The contributions of noradrenaline and ATP to the responses of the rabbit central ear artery to sympathetic nerve stimulation depend on the parameters of stimulation

The possibility that ATP and noradrenaline act as cotransmitters from sympathetic perivascular nerves was studied in the isolated rabbit central ear artery. Electrical stimulation of the perivascular nerves for either 1 s, or continuously until a maximum response was reached, produced frequency-dependent contractions that were sensitive to tetrodotoxin and guanethidine. Contractions to continuous stimulation were significantly greater than those to a 1 s train of stimulation. Prazosin (10(-6) M) significantly reduced, but did not abolish, all neurogenic contractions such that contractions to both a 1 s train and to continuous stimulation were now of a similar magnitude. A higher concentration of prazosin (10(-5) M) had no additional inhibitory effect on neurogenic contractions even though it further significantly inhibited contractions to exogenous noradrenaline. The greatest resistance to alpha-adrenoceptor blockade was seen at low frequencies. Desensitisation of the postjunctional P2-purinoceptor by repeated administration of alpha, beta-methylene ATP inhibited the non-adrenergic neurogenic contractions and contractions to exogenous ATP, but had no effect on contractions to exogenous noradrenaline. It is concluded that ATP and noradrenaline are excitatory cotransmitters from sympathetic perivascular nerves innervating the rabbit central ear artery. The relative contribution of each compound to neurogenic contractions of the ear artery is highly dependent on the parameters of stimulation used. Short pulse bursts (1 s) at low frequency (2-5 Hz) favour the prazosin-resistant (purinergic) component of the response.     

Influence of the endothelium on the amplification by serotonin of vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in rabbit isolated ear artery

The influence of the vascular endothelium on the amplifying effect of serotonin on steady-state responses to noradrenaline (NA) and sympathetic nerve stimulation (SNS) has been studied in the rabbit isolated ear artery. Removal of the endothelium abolished the dilator effect of acetylcholine (ACh, 1 microM) obtained in the presence of NA (0.3 microM). Responses to NA were increased after endothelium removal; however, responses to SNS were unaffected. Serotonin in a concentration which did not itself produce vasoconstriction (100 nM) increased responses of endothelium-intact preparations to NA and SNS and of endothelium-denuded preparations to SNS but had no effect on NA responses of endothelium-denuded preparations. If neuronal uptake was inhibited with cocaine (1 microM), serotonin markedly amplified responses to NA and SNS in both endothelium-intact and endothelium-denuded preparations. These results suggest that the endothelium influences the amplifying effect of serotonin on responses of rabbit ear artery to NA but not to SNS. Inhibition of removal of serotonin and NA by neuronal uptake may extend the facilitatory interaction between NA and serotonin to smooth muscle cells which are not subject to the inhibitory influence of an endothelium derived relaxing factor.     

The effects of histamine on responses of the rabbit ear artery to electrical stimulation and to exogenous noradrenaline

1 The effects of a subconstrictor dose of histamine (9 × 10^-7 mol/l) on the responses of the isolated perfused ear artery of the rabbit to electrical stimulation (E.S.) and to exogenous noradrenaline (NA) were investigated.

2 Both intraluminal (I/L) and extraluminal (E/L) histamine potentiated responses to E.S. and to I/L NA to the same extent.

3 Mepyramine alone (2.5 × 10^-6 mol/l) had no effect on the response of the ear artery to either stimulus, but in the presence of this concentration of mepyramine, the potentiation by histamine of the response to I/L NA was significantly decreased and that to E.S. was replaced by inhibition.

4 The H₁-receptor agonist, 2(2-pyridyl) ethylamine, applied I/L potentiated responses to I/L NA at both concentrations used (5.1 and 51 × 10^-7 mol/l), but only potentiated the effects of E.S. at the higher concentration.

