Adrenergic and purinergic neurotransmission in arterial resistance vessels of the cat intestinal circulation

European Journal of Pharmacology, 164 (1989) 23-33

23

Elsevier

EJP 50762

Adrenergic and purinergic neurotransmission in arterial resistance vessels of the cat intestinal circulation E d w i n M. T a y l o r * a n d Michael E. P a r s o n s Department of Pharmacology, Smith Kline & French Research Ltd., The Frythe, Welwyn, Hertfordshire AL6 9AR, U.K. Received 3 November 1988, revised MS received 30 January 1989, accepted 7 February 1989

The contribution of adrenoceptors and purine receptors in mediating neurogenic vasoconstriction was investigated in the autoperfused intestinal circulation of anaesthetised cats treated with atropine and propranolol. Prazosin (0.5 mg/kg) and yohimbine (1.5 mg/kg) reduced but did not abolish the vasoconstrictor responses to stimulation of the efferent sympathetic nerves. The inhibitory actions of the two antagonists were additive but even after cq- and az-adrenoceptor blockade nerve stimulation still elicited a residual, frequency-related vasoconstriction. The initial, rapid, phase of this response was completely abolished after desensitisation of Pzx-purinoceptors with a high dose (1.5 mg i.a.) of a,B-methylene ATP. In the absence of a-adrenoceptor antagonists, a,fl-methylene ATP reduced neurogenic vasoconstriction particularly at low frequency (1 Hz) nerve stimulation, but also caused a short-lasting decrease in noradrenaline and methoxamine responses which indicates that the drug may have some non-specific effects on arterial smooth muscle. The results suggest that neurotransmission in arterial resistance vessels of the cat intestinal circulation is predominantly under adrenergic control mediated by postsynaptic a 1- and a2-adrenoceptors, with a possible purine involvement in the initial rapid response of the blood vessels, particularly to low frequency nerve stimulation. Adrenergic/purinergic neurotransmission; a-Adrenoceptor antagonists; a, B-Methylene ATP; Resistance vessels (pre-capillary); (In vivo)

1. Introduction The vasoconstriction observed following stimulation of the sympathetic nerves is generally assumed to be mediated by the release of noradrenaline and its subsequent effects on postsynaptic a-adrenoceptors. In some blood vessels al-adrenoceptors are predominant while in others, particularly veins, stimulation of both al- and a2-adrenoceptors are involved (McGrath et al., 1982; Langer and Hicks, 1984; Docherty and Hyland, 1985; Steen et al., 1986; Bentley and Widdop, 1987; Bulloch et al., 1987). Noradrenaline also activates presynaptic a2-adrenoceptors to

* To whom all correspondence should be addressed.

cause inhibition of nervous activity (negative feedback). However, a number of recent publications have shown that the effects of sympathetic nerve stimulation in some vascular areas or specific blood vessels in vitro are not inhibited by a-adrenoceptor antagonists (Hirst and Neild, 1980; Holman and Surprenant, 1980; Sneddon and Burnstock, 1984b; Burnstock, 1986; Kennedy et al., 1986; Burnstock and Warland, 1987a,b; Bulloch and McGrath, 1988). One possible explanation to account for these results is that high local concentrations of noradrenaline may overcome the a-adrenoceptor blockade. This may be a plausible hypothesis when competitive a-adrenoceptor antagonists are used but would not explain the fact that non-competitive, irreversible, antagonists such as phenoxybenzamine are also ineffective.

0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

24 Hirst and Nield (1980) postulated that noradrenaline in these tissues was acting on novel postjunctional receptors distinct from a-adrenoceptors which they called 7-receptors. An alternative proposal which has gained considerable support in recent years is that other substances, including peptides, amines and purines, are released following autonomic nerve stimulation and may act as cotransmitters with noradrenaline in sympathetic nerves or with acetylcholine in parasympathetic nerves (recent review, Burnstock, 1986). There is a considerable amount of evidence to suggest that ATP or a closely related purine is involved in neurotransmission in the vas deferens (Sneddon and Burnstock, 1984a; Bulloch and McGrath, 1988), bladder (Hills et al., 1984; MacKenzie and Burnstock, 1984) and some blood vessels (Su, 1983; Sneddon and Burnstock, 1984b; Von Kt~gelgen and Starke, 1985; Kennedy et al., 1986; Burnstock, 1986; Burnstock and Warland, 1987a; Hirst and Lew, 1987; Muramatsu, 1987). The classification of purine receptors into P1 (adenosine sensitive)- and P2 (ATP-sensitive)-purinoceptors (Burnstock, 1978) and the further division of Pzpurinoceptors into two subtypes Pzx and Pzy (Burnstock and Kennedy, 1985) has helped in our understanding of the vascular actions of ATP and related purines. Stimulation of P2y-purinoceptors induces relaxation of smooth muscle, including vasodilatation mediated by the actions of E D R F and prostacyclin released from endothelial cells (Pearson et al., 1983; Hellewell and Pearson, 1984; Fleetwood and Gordon, 1987), although an endothelium-independent effect of ATP has been observed in the rabbit (Mathieson and Burnstock, 1985). The rank order of agonist potency at Pzypurinoceptors is 2-methylthio or 2-chloro ATP >> ADP > ATP >> a,B- or B,y-methylene ATP. In contrast, P2x-purinoceptors mediate contraction of smooth muscle (vasoconstriction) and are thought to be located on vascular smooth muscle cells possibly at nerve endings. For this receptor the rank order of agonist potency is very different: a,B-methylene ATP > B,y-methylene ATP > ATP = 2-methylthio ATP > ADP. In addition to being a selective P2x-purinoceptor agonist c~,B-methylene ATP at high concentrations or repeated high doses also causes a selective desensitisation of P2x-

