vasculature of the intact conscious rat

Transcription

1 3396 Journal of Physiology (1995), 485.3, pp Local modulation of adrenergic responses in the hindlimb vasculature of the intact conscious rat Stephen E. DiCarlo, Rahul D. Patil, Heidi L. Collins and Chao-Yin Chen Department of Physiology, Northeastern Ohio Universities, Rootstown, OH 44272, USA College of Medicine, 1. Local modulation of adrenergic responses was examined in the hindlimb vasculature of chronically instrumented intact conscious rats. Sprague-Dawley rats (n = 22) were instrumented with a Doppler flow probe around the right common iliac artery, a polyethylene catheter inserted just distal to the flow probe and a left carotid arterial catheter. 2. The effects of various concentrations of the a1-adrenergic receptor agonist phenylephrine ( jug kg-), the a2-adrenergic receptor agonist clonidine ( jug kg-'), and the endogenous adrenergic receptor agonist adrenaline ( jug kg-'), were investigated under control conditions, and in the presence of the nitric oxide (NO) synthase inhibitor N'-nitro-L-arginine methyl ester hydrochoride (L-NAME) (NO-X, 0-2 mg kg-) and the cyclo-oxygenase inhibitor indomethacin (CO-X, 10 mg kg-). Results were presented as dose-response curves. 3. Heart rate and arterial pressure were not altered by any of the agents because all were locally injected into the hindlimb vasculature and the selected doses were lower than those which elicited systemic responses. 4. Maximal vasoconstrictor responses to phenylephrine were enhanced in the presence of NO-X (50 + 6%) and CO-X (70 + 9%). Maximal vasoconstrictor responses to clonidine were also enhanced in the presence of NO-X ( %) and CO-X ( %). 5. The responses to adrenaline were biphasic; NO-X significantly attenuated the vasodilator response (87 + 6%), and enhanced the vasoconstrictor response (51 + 7%). CO-X attenuated the vasoconstrictor response (71 + 6%). 6. These results demonstrate local modulation of responses to al- and,b-adrenergic receptor agonists by receptor-mediated dose-dependent release of NO and prostaglandins. Endothelial cells play an important role in the local regulation of vascular smooth muscle tone by producing and releasing relaxing and contracting factors (Furchgott & Vanhoutte, 1989). These endothelium-derived substances are released tonically and in response to agonists (Katusic & Shepherd, 1991; Shepherd & Katusic, 1991). Endothelial modulation of adrenergic responses has been studied in the conduit vessels of rats (Carrier &; White, 1985; Topuzis, Schott & Stoclet, 1991), dogs (Young & Vatner, 1986), rabbits (Verrecchia, Sercombe & Seylaz, 1984) and pigs (Cocks & Angus, 1983). However, few studies have examined the role of the endothelium in modulating adrenergic responses in skeletal muscle resistance vessels (Nakamura & Prewitt, 1991; Ohyanagi, Nishigaki & Faber, 1992). Conduit vessels may differ in response from small arteries and arterioles (resistance vessels) and muscular venules, which are the main determinants of resistance and venous return in vascular regulation (Ohyanagi et al. 1992). The response of the resistance vessels may also vary between tissues (Nakamura & Prewitt, 1991; Ohyanagi et al. 1992). For example, vasoconstrictor responses to the az-adrenergic receptor agonist phenylephrine were enhanced after nitric oxide (NO) synthase inhibition in the rat cremaster muscle (Ohyanagi et al. 1992), but not in the arcade arterioles of the rat spinotrapezius muscles (Nakamura & Prewitt, 1991). Previous studies have examined the role of NO in modulating adrenergic responses in the skeletal muscle microvasculature (Nakamura & Prewitt, 1991; Ohyanagi et al. 1992) of acute anaesthetized denervated preparations, in which the drugs were given abluminally. Other investigators have examined the role of NO in modulating adrenergic responses in conscious animals with intact cardiovascular reflexes in which the drugs were given systemically (Gardiner, Kemp & Bennett, 1991 a, b). In such a situation, the direct effects of pharmacological agents on

