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Cardiovascular Research 1999 43(3):762-771; doi:10.1016/S0008-6363(99)00092-9
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

NOS inhibition potentiates norepinephrine but not sympathetic nerve-mediated co-transmission in resistance arteries

Karen M Smith, Joyce B Macmillan, Kirsty M McCulloch1 and John C McGrath*

Autonomic Physiology Unit and Clinical Research Initiative in Heart Failure, Division of Neuroscience & Biomedical Systems, Institute of Biomedical & Life Sciences, West Medical Building, University of Glasgow, Scotland, UK

* Corresponding author. Tel.: +44-141-330-3187; fax: +44-141-337-1651. joyce.macmillan{at}bio.gla.ac.uk

Received 2 December 1998; accepted 22 February 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Objective: The in vitro interaction between sympathetic nerves and basal nitric oxide release was studied in a resistance artery, since these interact powerfully in large vessels. Methods: The pharmacological interaction between L-NAME and vasoconstriction to field stimulation of sympathetic nerves or exogenous norepinephrine was studied in rabbit cutaneous resistance arteries in wire myographs. Results: Relaxation of norepinephrine-induced tone by acetylcholine, but not sodium nitroprusside, was blocked by N{omega}-nitro-L-arginine methyl ester (L-NAME: 100 µM), indicating that the agonist-induced release of nitric oxide could oppose the vasoconstrictor effect of norepinephrine and confirming that L-NAME had no effect on endothelium-independent vasodilatation. L-NAME increased norepinephrine potency indicating basal NO release. With short bursts of electrical field stimulation purinergic transmission was dominant at low frequencies and adrenergic at high frequencies. L-NAME had no effect on nerve-mediated responses, even after blocking the purinergic component with {alpha},β-methylene ATP (3 µM), suggesting that the influence of spontaneously released nitric oxide does not extend to the vascular smooth muscle cells under adrenergic nervous control. Conclusion(s): This resistance artery exhibits a highly effective nitric oxide-mediated vasodilatation to acetylcholine. It has basal release of nitric oxide which antagonises exogenous norepinephrine. However, basal nitric oxide did not influence adrenergic nerve transmission, which contrasts with previous studies of larger arteries and veins. We speculate that in small resistance arteries there may be a spatial limitation to the zones of vascular smooth muscle influenced by the adrenergic nerves and by basal nitric oxide from the endothelium, respectively. The role of endogenous nitric oxide in modulating vascular tone may thus be less in resistance arteries than in conducting arteries or capacitance vessels and purinergic transmission appears to be particularly resistant.

KEYWORDS Acetylcholine; Adrenergic; Autonomic nervous system; Nitric oxide; N{omega}-nitro-L-arginine methyl ester; Rabbit


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 
Control of peripheral vascular resistance and blood-pressure is under the opposing influences of autonomic nerves and the endothelium. Endothelium-derived factors are released continuously at a basal level and are modulated by many physical and chemical factors [1,2]. Although there are both excitatory [3] and inhibitory factors, the most widely encountered is the synthesis and release of nitric oxide, which exerts a depressant effect on vascular smooth muscle excitation. The interaction and balance of the contribution of sympathetic nerves and endothelium-derived vasodilators to resistance arterial vascular tone should be critical in the determination of vascular resistance [4].

In large arteries and veins blockade of basal nitric oxide synthesis with N{omega}-nitro-L-arginine methyl ester (L-NAME) or N-mono-methyl-L-arginine (L-NMMA) can potentiate responses to sympathetic nerve stimulation [5–9]. Surprisingly, little is known about the equivalent interaction of endothelial nitric oxide and sympathetic nerve stimulation in the resistance arteries, which are key to blood-pressure control. Gaining this was the primary objective of the present study.

Several studies have shown that acute nitric oxide synthase inhibition will enhance the actions of vasoconstrictor agents in large vessels both in vitro [8–13] and in vivo [14,15]. However, there are few studies on the effects of nitric oxide synthase inhibition versus vasoconstrictor nerve-mediated responses and these concentrate on Windkessel arteries, pulmonary arteries or capacitance veins [7–9]. In vivo, inhibition of endogenous nitric oxide with L-NAME in pithed rats increased blood-pressure and increased vasoconstrictor responses to sympathetic nerve stimulation and adrenal catecholamine release [16,17]. L-NMMA has similar actions in vivo on mesenteric arteries [18]. However, in vivo studies cannot determine whether these interactions occurred via a pharmaco-biochemical effect within the resistance vessel or reflected a more complex haemodynamic interaction [16]. In vitro, L-NAME or L-NMMA greatly potentiates (three- to six-fold increase in size) noradrenergic nerve-mediated contraction in the rabbit isolated lateral saphenous vein [8] and guinea pig pulmonary artery [7] indicating considerable scope for nitric oxide to override sympathetic nervous system control of capacitance and pulmonary blood flow.