5 The H₂-receptor antagonist, metiamide (4 × 10^-6 mol/l), alone did not alter the extent of potentiation of responses to either E.S. or I/L NA by histamine. This suggests relatively weak H₂-receptor activity in the rabbit ear artery. In the presence, but not the absence of metiamide, the potentiation by histamine of the I/L NA response was reversible, an observation suggesting an interaction between metiamide and the non-reversible component of the potentiating effect of histamine.

6 These results are interpreted in terms of postsynaptic H₁-receptors which potentiate and presynaptic H₂-receptors which inhibit contractile responses in the ear artery.

     

Effects of the muscarinic agonist McN-A-343 on responses to sympathetic nerve stimulation in the rabbit ear artery

1. Observations were made on the effects of 4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343) on responses of isolated segments of the central artery of the rabbit's ear to sympathetic nerve stimulation and noradrenaline.2. With low frequencies of nerve stimulation (2-5 Hz), McN-A-343 caused a decrease in responses to sympathetic nerve stimulation. This effect of McN-A-343 was abolished by dexamphetamine or atropine. In the presence of atropine, McN-A-343 caused an increase in responses to sympathetic nerve stimulation.3. With high frequencies of nerve stimulation (10-20 Hz), McN-A-343 caused an increase in responses. This effect was not qualitatively changed in the presence of atropine.4. When McN-A-343 had an inhibitory effect on responses to sympathetic nerve stimulation, responses to noradrenaline were unaffected.5. It is suggested that McN-A-343 acts on muscarinic receptors through which noradrenaline release may be inhibited; it may also act on the cholinergic stage in adrenergic transmission postulated by Burn & Rand (1959).     

Differential effects of calcium antagonists and Bay K 8644 on contractile responses to exogenous noradrenaline and adrenergic nerve stimulation in the rabbit ear artery.

1. The effects of three calcium antagonists (nifedipine, verapamil, diltiazem) and the calcium agonist Bay K 8644 were compared on contractile responses of similar amplitude elicited by noradrenaline (NA) and electrical nerve stimulation (ENS) in the rabbit isolated ear artery. 2. Contractions induced by both NA (3 x 10(-7) M) and ENS (10 Hz, 10s) were almost exclusively mediated by alpha 1-adrenoceptors, since 10(-7) M prazosin abolished (NA) or almost abolished (ENS) the responses, and prazosin was more than three orders of magnitude more potent than rauwolscine on both types of response. 3. ENS-induced contractions were considerably less inhibited by nifedipine, verapamil and diltiazem than were those elicited by NA. Bay K 8644 enhanced responses to NA more than those to ENS. 4. The inhibitory effect of nifedipine and Ca2+ deprivation on NA-induced contractions decreased with increasing NA concentration. Reduction of the NA response by prazosin or phenoxybenzamine increased the nifedipine inhibition. 5. Reduction of the ENS-induced contractions by prazosin or phenoxybenzamine, or by use of a lower stimulation frequency did not increase the inhibitory effect of nifedipine. 6. In conclusion, the differential effects of the calcium antagonists on NA- and ENS-induced contractions were not related to differences in alpha-adrenoceptor subtype (alpha 1/alpha 2), receptor reserve or response amplitude, but may rather reflect temporal and spatial differences in alpha-adrenoceptor activation between the responses.     

The electrical and mechanical responses of the rabbit saphenous artery to nerve stimulation and drugs.