2 818 S. E. DiCarlo and others J. Phy8iol heart rate, cardiac contractility, cardiac output, blood pressure and baroreceptor reflex-mediated compensatory mechanisms cannot be distinguished from the direct vascular effects. It is not known whether prostaglandins (PGs) and endothelium-derived NO modulate the response to adrenergic agonists independently of systemic responses, in the hindlimb vasculature in intact conscious animals. It has only recently (Patil, DiCarlo & Collins, 1993) become possible to effectively isolate hindlimb vascular responses from the systemic circulation and from baroreceptor reflexmediated compensatory mechanisms by close-arterial administration of small doses of vasoactive substances into the hindlimb. Therefore, the purpose of the present study was to determine the effects of NO synthase and cyclooxygenase inhibition on the responses to the a1-adrenergic receptor agonist phenylephrine, the a2-adrenergic receptor agonist clonidine and the endogenous adrenergic receptor agonist adrenaline in the hindlimb vasculature of chronically instrumented intact conscious rats (Patil et at. 1993). METHODS Experimental design Experiments were conducted in twenty-two Sprague-Dawley rats (13 males and 9 females; weight g, mean + S.E.M.). Common iliac blood flow velocity (IFV), heart rate (HR), pulsatile arterial blood pressure, and mean arterial blood pressure (MABP) were recorded under control conditions and during bolus injections of phenylephrine, clonidine and adrenaline into the hindlimb of conscious unrestrained rats (Patil et al. 1993). These experiments involved determination of vascular responses to the adrenergic agonists under three sets of experimental conditions: (1) under control conditions (control), (2) after NO synthase inhibition with N'-nitro-L-arginine methyl ester hydrochoride (L-NAME) (NO-X) and (3) after cyclo-oxygenase inhibition with indomethacin (CO-X). Instrumentation In the present study on intact conscious rats we investigated vascular responses evoked in an effectively isolated hindlimb, these responses were not affected by responses occurring in the general circulation. A cannula was implanted in the right common iliac artery so that close-arterial injections could be made into the hindlimb without altering pulsatile arterial pressure, pulse pressure, MABP or HR. For each substance a dose range was chosen that produced a local response, but no obvious systemic cardiovascular response (Patil et al. 1993). All instrumentation was performed using sterile surgical procedures. Anaesthesia was induced with sodium pentobarbitone (40 mg kg-' i.e.) and supplemental doses (5-10 mg kg-1 ip.) were administered as needed. Therats were instrumented with an epoxyresin cuff-type pulsed Doppler ultrasonic flow probe (Baylor College of Medicine, Houston, TX, USA) positioned around the right common iliac artery. A small opening was made in the right common iliac artery with a 25 gauge needle approximately 5 mm distal to the flow probe, and a fine polyethylene catheter (PE-10; Clay Adams, NJ, USA) was inserted to a minimal depth and held in place by a droplet of cyanoacrylate ester (Superglue, Duro, OH, USA). At the end of surgery the vessel was inspected for evidence of vasospasm or distortion of arterial configuration. The blood flow velocity was not altered by insertion of the catheter. In addition, a Teflon catheter was inserted into the descending aorta via the left common carotid artery for measurements of arterial pressure, MABP, and HR. Every day, catheters were flushed with physiological saline, filled with heparin (1000 units ml-'), and plugged with a paraffin-filled obturator. Animals were allowed to recover for 7 days before experimentation, monitored for signs of infection and weighed daily. At the time of the experiment, all animals had recovered, were healthy and gaining weight. Arterial pressure was determined by connecting the carotid arterial catheter to a pressure transducer (Gould P23XL) coupled to a recorder (Gould RS3600). Mean arterial blood pressure was derived electronically using a low-pass filter. Heart rate was determined using a Gould ECG/Biotach model system triggered from the arterial pressure pulse. The pulsed Doppler flow probe was connected to a multichannel ultrasonic flow dimension system (constructed by the instrumentation development laboratories, Baylor College of