We have now sought to examine the interaction of nerves and endothelial factors in an isolated resistance artery, the rabbit cutaneous, first demonstrating the necessary components, then their interaction. This vessel was chosen initially to provide an animal model equivalent to the human cutaneous resistance artery, which is now extensively employed in pathophysiological studies [19]. We were interested specifically in the extent to which basal endothelial release of nitric oxide would affect nerve-mediated adrenergic vasoconstriction. We approached this by investigating whether L-NAME could potentiate nerve-mediated responses using a similar protocol to that used previously with larger vessels [8,9]. It transpired that the rabbit cutaneous resistance artery exhibited purinergic/adrenergic co-transmission so this added an extra element.

Cotransmission involving norepinephrine and ATP (adenosine triphosphate) has been demonstrated in vitro in several species [20] and in vivo in the rat [21]. In the rabbit a substantial non-adrenergic component of sympathetic nerve-mediated vasoconstriction has been shown in vivo in the pithed preparation [22] and purinergic transmission has been identified in several rabbit isolated blood vessels [20,23,24]. The relative proportions of purinergic and noradrenergic components are vessel dependent, involving various combinations of P2X-purinoceptors, {alpha}1-adrenoceptors and {alpha}2-adrenoceptors [24]. The influence of basal nitric oxide on nerve-mediated vasoconstriction showed some relationship with type of transmission in these large vessels. In both saphenous artery and saphenous vein the noradrenergic {alpha}1-adrenoceptor-mediated response was potentiated by L-NAME, whereas the purinergic response in saphenous artery was unaffected [8,9]. No similar analysis is available for resistance arteries, so this was the prime objective.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 5 Conclusions
 References
 
2.1 Resistance arteries
Experiments were carried out in male New Zealand White rabbits weighing 3.0–4.5 kg. They were sacrificed by an overdose of pentobarbitone (100 mg kg–1) into the ear vein and a flap of skin from the area overlying the gluteal muscles was removed. Connective tissue was cleared from above the network of cutaneous arteries and resistance arteries (2 mm length) were isolated and excised under a dissecting microscope. The procedures were undertaken in accordance with Animals (Scientific Procedures) Act 1986 and the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

The arteries were mounted as ring preparations in a Mulvany-Halpern double myograph (J.P. Trading, Aarhus, Denmark), mounted on two 40 µm steel wires which were attached to a force transducer and a micrometer. The vessel was bathed in Krebs’-Henseleit solution (in mM: NaCl 118.4, KCl 4.7, MgSO4.H20 1.2, KH2PO4 1.2, NaHCO3 24.9, CaCl2 2.5, glucose 11.1, EDTA 0.023), maintained at 37°C and gassed with a 95% O2/5% CO2 mixture.

The drugs used were (-)-noradrenaline bitartrate (Sigma), acetylcholine chloride (Sigma), N{omega}-nitro-L-arginine methyl ester (Sigma), {alpha},β-methylene adenosine triphosphate (Sigma), and Hoechst 33342 (H1399 Molecular Probes, Leiden, The Netherlands).

After a rest period of 30 min the artery was stretched at 1 min intervals to determine the exponential passive wall tension–internal circumference (L) relationship. From the Laplace relationship, where P=T/r (P is the effective pressure, T is wall tension and r is the internal radius), the circumference (L100) was calculated by an iterative computer method that gave an equivalent transmural pressure difference of 100 mm Hg for each artery. The circumference at 0.9xL100 was calculated where the active force production is close to maximum (data not shown). Normalised vessel internal diameter for the remainder of the experiments was set at 0.9xL100. From the known length–tension relationship we calculated the equivalent wall tension at 0.9xL100 and the equivalent effective pressure P1 (mm Hg). Effective pressure is an estimate of the pressure which would be necessary to extend the vessel to the measured internal circumference. If arteries had a normalised internal diameter greater than 300 µm, they were excluded from our study.