purinoceptors and can be used as an effective antagonist. Thus a,fl-methylene ATP has been shown to inhibit the excitatory junction potentials seen with nerve stimulation in the vas deferens (Sneddon and Burnstock, 1984a) and blood vessels (Sneddon and Burnstock, 1984b) and abolish the a-adrenoceptor resistant component of sympathetic nerve stimulation (Von Ktigelgen and Starke, 1984; Kennedy et al., 1986; Burnstock, 1986; Burnstock and Warland, 1987a) supporting the role of ATP as a cotransmitter. All of the previously described studies have used in vitro techniques and very little work has been done on the effects of a,B-methylene ATP in vivo (Delbro et al., 1985). However, in a recent publication Parsons and Taylor (1988) described the effects of several ATP analogues, including ~,fl-methylene ATP, on pre-capillary resistance vessels of anaesthetised cats. a,B-Methylene ATP caused a powerful constriction of resistance vessels of the intestinal circulation but, conversely, had little effect on resistance vessels of the hindquarters and kidney. This suggests a very heterogeneous distribution of P2x-purinoceptors in the cat vasculature. Similar variations in P2xpurinoceptor density, both within and between species, probably accounts for the lack of consistency in the results of experiments designed to assess the importance of purines as neurotransmitters. In some blood vessels purines appear to play a dominant part in neurogenic vasoconstriction (Burnstock and Warland, 1987a) while in others no detectable purine component was observed (Bell, 1985). The present study was undertaken to evaluate the relative importance of c~adrenoceptors and P2x-purinoceptors in mediating the vasoconstrictor effects of sympathetic nerve stimulation in the intestinal circulation of anaesthetised cats. A preliminary account of this work was presented to the British Pharmacological Society meeting in December 1988. 2. Materials and methods

2.1. Surgical procedures Male or female cats (2.5-3.5 kg) were anaesthetised with pentobarbitone sodium (sagatal) 60

25 m g / k g i.p. The trachea was cannulated to ensure a clear airway and blood pressure recorded from a femoral artery. In some experiments heart rate was monitored using an instantaneous rate meter triggered from the blood pressure pulse. I.v. injections were given through a cannula in a superficial vein of the foreleg. About 2 cm of the left carotid artery was cleared of connective tissue and two loose ligatures placed around it. The abdomen was opened with a midline incision carefully ligating any points where bleeding occurred. The large intestines, caudal to the posterior mesenteric artery, and the duodenum, at the level of the anterior mesenteric artery and vein, were tied with double ligatures. The posterior mesenteric artery was also ligated. The sheets of connective tissue between the intestines and the trunk of the animal were cut, ligating any small blood vessels as necessary, leaving the intestines connected to the rest of the animal only by the anterior mesenteric artery and associated nerve plexus, the mesenteric vein and lymph ducts. The anterior mesenteric artery was freed from the nerve plexus and two loose ligatures placed around it. The nerve plexus was ligated. After completion of the surgery, the cats were left for about 1 h to allow seepage of blood to cease and clots to form. During this time the perfusion apparatus was prepared for use and primed with 0.9% saline. Heparin 1000 u / k g i.v. was administered and blood, taken from a cannula pushed down the left carotid artery into the descending aorta, was pumped around the extracorporeal circuit and into the cannulated anterior mesenteric artery to perfuse the intestinal vasculature at constant flow (20-30 m l / m i n ) . Perfusion pressure was monitored with a Bell and Howell (0-750 m m Hg) transducer connected to a T piece in the silicone-rubber tubing between the p u m p and the intestinal vasculature.

2.2. Perfusion apparatus Two peristaltic pumps in series were used in the autoperfusion experiments. Blood was taken from the left carotid artery and pumped through an extracorporeal circuit of silicone-rubber tubing (internal diameter 2 mm, wall thickness 1 mm, total volume approximately 5 ml). The first p u m p

was servo-controlled and set to operate at a constant pressure output which could be maintained at any chosen level between 0 and 250 m m Hg. Pressure was detected with a Bell and Howell transducer (0-750 m m Hg) connected to a T piece between the two pumps. Thus the second p u m p received blood at constant pressure irrespective of any changes in systemic blood pressure. The second peristaltic p u m p delivered blood at constant flow into the intestinal vasculature via the cannulated anterior mesenteric artery. Changes in perfusion pressure are directly proportional to changes in vascular resistance which, in turn, reflects changes in tone of the small pre-capillary blood vessels (small arteries, arterioles, pre-capillary sphincter vessels). Test compounds were either given i.v. or injected directly into the arterial inflow (i.a.) through a thick walled section of the extracorporeal circuit (Viton tubing, wall thickness 3 mm, length 2 cm) between the two peristaltic pumps.