Medicine), with 20 MHz high velocity modules. Blood flow velocity was measured in kilohertz Doppler shift, which is directly proportional to the absolute blood flow as determined with an electromagnetic system (Haywood, Shaffer, Fastenow, Fink & Brody, 1981). Phenylephrine, clonidine, adrenaline, indomethacin and L-NAME were administered as ,u bolus injections via the catheter placed in the common iliac artery. The dose-response curves for phenylephrine, clonidine and adrenaline were obtained by giving four or five bolus injections at 5-8 min intervals in random order until the entire curve was obtained. Physiological saline was used as the vehicle for agents and to flush the catheter. Indomethacin solution was buffered using sodium bicarbonate. Saline injections did not alter the measured variables indicating no vehicle or volume effect. Experimental protocol On the day of the experiment the rats were placed unrestrained in a large Plexiglass box. The animals were allowed to adapt to the laboratory environment for 1 h to attain resting haemodynamic variables. After the adaptation period, a phenylephrine dose-response curve was generated. Five doses ( jug kg-) of phenylephrine (in random order) were injected into the hindlimb. At least 5-8 min were allowed between doses, to allow haemodynamic parameters to return to baseline levels. The mean peak changes in IFV in response to bolus injections of phenylephrine were measured. The animal was allowed to recover for 1 h following drug administration. Subsequently, L-NAME ( mg kg-') was injected into the hindlimb at a dose selected to cause a decrease in IFV, without altering the systemic haemodynamic parameters. After 15 min, the procedure to obtain a dose-response curve for phenylephrine was repeated. At the end of the experiment, the vasodilator response to 0-1 ng kg-' acetylcholine was evaluated to check the effectiveness of NO synthase inhibition and the animals were returned to their cages. The entire data collection took approximately 4 h. On day 2 (after 48 h) the experiment was repeated, this time generating a dose-response curve for clonidine ( jug kg-1), rather than phenylephrine, before and after administration of L-NAME. Similarly, after a further 48 h, a

3 J Physiol Local modulation of adrenergic responses 819 dose-response curve for adrenaline (0' ,ug kg-') was generated before and after administration of L-NAME. Dose-response curves for phenylephrine, clonidine and adrenaline were also generated before and after administration of indomethacin (10 mg kg'); the dose was selected to eliminate the vasodilator response to arachidonic acid (3 mg kg-'). A series of experiments was performed to control for the effects of time (time controls) and the non-specific effects of NO-X and CO-X. Thus, dose-response curves were repeated in some animals (n = 6) before and after saline infusion, to control for a timecontrol-induced altered reponse to the second administration. There were no effects of time on the response to phenylephrine, clonidine or adrenaline. In another three animals, the non-specific effects of L-NAME and indomethacin were evaluated by substituting different inhibitors, NG-monomethyl-L-arginine (L-NMMA, an arginine analogue) and meclofenamic acid (a cyclooxygenase inhibitor), respectively. No differences were observed. Finally, the pharmacological antagonists prazosin hydrochloride (20 ug kg-) and yohimbine hydrochloride (50 jug kg-') were used in two animals to ensure that the responses to phenylephrine and clonidine were due to activation of a,- and a2-adrenergic receptors, respectively. Chemicals Arachidonic acid, adrenaline, indomethacin, clonidine, meclofenamic acid, L-NAME, L-NMMA and acetylcholine chloride were obtained from Sigma. Phenylephrine hydrochloride was obtained from Winthrop-Breon Laboratories (New York, USA). Calculations and statistical analysis The dose-response curves were constructed from the mean peak percentage change in IFV from baseline levels evoked by each dose of adrenergic agonist. The individual points are the means + standard error of the mean peak responses recorded at the various concentrations. The curves were analysed using a twoway analysis of variance (ANOVA) with