2.2 Contraction protocol
After the normalisation procedure the arteries were exposed to norepinephrine (10 µM), followed by KCl (125 mMx2). Thirty minutes later a cumulative concentration response curve (CRC) was generated to norepinephrine in half log unit concentration increments. The concentration response curve to norepinephrine was repeated following a 30 min exposure to the nitric oxide synthase competitive inhibitor, L-NAME (100 µM).

2.3 Relaxation protocol
Arteries were precontracted with sub-maximal concentrations of norepinephrine (1 µM) to produce a steady level of contraction. Cumulative concentrations of acetylcholine (0.001–30 µM) or sodium nitroprusside (SNP) (0.001–30 µM) were then added in log unit increments.

Acetylcholine CRCs were also measured in the presence of L-NAME (100 µM) and, at the end of this protocol, the integrity of the smooth muscle was examined using SNP. In a separate set of experiments relaxation curves to SNP were generated in arteries which had not been exposed to L-NAME.

Care was taken to preserve the endothelium as far as possible. Confocal microscopy of vessels, fixed after myograph mounting, and stained with a nuclear dye (Hoechst 33342) showed intact endothelium except where the vessel was in direct contact with the wires (see Fig. 1).


Figure 1
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  		Fig. 1 An extended focus (montage) view showing all cellular layers of the wall of a fixed rabbit cutaneous resistance artery stained by with the nuclear dye Hoechst 33342 (0.01 mg ml–1) using a laser scanning confocal microscope. The arrows show the axis of the blood flow. The elongated smooth muscle cell nuclei are arranged perpendicular to the axis of flow.

 
2.4 Nerve stimulation protocol
Cutaneous resistance arteries were mounted in wire myographs (as described above) with the exception that the 40 µm wires were attached to specially designed plastic myograph heads incorporating platinum stimulation electrodes. The arteries were normalised, as outlined above. After normalisation, vessels were precontracted with 10 µM norepinephrine; 3 µM acetylcholine was added after the response had reached a plateau to verify endothelial function. Following washout (4x) and return to baseline tension, the vessels were allowed to equilibrate for 20 min. Using a Grass S88 stimulator, the preparations were then exposed to three test trains of pulses at 16 Hz (stimulation parameters of 0.1 ms pulse width, 1 s train, at 35 V), with 5 min intervals between each train. Vessels were then washed (4x) with fresh Krebs’ and left to equilibrate for 30 min. Frequency response curves (FRC) were then constructed by stimulating at 4, 8, 16, 32 and 64 Hz (stimulation parameters as above).

The interval between FRCs (frequency response curves) was 30 min. Tissues were incubated for 30 min with prazosin (1, 10 or 100 nM), {alpha},β-methylene ATP (3 µM) or L-NAME (100 µM), before the construction of the second FRC. A combination of antagonists for 30 min was followed by construction of the third FRC. The addition of {alpha},β-methylene ATP always caused a transient contraction and L-NAME also caused small contractions, but not in every vessel. It had previously been shown that prazosin is a competitive antagonist of norepinephrine-induced vasoconstriction in this vessel with a pA2 value of 9.2 [25]: from the data in that study the decreases in norepinephrine sensitivity produced by prazosin were; at 1 nM, 1.3 fold; at 10 nM, 3 fold; at 100 nM, 44 fold. This justifies the use of these concentrations of prazosin against adrenergic nerve-mediated responses. {alpha},β-methylene ATP 3 µM has been shown to abolish the prazosin-resistant, i.e. non-adrenergic, nerve-mediated contraction in other rabbit blood vessels [24].

2.5 Vessel imaging. laser scanning confocal microscopy:
The objective of observing the structure of the resistance vessels in extended focus was to verify the integrity of the endothelium throughout the part of the vascular wall participating in the contractile response. At the end of the experimental protocols, arteries were fixed at 0.9xL100 for 30 min in 10% formal-saline solution. The arteries were cut open with a surgical blade, then exposed to the nuclear dye Hoechst 33342 (0.01 mg ml–1) for 30 min and washed for 5 min. They were then placed endothelial side-up on a slide and visualised with a laser scanning confocal microscope (LSCM) (Odyssey LSCM, Noran Instruments, Middleton, Wisconsin, USA) in conjunction with a Nikon Optiphot (Nikon, Kingston on Thames, Surrey, UK). The arteries were visualised with a x40 water immersion objective (Nikon, NA, 1.15), using the 364 nm line (400 nm barrier filter) of the LSCM, pinhole aperture 10mm. Stacks of 1 µm thick optical slices were then captured in z-axis and an extended focus (montage) image reconstructed from them with Metamorph Software (Universal Imaging Corporation) (Fig. 1).