2.3. Sympathetic nerve stimulation The cats were given atropine, 1 m g / k g i.v., to inhibit parasympathetic activity and propranolol, 1 m g / k g i.v. plus 3 m g / k g s.c. to prevent neuronally released noradrenaline from stimulating preor postsynaptic fl-adrenoceptors (Patel et al., 1981). A specially designed electrode, similar in shape to an electromagnetic flow probe, was fitted around the anterior mesenteric artery and nerve plexus distal to the cannulation of the artery. Transmural stimulation (30 V, 0.5 ms pulse duration) was applied across the anterior mesenteric artery and nerve plexus at different frequencies from 0.25 to 16 Hz. Each stimulation period was continued for 45-60 s until a peak vasoconstrictor response, to the given frequency, was obtained and intervals of 5-10 min were left between stimulation periods to allow the tissue to recover. Frequency-response curves were constructed before and after treatment with prazosin 0.5 m g / k g i.a., yohimbine 1.5 m g / k g i.a. or c~l- and a2adrenoceptor blockade with either prazosin and yohimbine or phenoxybenzamine 10 m g / k g i.a. Repeat frequency-response curves were also constructed in five cats given saline injections (con-

26 trols) to determine the reproducibility of the response. In a further series of experiments using phenoxybenzamine-treated cats, the residual vasoconstrictor response to 16 Hz nerve stimulation was determined before and after repeated high doses of a a,/3-methylene ATP (desensitisation of Pzx-purinoceptors). Altogether seven doses of a,/3-methylene ATP (50, 100, 100, 200, 200, 400 and 450/~g i.a.) were administered with less than 1 min between injections giving a total cumulative dose of 1.5 mg (equivalent to about 0.5 m g / k g ) . This procedure was adopted to build up the dose and desensitisation slowly and avoid the toxic effects (cardiac and respiratory arrest) of single high doses ( > 200 ~tg) of c~,/3-methylene ATP. The inhibitory effect of c~,/3-methylene A T P On 16 Hz nerve stimulation was determined at 1, 10, 20, 30 and 50 min after completion of the desensitisation procedure. The inhibitory effects of high doses of c~,/3-methylene ATP were also determined on the vasoconstrictor responses to 1 and 8 Hz stimulation of the sympathetic nerve plexus, 1 /~g i.a. noradrenaline and 50 #g i.a. methoxamine in cats not treated with c~-adrenoceptor antagonists. The inhibitory effects of a,/3-methylene ATP were measured at 1, 5, 10, 15, 30 and 45 min after completion of the desensitisation procedure.

2.4. Drugs used The following drugs were used: c~,/3-methylene ATP (Sigma U.K.), atropine sulphate (BDH), noradrenaline HC1 (Aldrich Chem. Co.), methoxamine HC1 (Burroughs Wellcome), phenoxybenzamine (Smith Kline and French), prazosin HC1 (Pfizer), propranolol HC1 (ICI) and yohimbine HC1 (Simes). Phenoxybenzamine was dissolved in hot polyethylene glycol '400' and diluted with an equal volume of 0.9% saline. All other drugs were dissolved in either distilled water or 0.9% saline. Dilutions were made in 0.9% saline.

2.5. Statistical analysis Results are expressed as mean values _+ S.E.M. with the number of determinations n given in parentheses. Statistical significance was tested

using Student's paired or unpaired t-test as appropriate. A probability level of P < 0.05 was considered statistically significant.

3. Results

3.1. Sympathetic neroe stimulation Intestinal perfusion pressure was relatively low in all experiments (mean value = 71.6 _+ 3.0 m m Hg, n = 30) because the efferent sympathetic nerves were ligated. Stimulation of the sympathetic nerves, distal to the ligation, caused pronounced and frequency-dependent increases in perfusion pressure (vasoconstriction) which reached peak values after 45-60 s of stimulation (fig. la,b). Perfusion pressure returned rapidly to resting levels after switching off the stimulation (fig. la). A stimulation frequency of 0.25 Hz caused a threshold vasoconstrictor response (an increase of 3-6 m m Hg in perfusion pressure) while 16 Hz stimulation elicited a maximal increase in perfusion pressure averaging 178.7 +_ 5.4 m m Hg (n = 20) which represents a 150% increase from mean resting levels (figs. l b and 2). Increasing the stimulation frequency to 32 Hz did not produce a greater vasoconstrictor response. The responses to sympathetic nerve stimulation were reproducible over a period of several hours and repeat frequency-response curves were not significantly different from the first curve (fig. lb). Each frequency-response curve took about 1 h to complete.