repeated measurements. A one-way ANOVA was used to determine differences in the resting haemodynamic variables immediately before each of the dose-response curves were generated (Table 1). When significant differences were obtained, post hoc analyses were performed using the Bonferroni correction for multiple comparisons (Wallenstein, Zucker & Fleiss, 1980). A level of P < 0 05 was considered significant. RESULTS Figure 1 shows an analogue recording of the response to adrenaline under control conditions and in the presence of NO-X. The response to adrenaline was biphasic with an initial vasoconstrictor response followed by a vasodilator response. NO-X significantly reduced resting IFV. In addition, NO-X significantly enhanced the vasoconstrictor responses and attenuated the vasodilator response to adrenaline. Note that all vascular responses were independent of changes in arterial pressure or heart rate Ei E 75- m O- E 500 w_ -- - _ - X N I Y'""" 1Jc [ [Adrenaline] (1ug kg-') Control NO-X (0-2 mg kg-1) Adrenaline/NO-X 008 Figure 1. Analogue recording of the response to adrenaline under the control condition and in the presence of NO-X The response to adrenaline was biphasic with an initial vasoconstrictor response followed by a vasodilator response. NO-X significantly reduced iliac blood flow (IFV). In addition, NO-X significantly enhanced the vasoconstrictor response and attenuated the vasodilator response. Note that all vascular responses were independent of changes in arterial blood pressure (ABP) or heart rate (HR).

4 820 S. E. DiCarlo and others J Phy8iol Table 1. Baseline haemodynamic variables of heart rate (HR), mean arterial blood pressure (MABP) and iliac blood flow velocity (IFV) before generation of nine dose-response curves Condition Phenylephrine Control NO-X CO-X Clonidine Control NO-X CO-X Adrenaline Control NO-X CO-X HR MABP IFV (beats min-) (mmhg) (khz) P ± 020t P t P t Values are given as means + S.E.M. under control conditions, and in the presence of NO-X and CO-X. NO-X and CO-X significantly reduced IFV (control vs. NO-X, P = 0-008; control vs. CO-X, tp = 0-01). HR and MABP were not significantly different. n Table 1 shows the baseline haemodynamic variables of HR, MABP and IFV before the dose-response curves for each agonist were generated. HR and MABP were not significantly different during generation of the nine dose-response curves. NO-X and CO-X significantly reduced IFV (25 + 2f4 (P = 0 008) and f3% (P = 0 01), respectively) without altering HR and MABP. At the end of the experiments, the vasodilator response to acetylcholine (0 1 ng kg-') was evaluated to check the effectiveness of NO-X. Acetylcholine increased IFV (by 3f1 + 0f2 khz). NO-X (0f2 mg kg-') attenuated the vasodilator response to acetylcholine by % (0f khz). At the end of the experiments, the vasodilator response to 3 mg kg-' arachidonic acid was evaluated to check the effectiveness of CO-X. Arachidonic acid increased IFV (by 1P khz). CO-X (10 mg kg-1) completely abolished the vasodilator response to arachidonic acid. Figure 2 shows the mean peak percentage changes in IFV during bolus injections of phenylephrine under control conditions, and in the presence of NO-X and CO-X. The vasoconstrictor responses to phenylephrine were significantly enhanced by both NO-X (P = 0 003) and CO-X (P = 0-001). There was also a significant treatmentby-dose interaction (see Discussion) between the curves: control vs. NO-X (P = 0-019) and control vs. CO-X (P = 0-001). Bonferroni post hoc analysis indicated that NO-X and CO-X significantly accentuated the vasoconstrictor responses to phenylephrine at all doses. The I--, Figure 2. Responses to the az-adrenergic receptor agonist phenylephrine Mean peak changes in IFV (AIFV) during bolus injections of phenylephrine under control conditions (0), and in the presence of NO-X (o) and CO-X (m). Both NO-X (P < 0 05) and CO-X (tp < 0 05) significantly enhanced the vasoconstrictor responses to phenylephrine compared with control. - vv Dose of phenylephrine (ug kg-1)

