM.

Do Schwann Cells Play a Role in ‘Upstream’ Regulation of the Release Probability in Sympathetic Nerve Varicosities?

The mechanisms which control the release probability (P) in sympathetic nerve varicosities were studied by extracellular recording in some model tissues. The method permits direct recording of different components of the action potential in the nerve terminals as well as of the excitatory junction current which reflects the release of quanta of ATP. The results indicate (i) that the action potential-induced Ca influx into the release site (ICaII) is necessary but not sufficient for release to occur, (ii) that P is controlled by a mechanism which depends on the resting Ca influx (ICaI) at a distance from (upstream of) the release site, and (iii) that Schwann cells may influence ICaI by controlling the Ca and K levels in the perineuronal ‘sub-Schwann space’.