3.2. Effects of c~-adrenoceptor antagonists Prazosin, 0.5 m g / k g i.a., had no significant effect on intestinal perfusion pressure (73.0 _+ 4.2 m m Hg before prazosin compared with 80.1 _+ 4.6 m m Hg after prazosin, n = 7) but did cause a fall in mean systemic blood pressure from 94.7 _+ 7.9 to 60.0 +_ 3.4 m m Hg (P < 0.001, n = 7). Prazosin had no effect on the vasoconstriction caused by low frequency (0.25-1 Hz) nerve stimulation but did reduce the responses to higher frequency (2-16 Hz) nerve stimulation (fig. 2). The time course of the vasoconstriction and the rate of recovery on

27 cessation of s t i m u l a t i o n were u n c h a n g e d after p r a z o s i n (fig. 3). This dose of p r a z o s i n was relatively ineffective against v a s o c o n s t r i c t i o n elicited

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by i.a. injections of n o r a d r e n a l i n e (fig. 4) whereas the responses to the a]-selective a d r e n o c e p t o r agonist, m e t h o x a m i n e (also given i.a.) were inhibited (fig. 4). Y o h i m b i n e , 1.5 m g / k g i.a., caused a small b u t statistically significant increase in intestinal perfusion pressure f r o m 73.7 + 7.8 to 91.7 _+ 11.3 m m H g (P < 0.05, n = 6) with a c o n c o m i t a n t decrease in m e a n systemic b l o o d pressure from 78.8 _+ 6.2 to 4 8 . 3 + 2 . 4 m m H g ( P < 0 . 0 1 , n = 6 ) . U n l i k e prazosin, y o h i m b i n e r e d u c e d the v a s o c o n s t r i c t o r effects of nerve s t i m u l a t i o n at all frequencies studied (fig. 2) a l t h o u g h the i n h i b i t i o n was m o r e p r o n o u n c e d at the higher frequencies. In some e x p e r i m e n t s y o h i m b i n e a p p e a r e d to slow b o t h the rate of onset of v a s o c o n s t r i c t i o n a n d the rate of recovery after switching off the s t i m u l a t i o n (fig. 3). N o a t t e m p t was m a d e to q u a n t i f y the effects of y o h i m b i n e on the time course of neurogenic vasoconstriction. Y o h i m b i n e given alone or after p r a z o s i n c a u s e d a m a r k e d i n h i b i t i o n of n o r a d r e n a line v a s o c o n s t r i c t o r responses (fig. 4). Even after i n h i b i t i o n of b o t h a]- a n d c~2-adreno c e p t o r s with either p r a z o s i n 0.5 m g / k g i.a a n d y o h i m b i n e 1.5 m g / k g i.a. (n = 4) or a high dose of p h e n o x y b e n z a m i n e , 10 m g / k g i.a. (n = 3), a residual f r e q u e n c y - r e l a t e d v a s o c o n s t r i c t o r response was seen on s t i m u l a t i o n of the s y m p a t h e t i c nerves (fig. 2) which at 16 H z resulted in an increase in

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Fig. 1. Anaesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal circulation. (a) Original tracing showing increases in intestinal perfusion pressure (PP, lower record) elicited by transmural stimulation (30 V, 0.5 ms pulse duration) of the sympathetic nerve plexus around the anterior mesenteric artery at different frequencies (0.5-16 Hz). Stimulation was continued for 45-60 s until a peak vasoconstrictor response was obtained. On switching off the stimulation perfusion pressure returned rapidly to resting levels. The associated increases in systemic blood pressure (BP, upper record) were probably caused by increases in venous return resulting from splanchnic venoconstriction. (b) Three sequential frequency-response (F/R) curves to sympathetic nerve stimulation (0.25-16 Hz) with no intervening drug treatment to demonstrate the reproducibility of the responses. (e) 1st F / R curve, ( 0 ) 2nd F / R curve, (©) 3rd F / R curve. Each curve took about 1 h to complete. There was no significant difference between the F / R curves. Points shown are mean values+ S.E.M. (n = 6).

28 T

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ing recovery from the effects of high doses of a,fl-methylene ATP (see next section, fig. 3).

3.3. Effects of a, fl-methylene A TP The residual vasoconstrictor response to 16 Hz nerve stimulation in phenoxybenzamine-treated cats was compared before and after repeated high doses of c~,fl-methylene ATP (desensitisation of Pzx-purinoceptors). The first dose (50 /~g i.a.) of

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Frequency (Hz) Fig. 2. Anesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal circulation. Increases in intestinal perfusion pressure (PP) elicited by stimulation of the sympathetic nerves at different frequencies (0.25-16 Hz) before (©, n = 13) and 10-15 min after prazosin 0.5 m g / k g i.a. ( . , n = 7), yohimbine 1.5 m g / k g i.a. (e, n = 6) or complete a-adrenoceptor blockade with either prazosin+ yohimbine (n = 4) or phenoxybenzamine 10 m g / k g i.a. (n = 3) (@, n = 7). Points shown are mean values_+ S.E.M. * Significantly different (P < 0.05 or better) when compared to control responses. * * Significantly different (P < 0.05 or better) when compared to responses after prazosin or yohimbine alone.