5 J Phy8iol Local modulation of adrenergic responses 821 0o Figure 3. Responses to the a2-adrenergic receptor agonist clonidine Mean peak changes in IFV (AIFV) during bolus injections of clonidine under control conditions (0), and in the presence of NO-X (ol) and CO-X (U). Both NO-X (P < 0 05) and CO-X (tp < 0 05; lowest dose only) enhanced the vasoconstrictor responses to clonidine compared with control U-! -60' -80' -0. vi Dose of clonidine (jug kg-1) 0-8 maximal vasoconstrictor response to phenylephrine was enhanced % after NO-X and % after CO-X. Figure 3 shows the mean peak percentage changes in IFV during bolus injections of clonidine under control conditions, and in the presence of NO-X and CO-X. The vasoconstrictor response to clonidine was significantly enhanced by both NO-X (P = 0 005) and CO-X (P = ). Bonferroni post hoc analysis revealed that the vasoconstrictor response to clonidine was enhanced at all doses of NO-X, but only at the lowest dose of CO-X. There was no significant treatment-by-dose interaction between the curves: control vs. CO-X (P = 0 92) and control vs. NO-X (P = 0 84). The maximal vasoconstrictor response to clonidine was enhanced % after NO-X and % after CO-X. The responses to adrenaline were biphasic with an initial vasoconstrictor response followed by a vasodilator response. The effects of CO-X and NO-X on the vasoconstrictor and vasodilator responses to adrenaline were analysed separately. Figure 4 shows the mean peak percentage decreases in IFV during bolus injections of adrenaline under control conditions, and in the presence of NO-X and CO-X. NO-X significantly enhanced (P = 0 005) the vasoconstrictor responses to adrenaline, while CO-X significantly attenuated (P = 0 004) the vasoconstrictor response to adrenaline. There was a significant treatmentby-dose interaction between the control vs. CO-X curves (P = 0 004), but there was no significant treatment-bydose interaction between the control vs. NO-X curves (P = 0 53). Bonferroni post hoc analysis revealed that NO-X enhanced the vasoconstrictor responses at all doses and CO-X attenuated the vasoconstrictor responses at all except the lowest dose. The maximal vasoconstrictor response was enhanced % after NO-X and was attenuated % after CO-X. 0 1t t -20 Figure 4. Vasoconstrictor responses to adrenaline Mean peak decreases in IFV (AIFV) during bolus injections of adrenaline under control conditions (0), and in the presence of NO-X (o) and CO-X (i). NO-X significantly (P < 005) enhanced, while CO-X significantly (tp < 005) attenuated, the vasoconstrictor responses to adrenaline compared with control. I--, 0- >L Dose of adrenaline (jug kg-1) 0-1-0

6 822 S. E. DiCarlo and others J Physiol "- al Figure 5. Vasodilator responses to adrenaline Mean peak increases in IFV (AIFV) during bolus injections of adrenaline under control conditions (A), and in the presence of NO-X (5) and CO-X (-). NO-X significantly (P < 005) attenuated the vasodilator responses to adrenaline, while CO-X did not significantly alter the response compared with control. v Dose of adrenaline (jig kg-') Figure 5 shows the mean peak percentage increases in IFV during bolus injections of adrenaline under control conditions, and in the presence of NO-X and CO-X. The vasodilator response to adrenaline was significantly attenuated by NO-X (P = 0-001), while CO-X had no significant effect (P = 0O29). There was a significant treatment-by-dose interaction between the control vs. NO-X curves (P = 0-000). Bonferroni post hoc analysis revealed that NO-X significantly attenuated the vasodilator responses to adrenaline at all doses. The maximum vasodilator response was attenuated % after NO-X. DISCUSSION The present study examined the modulating role of PG and endothelium-derived NO to adrenergic agonists in the hindlimb vasculature of intact conscious rats. The major findings from this study were as follows. (1) Vasoconstrictor responses to phenylephrine and clonidine were enhanced after CO-X and NO-X administration. (2) NO-X significantly attenuated the vasodilator responses and enhanced the vasoconstrictor responses to adrenaline, while CO-X significantly attenuated the vasoconstrictor responses to adrenaline. The experimental model Before discussing the results further, it is important to consider the experimental techniques employed. Blood flow velocity records responses in resistance vessels rather than in