1. Electrical and mechanical responses to field stimulation (1-64 Hz, 0.5 ms supramaximal voltage) were recorded simultaneously in the rabbit saphenous artery. The electrical response consisted entirely of excitatory junction potentials (e.j.ps) which were abolished by alpha, beta methylene ATP (alpha, beta MeATP, 10(-6) M) and by tetrodotoxin (TTX, 10(-6) M) but were unaffected by the alpha 1-adrenoceptor antagonist, prazosin (10(-6) M). No additional electrical response was evoked by field stimulation, even in the presence of normetanephrine (NMN) and desmethylimipramine (DMI, each 10(-6) M), which block neuronal and extraneuronal uptake of noradrenaline (NA) respectively. Action potentials to field stimulation were produced only in the presence of tetraethylammonium (10(-3) M) which also enhanced the contraction. 2. Contractions to field stimulation were reduced (by some 50%) by prazosin (10(-6) M) and abolished by the additional presence of alpha, beta MeATP (10(-6) M), which blocks purinoceptors by desensitization, suggesting the involvement of both NA and an ATP-like substance in the contractile response. 3. Idazoxan (10(-6) M) which blocks prejunctional alpha 2-adrenoceptors, significantly increased the amplitude of both e.j.ps and the contraction to field stimulation (10 pulses, 1-4 Hz, 0.5 ms, supramaximal voltage). 4. NA (10(-2) M by pressure ejection) did not alter membrane potential even in the presence of NMN and DMI (each 10(-6) M). ATP (10(-2) M by pressure ejection) produced a concentration-dependent, alpha, beta MeATP-sensitive depolarization. 5. In tissues desensitized by constant infusion of alpha, beta MeATP (10(-6) M contractions to NA (10(-7) - 3 x 10(-5) M), histamine (10(-7) - 3 x 10(-5) M) and KCl (1-1.6 x 10(-2) M) were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effects of capsaicin on responses of the rabbit ear artery to field stimulation.

1. The effects of capsaicin (Cap) on contractions of ring segments of rabbit ear artery induced by field stimulation were studied. 2. At low concentrations (0.3-3 microM) Cap caused transient enhancement and at higher concentrations (above 3 microM) inhibition of stimulation-induced contractions, without affecting noradrenaline (NA)-induced contractions. 3. In the continuous presence of high concentrations of Cap, rebound facilitation was observed after inhibition, and at this stage, Cap elicited less inhibition of the response. 4. Repeated application of Cap at 60 min intervals irreversibly desensitized the artery to the inhibitory effect of Cap. 5. Functional removal of the endothelium enhanced the facilitatory effect of low concentrations of Cap and attenuated its inhibitory effect. 6. Pretreatment with indomethacin abolished the facilitatory effect of Cap and enhanced its inhibitory effect, indicating that prostaglandins are involved in the action of Cap. The effect of indomethacin was more marked in preparations from which the endothelium had been removed. 7. Desensitization to substance P (SP) or substance K (SK), did not affect either the inhibitory or the facilitatory effect of Cap. 8. These results suggest that the dual effects of Cap on stimulation-induced contractions of rabbit ear artery may arise from the release of multiple mediators that act prejunctionally to modulate NA release. The stimulant effect seems to be mediated by prostanoids, while the inhibitory effect seems to be caused by a substance(s) that is not SP or SK. The possibility that the mediator is calcitonin gene-related peptide requires further study.     

Effects of tyramine on noradrenaline outflow and electrical responses induced by field stimulation in the perfused rabbit ear artery.