perfusion pressure of around 50 m m Hg. There was no difference between the effects of prazosin and yohimbine or phenoxybenzamine so the results were combined (n = 7) to give the single curve shown in fig. 2. In some tracings the vasoconstrictor response appeared to be biphasic consisting of an initial, rapid but probably not well sustained increase in perfusion pressure followed by a secondary, slower onset but more sustained increase in perfusion pressure. It was not possible, however, to accurately define and measure the two phases of the vasoconstrictor response except dur-

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Fig. 3. Anaesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal vasculature. Upper record: original tracing showing the increases in intestinal perfusion pressure (PP) elicited by 8 Hz stimulation of the sympathetic nerves (S) for 60 s before and 10-15 min after prazosin 0.5 m g / k g i.a. (Pr), yohimbine 1.5 m g / k g i.a. (Yo) and prazosin_+yohimbine ( P r + Y o ) . The recording showing the effects of yohimbine alone was taken from a separate experiment in which the control response to 8 Hz stimulation of the sympathetic nerves closely matched (both in shape and peak response) the response shown in the figure. Lower record: original tracing showing the increases in intestinal perfusion pressure (PP) elicited by 16 Hz stimulation of the sympathetic nerves (S) in a phenoxybenzamine (10 m g / k g i.a.)-treated cat before and after desensitisation of P2x-purinoceptors with a high dose of c~,fl-methylene ATP (AMPCPP). Doses given (O) in rapid succession were 50, 100, 100, 200, 200, 400 and 450 ~g i.a. (total 1.5 mg). Sympathetic nerve stimulation was applied at 1, 6, 15 and 30 rain after desensitisation. * Recording run at twice normal speed to show the biphasic nature of the vasoconstrictor response to sympathetic nerve stimulation.

29

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sensitisation. Thereafter the response to nerve stimulation recovered slowly reaching pre-dose levels within 40-50 min. At this time there was also a return of the vasoconstrictor response to lower doses of a,fl-methylene ATP. The inhibitory actions of a,fl-methylene ATP were also evaluated in cats which were not treated

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pg i.a. methoxamine Fig. 4. Anaesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal circulation. Upper record: original tracing showing the increases the intestinal perfusion (PP) elicited by noradrenaline (0.02-20 /~g i.a.) before and 10-15 min after prazosin 0.5 m g / k g i.a. and prazosin+yohimbine (1.5 m g / k g i.a.). Lower record: original tracing showing the increases in intestinal perfusion pressure (PP) elicited by methoxamine (10-200/~g i.a.) before and after prazosin 0.5 m g / k g i.a. (continuous tracing).

~,fl-methylene A T P caused a substantial increase in perfusion pressure (fig. 3), whereas the final dose (450 #g i.a.) caused little change in perfusion pressure (fig. 3) indicating adequate desensitisation of the P2x-purinoceptors. By the conclusion of the desensitisation period there was a significant fall in mean systemic blood pressure from 69.0 + 2.6 to 50.3 + 1.7 m m Hg (P < 0.01, n = 6) and intestinal perfusion pressure from 72.5 + 6.9 to 50.0 + 2.4 m m Hg (P < 0.01, n = 6). The rapid increase in perfusion pressure caused by 16 Hz nerve stimulation as completely abolished by c~,flmethylene A T P (figs. 3, 5) but a small, slower onset, more sustained vasoconstriction persisted (fig. 3). The mean increase in perfusion pressure was 13.0 + 4.3 m m Hg (n = 6) with a range of 3 to 30 m m Hg indicating considerable between-animal variation. The maximal inhibitory effect of ~,flmethylene A T P was seen immediately after de-

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Fig. 5. Anaesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal circulation. Histogram showing the increases in intestinal perfusion pressure (PP) elicited by 16 Hz stimulation of the sympathetic nerves before (C, n =13) and 10-15 min after prazosin 0.5 m g / k g i.a. (Pr, n = 7), yohimbine 1.5 m g / k g i.a. (Yo, n = 6) or blockade of ~1o and ~2-adrenoceptors with either prazosin+ yohimbine ( P r + Y o , n = 4) or phenoxybenzamine 10 m g / k g i.a. (Ph, n = 4), in total (n = 7). The final column represents the effect of desensitisation of P2x-purinoceptors with repeated high doses (total dose 1.5 mg i.a.) of ct,fl-methylene ATP (AMPCPP) on the residual vasoconstrictor response to sympathetic nerve stimulation in phenoxybenzamine-treated cats (n = 6) ~,fl-methylene ATP abolishes the initial rapid phase (R) of the residual vasoconstrictor response but a relatively small, slower onset (S) and more sustained vasoconstriction persists. It was not always possible to accurately separate the two phases prior to ~,fl-methylene ATP treatment and so only the overall peak response is represented. Values shown are mean increases+S.E.M. * Significantly different from control response (P < 0.01). ** Significantly different from the response after prazosin or yohimbine alone (P < 0.01). * * Significantly different from the response after prazosin+ yohimbine or phenoxybenzamine (P < 0.01).