the large blood vessels. Therefore, in contrast to in vitro studies on sections of large blood vessels, our results concern the role of the local modulators, NO and cyclooxygenase products, in the responses evoked by adrenergic substances in resistance vessels. Blood flow velocity, measured by the Doppler ultrasonic flow probe, records changes in the resistance vessels and does not reflect changes in the large blood vessels (Hartley & Cole, 1974; Haywood et at. 1981; DiCarlo, Blair, Bishop & Stone, 1989). Indeed, the diameter of the common iliac artery (where the flow probe is positioned) cannot change because the walls of the artery adhere to the cuff of the probe. Changes in the hindlimb resistance vasculature can therefore be recorded by measuring blood flow velocity at the probe. By using this technique, we were able to investigate the role of local modulators of adrenergic agents in the hindlimb resistance vessels of intact conscious rats without causing systemic responses. This model also allowed us to avoid the compounding effects of abluminal drug administration, anaesthesia and denervated vessels that have been present in other studies. Matsuda, Kuon, Holtz & Busse (1985) showed that a2-adrenergic receptor-mediated endothelium-dependent dilatation was only present when UK (an a2-adrenergic receptor agonist) was delivered in the luminal perfusate; abluminal administration of the drug did not elicit the response. General anaesthesia also modifies autonomic control of circulation (Vatner & Braunwald, 1975). Since our studies were performed on conscious animals, the complicating influences of anaesthesia upon the evoked vascular responses were avoided. Previous studies on intact conscious rats have examined the modulatory influence of NO by giving drugs that interfere with NO synthesis at a large dose into the general circulation. Therefore, the direct effects of such drugs on the vasculature were complicated by their effects on arterial pressure and by any consequent baroreceptor reflex responses. We avoided this problem by injecting the substances locally into the hindlimb. Indeed, we believe that we have carried out the first direct investigation of the role of these local modulators in adrenergic responses evoked in the resistance vessels of conscious animals. Other investigators have examined the role of NO in modulating adrenergic responses in conscious animals with intact cardiovascular reflexes but with the drugs administered systemically (Gardiner et al a, b). In such a situation, direct effects of the pharmacological agents on heart rate, cardiac contractility, cardiac output, blood pressure and baroreceptor-mediated compensatory mechanisms cannot be distinguished from the direct vascular effect. Our

7 J Physiol Local modulation of adrenergic responses 823 experimental model made it possible to examine vascular function, under intact physiological conditions, independently of systemic effects or compensation from baroreceptors. One potential limitation of our study is the lack of a control for the baseline haemodynamic effects of NO-X or CO-X. That is, NO-X and CO-X significantly reduced resting IFV (25 + 2A4 and P3%, respectively) without altering HR or MABP. The possibility exists that any subsequent change in IFV due to the adrenergic agonist may simply be the result of a change in IFV. However, for two reasons, we do not believe that changes in the baseline level explain the results obtained. First, we allowed for any baseline change by calculating the percentage change in IFV rather than taking just the absolute change. Second, NO-X and CO-X had differential effects on the responses evoked by the different adrenergic agonists. Therefore, within the confines of this potential limitation, we are confident with our results. Local modulation of resting blood flow CO-X and NO-X significantly reduced resting IFV (14 + 1P3 and A4%, respectively) without altering HR and MABP by inhibition of the basal release of NO and a cyclo-oxygenase-derived vasodilator PG. Similarly, NO inhibition caused a dose-dependent increase in arteriolar tone, in the rat spinotrapezius muscle (Nakamura & Prewitt, 1991). These findings suggest that the resting tone of hindlimb