In the perfused rabbit ear artery the basal outflows of noradrenaline (NA) and 3,4-dihydroxyphenylglycol (DOPEG) were less than 1 ng g-1 and 1-2 ng g-1 wet weight of tissue respectively. Field stimulation increased outflows of NA and DOPEG in a frequency-dependent manner, and they reached the maximum value at frequencies over 5 Hz. Tyramine (1 X 10(-6) -1 X 10(-4) M) increased basal outflow of NA and DOPEG, in a dose-dependent manner. This effect was not blocked by tetrodotoxin (TTX, 3 X 10(-7) M), but was prevented by pretreatment with 6-hydroxydopamine (6-OHDA). Tyramine increased the field stimulation-induced outflow of NA but not that of DOPEG in a dose-dependent manner. Cocaine (1 X 10(-5) M) reduced the increased outflow of NA induced by tyramine at rest and during field stimulation, without modifying DOPEG-outflow. Guanethidine (5 X 10(-6) M), increased outflows of NA and DOPEG at rest, and reduced the NA outflow induced by field stimulation. Pretreatment with guanethidine (5 X 10(-6) M) did not block the action of tyramine on NA and DOPEG basal outflows. Additional application of guanethidine during the presence of tyramine did reduce the outflow of NA induced by field stimulation, but did not modify the outflow of NA and DOPEG at rest. Tyramine at concentrations over 1 X 10(-5) M depolarized the smooth muscle membrane of the rabbit ear artery. After chemical denervation with 6-hydroxydopamine (6-OHDA) the depolarizing action of tyramine was reduced. Tyramine-induced depolarization was attenuated by prazosin (5 X 10(-6) M) or phentolamine (5 X 10(-6) M), but not by guanethidine (5 X 10(-6) M). In 6-OHDA-denervated tissues, tyramine-induced depolarization was attenuated by phentolamine but not by prazosin. Field stimulation evoked excitatory junction potential (e.j.p.), slow depolarization and spike potential in the rabbit ear artery. Tyramine reduced, while guanethidine blocked these electrical responses. Tyramine did not alter the facilitation process of e.j.ps. In tissues pretreated with guanethidine, tyramine evoked either no electrical response or a slow depolarization during field stimulation. The slow depolarization was blocked by prazosin. Tyramine reduced the NA content of tissues in a dose-dependent manner (by 31% at 10(-4) M). Guanethidine (5 X 10(-6) M) reduced the NA content by 20%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of reduced calcium ion concentration and of diltiazem on vasoconstrictor responses to noradrenaline and sympathetic nerve stimulation in rat isolated tail artery.

In isolated, perfused proximal segments of Sprague-Dawley rat tail artery, idazoxan (100 nmol l-1) displaced the concentration-response curve to noradrenaline (NA) to the right. The log shift of the NA concentration-response curve was greater at lower concentrations than at higher concentrations of NA. Idazoxan (100 nmol l-1) had no effect on responses to electrical stimulation. Prazosin (10 nmol l-1) displaced the concentration-response curve to NA to the right as well as markedly reducing responses to sympathetic nerve stimulation. The concentration-response curve to NA, obtained after reducing the concentration of calcium ions in the Krebs solution from 2.5 to 0.6 mmol l-1, was significantly displaced to the right. Responses to sympathetic nerve stimulation were not affected by this reduction in the concentration of calcium ions. Diltiazem (1 and 10 mumol l-1) significantly displaced the concentration-response curve to NA to the right but had no effect on sympathetic nerve stimulation. These in vitro results in peripheral arterial smooth muscle confirm the findings of previous in vivo studies which suggest that alpha2-adrenoceptors contribute to the vasoconstrictor responses elicited by alpha-adrenoceptor agonists and that these responses but not those mediated by alpha1-adrenoceptors are dependent on extracellular calcium.     

Effects of the inhibition of noradrenaline uptake and synthesis on the maintenance of the response to continuous nerve stimulation in the central artery of the rabbit ear

1. The central artery of the rabbit ear was perfused through its lumen in vitro, with a constant pressure technique, and stimulated continuously via its periarterial sympathetic nerves at the physiological frequency of 5 Hz.2. The vasoconstrictor response, which led initially to an almost complete cessation of intraluminal flow, deteriorated steadily over a period of hours. The involvement of presynaptic mechanisms in this effect was indicated by the finding that noradrenaline, administered extraluminally, produced a similar response before the onset of continuous stimulation and at a late stage when the constriction had decreased markedly. In addition, the noradrenaline precursor DOPA, restored the depressed responses towards their original values, indicating that failure involved depletion of mediator for release.3. Responses to continuous stimulation declined significantly faster after inhibition of tyrosine hydroxylase with alpha-methyl-p-tyrosine. However, inhibition of the uptake of noradrenaline with cocaine did not enhance the decline of the response, even when the sensitization produced by the compound was taken into account.4. It is concluded that synthesis, along with the mobilization of stored mediator, rather than uptake and re-use of noradrenaline maintain the effector response in the central artery of the rabbit ear stimulated continuously at a frequency within the physiological range.     