In these vessels it was technically difficult to deliberately physically remove the acetylcholine-mediated vasodilatation without damaging the smooth muscle contraction. This was attempted by air perfusion and internal rubbing with a human hair. The latter was the more successful and satisfied us that the acetylcholine-mediated response required the presence of the endothelium. This was confirmed by LSCM. In the standard experiments, despite the manipulation of wires through the vascular lumen, confocal microscopy revealed that the endothelial integrity was well maintained except for the parts in direct contact with the wires during the experiment (Fig. 1).

2.6 Study design and statistical analysis:
Contractile responses of arteries were expressed as increase in active effective pressure (P, mmHg), calculated as increase in isometric tension (T) above resting divided by the normalised internal radius. Relaxation responses were calculated in terms of active effective pressure loss from the precontracted level. Agonist potency was expressed in terms of pD2 values which represent the negative logs of the concentrations of the agonist required to produce 50% of the maximum response. Values were presented as mean±s.e. mean and paired two-tailed t tests were performed with a commercially available statistical package for the PC (Microsoft Excel version 7.0) to evaluate the effects of L-NAME on norepinephrine, acetylcholine or nerve-mediated responses; i.e. to compare potencies at the pD2 levels and at concentrations producing 10% and 25% of maximum or at individual frequencies. The effects of antagonists on the FRCs were evaluated using Graph Pad Prism 2.01 (Institute for Scientific Information, San Diego, California, USA.) by a one way analysis of variance (ANOVA) followed by a Bonferroni post test allowing multiple comparisons. Differences were considered significant at a level of P<0.05


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 5 Conclusions
 References
 
3.1 Vascular responses to exogenous acetylcholine and norepinephrine
Acetylcholine produced a maximal relaxation of 58.2±4.4% (n=36/21) (n=number of arteries/number of rabbits) in arteries vs. norepinephrine (1 µM) induced tone, which L-NAME (100 µM) virtually abolished (P<0.001) (Fig. 2A). In contrast, L-NAME (100 µM) had no significant effect on sensitivity or maximal vasodilatation to the endothelium-independent vasodilator sodium nitroprusside (Fig. 2B). This shows high sensitivity of the vessel’s smooth muscle cells to agonist-released nitric oxide and that L-NAME (100 µM) influences the production, and not the action, of nitric oxide. On addition to the bath, L-NAME (100 µM) produced a transient contraction which was variable in size returning to baseline within 15 min and if not, after further washing and reapplication of L-NAME. Any reapplication of L-NAME caused no further contraction.


Figure 2
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  		Fig. 2 Responses in the rabbit cutaneous resistance artery to, A: acetylcholine in the absence and presence of L-NAME 100 µM (n=36/21), B: sodium nitroprusside in the absence (n=8/5) and presence of L-NAME 100 µM (n=36/21) expressed as% relaxation to tone induced by norepinephrine 1 µM. Data are shown as mean±s.e.m. Open circles, control; closed circles, with L-NAME.

 
Norepinephrine produced a sigmoidal concentration response curve in the rabbit cutaneous resistance arteries with a pD2=6.69±0.05 (n=36/21). L-NAME (100 µM) increased the sensitivity to norepinephrine at the 10% of maximum (P<0.001), 25% of maximum (P<0.001) and 50% of maximum (pD2) (P<0.001) levels (Fig. 3) indicating a population of smooth muscle cells whose response to low concentrations of norepinephrine is attenuated by endogenous nitric oxide.


Figure 3
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  		Fig. 3 Responses in the rabbit cutaneous resistance artery to norepinephrine in the absence and presence of L-NAME 100 µM in active effective pressure. The inset graph plots the negative log of the concentrations achieving 10%, 25% and 50% of maximum for the same data in the presence and absence of L-NAME 100 µM. Data are shown as mean±s.e.m. (n=36/21). * P<0.05; ** P<0.01; *** P<0.001 for±L-NAME, Student’s paired t-test. Open circles, control; closed circles, with L-NAME.