30

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c o n s t r i c t o r response to n o r a d r e n a l i n e a n d a 60.7 ___6.4% (P < 0.001) r e d u c t i o n in the v a s o c o n s t r i c tion elicited b y m e t h o x a m i n e . T h e i n h i b i t o r y effects of c~,B-methylene A T P were transient with recovery of the responses within 10-15 m i n (fig. 6). a , B - M e t h y l e n e A T P also c a u s e d a significant decrease in m e a n systemic b l o o d pressure f r o m 80.9 ___2.2 to 56.4 + 2.3 m m H g (P < 0.001, n = 8) a n d intestinal p e r f u s i o n p r e s s u r e from 72.0 + 3.0 to 55.6 + 2.3 m m H g (P < 0.001, n = 8).

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Fig. 6. Anaesthetised cat pretreated with atropine and propranolol; constant flow perfusion of the intestinal circulation. Effect of a,fl-methylene ATP (total dose 1.5 mg i.a.) on the increases in intestinal perfusion pressure (vasoconstriction) elicited by 1 Hz ( . , n = 5) and 8 Hz (~, n = 5) stimulation of the sympathetic nerves, noradrenaline 1 btg i.a. (O, n = 4) and methoxamine 50 /~g i.a. (o, n = 4). Results are presented as percentage of control responses at given times after a,/3-methylene ATP. Points shown are mean values+S.E.M. * Significant (P < 0.05 or better) change from control values.

with c~-adrenoceptor antagonists. S t i m u l a t i o n of the s y m p a t h e t i c nerve plexus at frequencies of 1 a n d 8 H z caused increases in intestinal p e r f u s i o n pressure of 25.6 + 2.2 a n d 153.2 + 10.2 m m H g respectively, from a m e a n resting value of 65.0 + 3.9 m m H g (n = 5). I m m e d i a t e l y after the desensiting doses of c~,fl-methylene A T P (total dose = 1.5 mg i.a.) therc was a 7 4 _ 2.4% (P < 0.001) decrease in the v a s o c o n s t r i c t o r response elicited b y 1 H z nerve s t i m u l a t i o n b u t only a 31.0 + 2.0% (P < 0.001) r e d u c t i o n of the response to 8 H z nerve stimulation. T h e i n h i b i t o r y effects of a, flm e t h y l e n e A T P were relatively short-lived lasting less than 30 min (fig. 6). I n t r a a r t e r i a l injections of n o r a d r e n a l i n e (1 /~g) a n d m e t h o x a m i n e (50 /zg) caused increases in p e r f u s i o n pressure of 56.7 + 2.8 a n d 59.7 + 1.8 m m H g respectively from baseline values of 71.2 + 6.6 a n d 96.2_+ 14.1 m m H g respectively (n = 4). I m m e d i a t e l y after the desensitising dose of c~,fl-methylene A T P there was a 54.7 + 4 . 2 % (P < 0.001) decrease in the vaso-

T r a n s m u r a l s t i m u l a t i o n of the s y m p a t h e t i c nerve plexus a r o u n d the a n t e r i o r m e s e n t e r i c a r t e r y elicited f r e q u e n c y - d e p e n d e n t increases in perfusion pressure (vasoconstriction) which were highly r e p r o d u c i b l e over a 3-4 h p e r i o d thus allowing the testing of a n t a g o n i s t s after the e s t a b l i s h m e n t of c o n t r o l responses in each e x p e r i m e n t . E a c h stimulation p e r i o d was c o n t i n u e d for j u s t long e n o u g h ( a p p r o x i m a t e l y 45-60 s) to allow a p e a k r e s p o n s e to be o b t a i n e d a n d sequential r a t h e r t h a n c u m u l a tive f r e q u e n c y - r e s p o n s e curves were c o n s t r u c t e d to m i n i m i s e the effects of p r e s y n a p t i c i n h i b i t i o n a n d to avoid fade or ' e s c a p e ' of the v a s o c o n s t r i c t o r response caused b y o t h e r factors (Ross, 1971; G r e e n w a y et al., 1976). In s o m e e x p e r i m e n t s the a2-selective a d r e n o c e p t o r a n t a g o n i s t , y o h i m b i n e (Weitzell et al., 1979), caused a slowing in the rate of recovery from v a s o c o n s t r i c t i o n after switching off the stimulation. This m a y b e c a u s e d b y an increase in n o r a d r e n a l i n e efflux f r o m the symp a t h e t i c nerve endings as a result of i n h i b i t i o n of p r e s y n a p t i c a 2 - a d r e n o c e p t o r s . It follows, therefore, that the r e d u c t i o n in n e u r o g e n i c vasoconstriction o b s e r v e d after y o h i m b i n e m i g h t b e a slight u n d e r e s t i m a t e as the c o m p o u n d will also c o u n t e r a c t the effects of p r e s y n a p t i c inhibition. The results o b t a i n e d with the a l - s e l e c t i v e a d r e n o c e p t o r antagonist, p r a z o s i n ( C a m b r i d g e et al., 1977), a n d y o h i m b i n e , given s e p a r a t e l y o r together, suggest that p o s t s y n a p t i c a 1- a n d a 2a d r e n o c e p t o r s are involved in m e d i a t i n g vasoc o n s t r i c t o r responses to s y m p a t h e t i c nerve stimulation in resistance vessels of the cat intestinal vasculature. T h e failure of p r a z o s i n to i n h i b i t low