vasculature is modulated by endogenous biosynthesis and basal release of NO, and a vasodilator PG. Local modulation of the response to phenylephrine In the present study, NO synthase inhibition enhanced the vasoconstrictor responses to phenylephrine. Accentuation of responses to phenylephrine after NO inhibition has been previously demonstrated in conduit vessels of rats (Carrier & White, 1985; Topuzis et al. 1991) and dogs (Young & Vatner, 1986), and in the skeletal muscle vasculature of rats (Ohyanagi et al. 1992). In the present study the enhanced response to phenylephrine was probably due not only to the inhibition of the basal release of NO, but also to the inhibition of an a1-adrenergic receptor-stimulated dosedependent release of NO. This is suggested because a two-way ANOVA revealed that there was a significant treatment-by-dose interaction. That is, the control and NO-X curves were not parallel (i.e. they had dissimilar shapes) and the slopes of the curves were significantly different (P = 0019). Several studies have failed to observe an ac-adrenergic receptor-mediated NO release in preconstricted vascular preparations (Cocks & Angus, 1983; Vanhoutte & Miller, 1989). However, significantly preconstricting the vascular ring may mask the effect of NO (Martin, Furchgott, Villani & Jothianandan, 1986). Species and tissue specificity may also account for these differences (Cocks & Angus, 1983). Furthermore, since ac-adrenergic agonists have greater efficacy for phenylephrine than for a2-adrenergic agonists, and have a larger receptor reserve, it may be difficult to demonstrate this effect in isolated vessels. The vasoconstrictor responses to phenylephrine were also enhanced by cyclo-oxygenase inhibition. There was a significant treatment-by-dose interaction, suggesting receptor-stimulated dose-dependent release of a vasodilator PG. Other vasoconstrictors such as angiotensin II also release a vasodilator PG (Toda, 1984). Endothelial cells are known to respond to changes (Rubanyi, 1991; Davies & Tripathi, 1993) in the local milieu (catecholamine, flow, pressure, P02, [ATP], [ADP], [serotonin], [bradykinin], [thrombin]) by producing and releasing factors that modulate the tone of the underlying vascular smooth muscle. This response may be an important mechanism in regulating local perfusion during sympathetic nerve activation (Miller, 1991). Local modulation of the response to clonidine NO synthase inhibition enhanced the vasoconstrictor responses to clonidine. Enhanced vasoconstrictor responses to a2-adrenergic activation after NO synthase inhibition has been previously demonstrated in the resistance vessels (Nakamura & Prewitt, 1991; Ohyanagi et al. 1992). In our study there was no significant treatment-by-dose interaction, suggesting that there was no receptorstimulated dose-dependent release of NO. This finding contrasts with results previously obtained in canine and porcine coronary arteries (Cocks & Angus, 1983) and in dog femoral arteries (Miller & Vanhoutte, 1985), in which an a2-adrenergic receptor-mediated release of NO was demonstrated. However, the a2-activated endothelial release of NO shows significant species and tissue variability. For example, a2-adrenergic receptor-mediated release of NO could not be demonstrated in bovine coronary vessels, canine mesenteric or renal arteries (Angus, Cocks & Satoh, 1986), nor in canine femoral vein (Vanhoutte & Miller, 1989). Although cyclo-oxygenase inhibition enhanced the vasoconstrictor responses to clonidine, there was no significant treatment-by-dose interaction. This suggests that the enhanced vasoconstrictor responses to clonidine were entirely due to inhibition of the basal release of vasodilator PGs and not due to a receptor-stimulated dose-dependent release of PG. The suggestion that al1- rather than a2 -adrenergic receptors mediate the release of NO and vasodilator PGs in hindlimb resistance vasculature is in accordance with the relative functional importance of a,-adrenergic receptors compared with a2-adrenergic receptors in the peripheral vasculature (Ruffolo, 1986). Postsynaptic vascular ac-adrenergic receptors in the peripheral arterial circulation are located at the neuroeffector junction (i.e. junctional or synaptic receptors), while the postsynaptic vascular a2 -adrenergic receptors are located away from the neuroeffector junction