 
3.2 Nerve stimulation:
Prazosin (0.1 µM) caused a reduction in the nerve-mediated responses throughout the frequency range, reaching significance at ≥16 Hz (Fig. 4A).


Figure 4
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  		Fig. 4 A: Frequency response curve in the absence (light stipple) and presence (dark stipple) of prazosin 100 nM(n=6/6). B: Frequency response curves in control (n=19/7) (white bars), and in the presence of {alpha},β, mATP 3 µM (n=19/7) (hatched); {alpha},β, mATP 3 µM+prazosin 1 nM (n=4/4) (dark stipple); {alpha},β, mATP 3 µM+prazosin 10nM (n=7/5) (light stipple); {alpha},β, mATP 3 µM+prazosin 100 nM (n=8/7) (dark). Data are shown as mean±s.e.m. (n=36/21). A: ±Prazosin Student’s paired t-test. B: one way ANOVA with a Bonferroni multiple range test. *against control values, {dagger}against {alpha},β, mATP 3 µM. P <0.05

 
{alpha},β-methylene ATP (3 µM) reduced nerve-mediated vasoconstriction at the lower frequencies, significantly at 8 Hz (P<0.05) (Fig. 4B). Taken together this indicates a dominance for the purinergic component at low frequencies and for the adrenergic component at higher frequencies.

The effect of prazosin (1, 10 and 100 nM) after {alpha},β-methylene ATP (3 µM) was examined to test whether the remaining response was adrenergic; prazosin produced concentration-dependent inhibition with virtually complete blockade by 100 µM (Fig. 4B). Thus after elimination of the purinergic component with {alpha},β-methylene ATP (3 µM), the residual response shows sensitivity to prazosin consistent with transmission via {alpha}1-adrenoceptors, indicating co-transmission with a tendency to favour P2X at low and {alpha}1 at higher frequencies, respectively.

L-NAME (100 µM) had no significant effect on nerve-mediated responses at any frequency, contrasting with its marked potentiation of equivalent sized responses to norepinephrine (Fig. 5A).


Figure 5
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  		Fig. 5 A: Frequency response curve in the absence (light stipple) and presence (dark stipple) of L-NAME 100 µM (n=9/9), in the separate graph agonist induced responses to norepinephrine at concentrations of 10nM, 30 nM and 100 nM in the absence and presence of L-NAME 100 µM (n=36/21). B: Frequency response curve in the presence of {alpha},β, mATP 3 µM in the absence (light stipple) and presence (dark stipple) of L-NAME 100 µM (n=5/5). Data are shown as mean±s.e.m. * P<0.05; ** P<0.01; ***P<0.001 for±L-NAME, Student’s paired t-test.

 
In order to examine specifically whether L-NAME (100 µM) was having an effect on adrenergic nerve-mediated vasoconstriction, we examined its effect in the presence of the desensitising purinergic agonist {alpha},β-methylene ATP (3 µM); L-NAME (100 µM) had no effect on nerve-mediated responses after {alpha},β-methylene ATP (3 µM) (Fig. 5B).

It would have been useful to examine the effect of agonist-induced endothelial nitric oxide release versus nerve-induced vasoconstriction. This was attempted by administering increasing concentrations of acetylcholine during electrical field stimulation with 1 s trains at 8 Hz at 5 min intervals. Acetylcholine (≥10–9 M) produced concentration-related inhibition of the nerve-induced vasoconstriction but this was unaffected by L-NAME (data not shown). It was concluded that it was impractical to observe the effects of acetylcholine-induced nitric oxide release versus nerves due to the over-riding influence of the direct effect of acetylcholine on neurotransmitter release [26]. Similar considerations would apply to other agonists. It should be noted that acetylcholine’s non-nitric oxide-mediated action at blocking nerve-mediated responses was more potent than its nitric-oxide-mediated action to relax norepinephrine-induced tone.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
5 Conclusions
 References
 
The rabbit cutaneous resistance artery is sensitive to vasoconstriction by norepinephrine and its sympathetic nerve-mediated response exhibits noradrenergic:purinergic co-transmission. It is thus a useful addition to the preparations available for the study of resistance artery biology. It is also sensitive to acetylcholine-induced vasodilatation mediated by nitric oxide.