31 frequency nerve stimulation is surprising as a 1adrenoceptors are claimed to be predominant in neurotransmission (Langer, 1974; Langer et al., 1981; Docherty and McGrath, 1980). Clearly mediation via a2-adrenoceptors may be particularly important for low frequency nerve stimulation and low doses of injected noradrenaline. It follows that a2-adrenoceptors are not only extrajunctional, as suggested by Docherty and McGrath (1980) and Langer et al. (1981), but also intrajunctional and involved in vasomotor control. A similar conclusion has been reached by a number of investigators including Drew and Whiting (1979), Patel et al. (1981), Gardiner and Peters (1982), Alabaster and Davey (1984), Docherty and Hyland (1985) and Bulloch et al. (1987). It is clear from the preceding discussion that the failure of prazosin in certain tissues to block the effects of sympathetic nerve stimulation may be due to a significant a2-adrenoceptor-mediated component of the response. However, in a number of in vitro preparations non-selective a-adrenoceptor antagonists such as phentolamine or phenoxybenzamine still failed to inhibit the responses to sympathetic nerve stimulation (Hirst and Neild, 1980; Holman and Surprenant, 1980; Sneddon and Burnstock, 1984a,b; Kennedy et al., 1986; Burnstock and Warland, 1987a,b). The non-adrenergic component of neurotransmission was assumed to be mediated either by noradrenaline acting on putative y-receptors (Hirst and Neild, 1980), or by the release of other substances, including purines and peptides, which act as cotransmitters with noradrenaline (Burnstock, 1986). In vivo, the situation is less clear. Bulloch and McGrath (1988) found that high doses of a,fl-methylene ATP reduced the pressor responses to sympathetic nerve stimulation in the pithed rat suggesting a purine involvement via stimulation of postsynaptic P2x-purinoceptors, while Hicks et al. (1985) demonstrated an increased purine component of sympathetic neurotransmission in the tail artery of hypertensive rats compared with normotensive controls. In contrast, Bell (1985) was able to completely inhibit neurogenic vasoconstriction in the dog hindlimb with phentolamine. In the present series of experiments a residual frequency-related vasoconstrictor response to

sympathetic nerve stimulation remained even after a 1- and az-adrenoceptor blockade with either prazosin and yohimbine given together or a high dose of the irreversible antagonist phenoxybenzamine. The residual vasoconstrictor response appeared to be biphasic with an initial increase in perfusion pressure, which was not well sustained, followed by a variable, slowly developing increase in perfusion pressure. Since stimulation of P2xpurinoceptors has been shown to elicit rather transient effects on cell membranes (Sneddon and Burnstock, 1984b; Kennedy et al., 1986; Parsons and Taylor, 1988), it was suspected that the initial response to sympathetic nerve stimulation might be mediated by the release of ATP or a related purine and as such should be sensitive to blockade by high doses of a, fl-methylene ATP (Sneddon and Burnstock, 1984a,b). However, it proved to be more difficult to achieve adequate desensitisation of P2×-purinoceptors under in vivo conditions. The dose of a,fl-methylene ATP had to be built up progressively, by giving repeated intraarterial injections, because single high doses, above 200/~g, were toxic causing respiratory and cardiac arrest. In addition, desensitisation, once achieved, was relatively short-lasting compared with the prolonged effects observed in vitro, a finding recently confirmed by Bulloch and McGrath (1988). Intraarterial infusions of a,fl-methylene ATP were avoided because of the possibility of sustained increases in the release of E D R F and the resultant indirect effects on vasoconstricting agents. Because of these problems, the investigation was restricted to studying the effects of a,fi-methylene ATP on the residual vasoconstrictor response to 16 Hz nerve stimulation. The initial phasic response to nerve stimulation was completely abolished by a,/3-methylene ATP whereas the slower onset vasoconstriction was still present although reduced, when compared with response observed 30 min after desensitisation. These results suggest that the initial, rapid increase in vascular resistance following nerve stimulation in phenoxybenzamine-treated cats is the result of stimulation of postsynaptic Pzx-purinoceptors b y neuronally released ATP or a closely related purine. Further experiments will be needed to determine if the a,fi-methylene ATP-resistant in-