8 824 S. E. DiCarlo and others J Phy8iol (extrajunctional or extrasynaptic receptors). The physiological role of the postsynaptic junctional az-adrenergic receptors appears to be in maintaining resting vascular tone and responding to changes in sympathetic tone, as suggested by the fact that these receptors are located in the vascular neuroeffector junction and can therefore interact with nerve-released noradrenaline (NA). The physiological role of the extrajunctional a2-adrenergic receptors is not fully understood. It has been suggested that extrajunctional a2-adrenergic receptors do not normally interact with nerve-released NA because they are located at a distance from the adrenergic nerve terminal, and the highly efficient neuronal uptake mechanism keeps synaptic levels of NA low so preventing diffusion of the neurotransmitter to the extrajunctional sites (Langer & Shepperson, 1982). The extrajunctional a2-adrenergic receptors may therefore respond to circulating catecholamines (Ruffolo, 1986). Our results suggest that the vasoconstrictor influence of these receptors may be moderated by NO and CO products. Local modulation of the response to adrenaline Responses to adrenaline were biphasic. There was an initial vasoconstrictor response followed by vasodilatation. NO-X significantly attenuated the vasodilator response, but enhanced the vasoconstrictor responses. The significant treatment-by-dose interaction suggests that there is receptor-stimulated dose-dependent release of NO. These results are consistent with previous studies (Rubanyi & Vanhoutte, 1985; Young & Vatner, 1986). Thus, endothelial denudation of the iliac artery of the conscious dog converted a vasodilator response to adrenaline into a vasoconstrictor response (Young & Vatner, 1986) and attenuated the response to isoprenaline in canine coronary arteries (Rubanyi & Vanhoutte, 1985), suggesting that an endothelium-derived factor contributes to /6-adrenergic receptor-mediated vasodilatation. In addition, Gardiner and co-workers reported that systemic administration of salbutamol (Gardiner et al a) and adrenaline (Gardiner et al b) caused hyperaemic vasodilatation in the vascular bed of the hindquarters of conscious rats with intact cardiovascular reflexes; this response was attenuated in the presence of L-NAME. The fact that CO-X significantly attenuated the vasoconstrictor response to adrenaline is consistent with the observation that CO-X enhanced the relaxation of canine coronary arteries mediated by the fl-adrenergic agonist isoprenaline (Rubanyi & Vanhoutte, 1985), and suggests that CO-X inhibits a vasoconstrictor PG. However, it is also possible that the vasoconstrictor thromboxane could be involved since inhibition of cyclo-oxygenase would block the production of thromboxanes as well as PG. The significant treatment-by-dose interaction supports the suggestion that there was a receptor-stimulated dosedependent release of a vasoconstrictor PG. Conclusion Local modulation of adrenergic responses was examined in the hindlimb vasculature of chronically instrumented, intact conscious rats. Dose-response curves for the a,-adrenergic receptor agonist phenylephrine, the a2-adrenergic receptor agonist clonidine and the endogenous adrenergic receptor agonist adrenaline were generated under control conditions, in the presence of the NO synthase inhibitor L-NAME and in the presence of the cyclo-oxygenase inhibitor indomethacin. Heart rate and arterial pressure were not altered by any of the agents because we selected doses below those which elicited systemic responses. Maximal vasoconstrictor responses to phenylephrine were enhanced in the presence of NO-X (50 ± 6%) and CO-X (70 + 9%). Maximal vasoconstrictor responses to clonidine were also enhanced in the presence of NO-X ( %) and CO-X ( %). The responses to adrenaline were biphasic, NO-X significantly attenuated the vasodilator response (87 ± 6 %) and enhanced the vasoconstrictor response ( %). CO-X attenuated the vasoconstrictor response to adrenaline (71 + 6%). 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