Although contraction to exogenous norepinephrine showed evidence of coincidental inhibition by nitric oxide, responses to nerve stimulation did not. This contrasts markedly with a previous study, under the same experimental conditions, in a venous preparation from the same species, whose nerve-mediated contractions were greatly increased [8] as in many similar studies [5–9]. The key issues are therefore why the resistance artery and capacitance vein differ so markedly in their properties and, in the resistance artery, why there is a greater influence of endogenous nitric oxide versus norepinephrine than versus nerves.

4.1 Comparison of vessels
On the physiological level the relative lack of effect of endogenous nitric oxide on nerve-mediated responses in resistance vessels could be important for central control of peripheral resistance and hence of blood-pressure, allowing the sympathetic vasoconstrictor nerves to override local factors in resistance arteries. The contrast with rabbit saphenous vein may indicate a relative importance of local factors in capacitance vessels; similarly for flow-induced vasodilatation in pulmonary arteries or conducting arteries.

It is also interesting that, in rabbit saphenous vein, L-NAME increased the size of contraction over a range of concentrations of norepinephrine and indeed increased the maximum response [8] but, in contrast to the cutaneous resistance artery, the sensitivity to norepinephrine was not altered. This suggests a difference between the artery and vein in the cross-talk at the vascular smooth muscle cell signalling level.

4.2 Comparison of the relative influence of nitric oxide versus norepinephrine and nerves
In the cutaneous resistance artery the nitric oxide synthase inhibitor L-NAME (100 µM) produced a significant increase in the sensitivity to norepinephrine indicating that endogenous nitric oxide can influence vascular tone in these resistance arteries. However, the same concentration of L-NAME had no significant effect on similarly-sized nerve-mediated responses at any of the frequencies tested (see Fig. 5A), which also stands in marked contrast to rabbit saphenous vein and saphenous artery [8,9].

The ability of inhibition of nitric oxide synthase to increase sensitivity to norepinephrine is of fundamental importance to the study of adrenergic mechanisms. The increased sensitivity to norepinephrine induced by L-NAME suggests that vascular smooth muscle cells of this resistance artery have a higher sensitivity to exogenous norepinephrine than is uncovered in a straightforward concentration response curve, but that this is "suppressed" either by basal nitric oxide release or by norepinephrine-induced nitric oxide release (see later). However, the lack of such an influence versus endogenous adrenergic nerve stimuli suggests that neither basal nor nerve-induced nitric oxide influences the vascular smooth muscle cells activated by the nerves.

If there is basal nitric oxide release then the greater effects of L-NAME versus norepinephrine than versus nerves suggests that there is a zone within the vascular medial layer in which vasoconstriction (e.g. to exogenous norepinephrine) is influenced by endothelium and that there is a separate zone influenced by sympathetic nerves but not by the endothelium. There is further evidence of an influence of basal nitric oxide release since L-NAME produces contraction, albeit transient.

It has been proposed that, in the mesenteric circulation, endogenous nitric oxide from the endothelium or from specific neurones might inhibit norepinephrine release from perivascular nerves [27]. The lack of potentiation by L-NAME in cutaneous resistance arteries argues against neuronally produced nitric oxide or nitric oxide-induced inhibition of transmitter release in this preparation. This may be consistent with the requirement for the dominance of central control of the cutaneous but not mesenteric circulations.

With regard to size of vessel and distances for diffusion, it appears to be counter-intuitive that the small resistance vessel shows relatively little interaction between adventitial nerves and intimal nitric oxide. Our observations may thus indicate that the properties of the particular smooth muscle cells in the respective zones influenced by the nerves or by nitric oxide are more important than diffusion distance per se and that smooth muscle cell phenotypic variation in different blood vessels is the important factor for interaction of these important modulators. This is not to deny a possible role of endothelium-to-nerve proximity in facilitating interaction. Indeed, amongst the best examples of potentiation of nerve responses by NO synthase-inhibition are veins and pulmonary arteries in which the sympathetic nerves penetrate deep into the medial layer in close proximity to the endothelium.