32

crease in vascular resistance is caused by high local concentrations of noradrenaline overpowering the a-adrenoceptor blockade or an increase in other transmitter substances (e.g. peptides). What is not clear from the experiments in phenoxybenzamine-treated cats is the relative importance of purines in neurotransmission and sympathetic nervous control of vascular resistance under normal conditions. To address this question the inhibitory actions of a,/3-methylene ATP were also evaluated in cats which were not treated with a-adrenoceptor antagonists. Desensitising doses of a,/3-methylene ATP caused a 74% reduction in the vasoconstrictor response to 1 Hz nerve stimulation but only a 30% reduction of the response to 8 Hz nerve stimulation suggesting a greater purinergic involvement in neurotransmission at low frequency stimulation. However, the vasoconstrictor responses to the al-selective adrenoceptor agonist, methoxamine, and to noradrenaline, which acts largely by stimulation of az-adrenoceptors in this preparation, were reduced by about 60% following treatment with a,/3-methylene ATP. Although a,/3-methylene ATP appears to have a relatively more pronounced and prolonged effect on low frequency nerve stimulation in the absence of a-adrenoceptor antagonists and on the initial response to nerve stimulation in phenoxybenzaminetreated cats in comparison to the inhibition of methoxamine and noradrenaline, the results must be interpreted with caution because of the clear non-specific effects of the drug on vascular smooth muscle which may also be partly responsible for the hypotension and fall in intestinal perfusion pressure. Further proof will be required before more concrete conclusions can be drawn regarding the importance of purines in neurotransmission in vivo, particularly considering the inadequacies of using a,/3-methylene ATP as a P2×-purinoceptor antagonist. Obviously, the discovery of potent, selective and preferably competitive P2×-purinoceptor antagonists will greatly advance our understanding of the actions of purines mediated by P2-purinoceptors and their importance in normal physiology and in pathological states. In conclusion, neurotransmission at sympathetic nerve endings in resistance vessels of the cat intestinal circulation is largely under adren-

ergic control. The effects of neuronally released noradrenaline are mediated by postsynaptic a aand a2-adrenoceptors, the latter being particularly involved in mediating the vasoconstrictor effects of low frequency firing of the sympathetic nerves and injected noradrenaline. A purinergic component of neurotransmission also appears to exist but its effects are modest and transient in comparison with the adrenergic component. However, as cotransmitters noradrenaline and ATP may have synergistic effects which might be important under certain conditions, for example, in the splanchnic response to stress (fight or flight response) or rapid changes in posture. Other neurotransmitters (e.g. peptides) may also be involved in mediating neurogenic vasoconstriction.

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Langer, S.Z., 1974, Presynaptic regulation of catecholamine release, Biochem. Pharmacol. 23, 1793. Langer, S.Z. and P.E. Hicks, 1984, Alpha-adrenoceptor subtypes in blood vessels: physiology and pharmacology, J. Cardiovasc. Pharmacol. 6, 5547. Langer, S.Z., N.B. Shepperson and R. Massingham, 1981, Preferential noradrenergic innervation of alpha-adrenergic receptors in vascular smooth muscle, Hypertension, 3 (Suppl. 1), 112. MacKenzie, I. and G. Burnstock, 1984, Neuropeptide action on the guinea-pig bladder: a comparison with the effects of field stimulation and ATP, European J. Pharmacol. 105, 85. Mathieson; J.J.I. and G. Burnstock. 1985, Purine-mediated relaxation and contraction of isolated rabbit mesenteric artery are not endothelium dependent, European J. Pharmacol. 118, 221. McGrath, J.C., N.A. Flavahan and C.E. McKean, 1982, a tand a2-adrenoceptor-mediated pressor and chronotropic effects in the rat and rabbit, J. Cardiovasc. Pharmacol. 4, 5101. Muramatsu, I., 1987, The effect of reserpine on sympathetic, purinergic neurotransmission in the isolated mesenteric artery of the dog: a pharmacological study, Br. J. Pharmacol. 91,467. Parson, M.E. and E.M. Taylor, 1988, Effect of a, /3 methylene ATP on pre-capillary resistance vessels in vivo, Br. J. Pharmacol. 93, 251P. Patel, P., D. Bose and C. Greenway, 1981, Effects of prazosin and phenoxybenzamine on c~- and B-receptor mediated responses in intestinal resistance and capacitance vessels, J. Cardiovasc. Pharmacol. 3, 1050. Pearson, J.D., L.L. Slakey and J.L. Gordon, 1983, Stimulation of prostaglandin production through purinoceptors in cultured porcine endothelial cells, Biochem, J. 214, 273. Ross, G., 1971, Escape of mesenteric vessels from adrenergic and nonadrenergic vasoconstriction, Am. J. Physiol. 221, 1217. Sneddon, P. and G. Burnstock, 1984a, Inhibition of excitatory junction potentials in guinea-pig vas deferens by a,B-methylene ATP: further evidence for ATP and noradrenaline as cotransmitters, European J. Pharmacol. 100, 85. Sneddon, P. and G. Burnstock, 1984b, ATP as a neurotransmitter in rat tail artery, European J. Pharmacol. 106, 149. Steen, S., J. Castenfors, T. Sjoberg, T. Skarby, K. Andersson and L. Norgren, 1986, Effects of a-adrenoceptor subtypeselective antagonists on the h u m a n saphenous vein in vivo, Acta Physiol Scand. 126, 15. Su, C., 1983, Purinergic neurotransmission and neuromodulation, Ann. Rev. Pharmacol. Toxicol. 23, 397. Von Kiigelgen, I. and K. Starke, 1985, Noradrenaline and adenosine triphosphate as co-transmitters of neurogenic vasoconstriction in rabbit mesenteric artery, J. Physiol. 367, 435. Weitzell, R., T. Tanaka and K. Starke, 1979, Pre- and postsynaptic effects of yohimbine stereoisomers on noradrenergic transmission in the pulmonary artery of the rabbit, Naunyn-Schmiedeb. Arch. Pharmacol. 308, 127.