Hydralazine: effect on the outflow of noradrenaline and mechanical responses evoked by sympathetic nerve stimulation of the rat tail artery.

1 The effects of hydralazine on the vasoconstrictor responses to field stimulation of sympathetic nerves were studied in the isolated proximal segments of the rat tail artery. Vasoconstrictor responses to transmural stimulation were depressed by superfusion of hydrazine (0.3, 3 and 30 muM) in a concentration-dependent manner. The inhibition appeared slowly and was not easily reversed by washing. 2 Hydralazine (30 nM, 0.3 and 3 muM) reduced the stimulation-induced overflow of tritium from proximal and distal segments of the tail artery labelled with [3H]-noradrenaline in a concentration-dependent manner. This phenomenon appeared rapidly and was easily reversed by washing. 3 Theophylline (0.5 mM) did not affect the inhibitory effect of hydralazine on the stimulation-induced tritium efflux from the distal segment of the rat tail artery. 4 The present results indicate that hydralazine has, in addition to its action on vascular smooth muscle, a very marked effect on sympathetic nerve terminals. The mechanism of this presynaptic inhibition appears to be different from the postsynaptic effect, in view of the much shorter delay, the shape of the dose-effect curve, and the lack of interaction with theophylline.     

Inhibitory effects of clonidine on responses to sympathetic nerve stimulation in the pithed rat.

1. The spinal sympathetic outflow to the eyelid, heart, splanchnic blood vessels, vas deferens and anococcygeus muscle was stimulated in pithed rats. 2. Clonidine inhibited sympathetic outflow to all of the tissues studied. The inhibitory effects of clonidine on cardiac nerves and hypogastric nerves were antagonized by phentolamine. 3. Clonidine produced a postsynaptic alpha-adrenoceptor agonist action on the eyelid, splanchnic blood vessels and the anococcygeus muscle. These effects were also antagonized by phentolamine. 4. The effects of clonidine, naphazoline and oxymetazoline on pre- and postsynaptic alpha-adrenoceptors were determined. 5. The presynaptic alpha-adrenoceptors employed were situated in either the sympathetic cardiac or hypogastric nerve terminals. Increases in diastolic blood pressure were used to assess concurrent postsynaptic alpha-adrenoceptor agonist activity. 6. The presynaptic alpha-adrenoceptor agonist potencies of clonidine, naphazoline and oxymetazoline were very similar on cardiac nerve terminals whereas on the hypogastric nerve terminals oxymetazoline was about 6 times more potent than either naphazoline or clonidine. 7. The results support the view that presynaptic alpha-adrenoceptors regulate transmitter release in sympathetic nerves. There appear to be subtle differences between the presynaptic alpha-adrenoceptors of different sympathetic nerve endings.     

The alpha 2-adrenoceptor agonists clonidine, TL99 and DPI enhance vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline in the rat tail artery preparation

1. Clonidine (1-30 nmol/l) enhanced the vasoconstrictor responses to sympathetic nerve stimulation in isolated perfused artery preparations from the rat tail. Higher concentrations of clonidine produced vasoconstriction in most artery preparations and reduced the response to sympathetic nerve stimulation. 2. The enhancing effect of clonidine was not affected by prazosin but was blocked by idazoxan, indicating that it was due to activation of alpha 2-adrenoceptors. 3. The alpha 2-adrenoceptor agonists DPI and TL99, like clonidine, enhanced vasoconstrictor responses to sympathetic nerve stimulation. 4. Vasoconstrictor responses to noradrenaline were enhanced by clonidine and TL99. 5. The findings suggest that activation of postjunctional alpha 2-adrenoceptors results in facilitation of the contractile response elicited by activation of alpha 1-adrenoceptors.