It would be of interest to know whether agonist-activated nitric oxide release (as opposed to spontaneous, basal release) can influence the nerve-mediated response. An attempt was made to test this by examining the effect of acetylcholine versus nerve-mediated responses. This carried the risk, which proved well-founded, of a direct effect of acetylcholine on the nerve endings, which inhibits transmitter release [26]. The question of interest was whether, in our experiments, acetylcholine inhibited nerve-mediated contractions (16 Hz) in a dose-dependent manner which was susceptible to nitric oxide synthase inhibition. Acetylcholine did induce a dose-dependent inhibition, but exposure to L-NAME (100 µM) for 30 min did not block it, indicating that the influence of presynaptic muscarinic receptors in this preparation was too powerful to allow exposure of any part of the acetylcholine-mediated inhibition of the nerve-induced contraction which is susceptible to L-NAME.

Although it is theoretically possible that norepinephrine might be releasing nitric oxide from the endothelium and so inhibiting its own contraction, we find this intuitively unlikely since it is the lowest concentrations of norepinephrine whose contractions were most potentiated by L-NAME. Nevertheless we made an attempt to uncover such an effect using the supposedly selective {alpha}2-adrenoceptor agonist UK14304 since such receptors have been postulated to release endothelial nitric oxide in coronary vessels [28]. We could not uncover such a response. We were not able to demonstrate the existence of {alpha}2-adrenoceptors either with respect to producing direct vascular smooth muscle cell contraction or releasing endothelial nitric oxide. Furthermore there was no evidence for a contribution from vascular smooth muscle cell {alpha}2-adrenoceptors in the nerve-mediated contractions in rabbit cutaneous resistance arteries since the combination of {alpha},β-methylene ATP (3 µM) and prazosin (100 nM) abolishes these responses, even at the highest frequencies. In our previous experiments in rabbit cutaneous resistance arteries, UK14304 had a pD2 of 5.34±0.40 (n=6/6); this potency indicates the presence of {alpha}1- as opposed to {alpha}2-adrenoceptors [29]. UK14304-induced vasoconstriction was not consistently potentiated by L-NAME (100 µM) and UK14304 did not produce any relaxation of tone produced by a variety of agents, i.e., norepinephrine, phenylephrine, vasopressin, angiotensin II or KCl (50 mM) (data not shown). In summary, no evidence could be found for {alpha}2-adrenoceptor-activated release of nitric oxide in this vessel.

4.3 Co-transmission
Purinergic and adrenergic components of the nerve-mediated responses could be defined in rabbit cutaneous resistance arteries. Co-transmission in resistance vessels was first shown in vivo in the pithed rat; noradrenergic:purinergic co-transmission was demonstrated within sympathetic nerve-induced rises in blood-pressure, presumably due to stimulation of resistance arteries [21]. This was confirmed in vitro by Sjoblom-Widfeldt & Nilsson [30], who demonstrated adrenergic and purinergic components within the response to electrical field stimulation of rat mesenteric resistance arteries. As in the present study they found that purinergic and adrenergic components dominated at low and high frequencies respectively. These observations, now covering two species, different resistance beds, in vitro and in vivo emphasise the potential importance of co-transmission in resistance vessels, which has been studied mainly in large vessels [20,31].

Resistance of sympathetic nerve mediated vasoconstriction to endogenous nitric oxide influence may not be a ubiquitous feature of small resistance vessels. Nase et al. [18], in their investigation of sympathetic nerve activity in rat intestinal arterioles concluded that endogenous nitric oxide activity could attenuate sympathetic neurogenic constriction in the intestinal microvasculature since the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 100 µM) increased the magnitude of sympathetic vasoconstriction in the arterioles (although since this is in vivo the interaction might be between NO and another vasoconstrictor synergising with the nerve response). For example, in an analogous situation in the pithed rat sympathetic vasoconstrictor responses are considerably enhanced by circulating angiotensin II, which is a secondary factor in the response to sympathetic nerve stimulation. Inhibition of angiotensin II by nitric oxide would therefore have an indirect inhibitory effect on nerve responses. This vasoconstriction in intestinal microvasculature was abolished by phentolamine (1 µM), so, unlike rabbit cutaneous resistance arteries there was no significant purinergic component. Interestingly the nerve-mediated response in rabbit saphenous vein, which is greatly potentiated by L-NAME, has no purinergic component [24] and in rabbit saphenous artery the noradrenergic but not the purinergic response was potentiated by L-NAME. We know of no example where the purinergic response has been shown to be under the influence of endogenous nitric oxide. However, it is not only purinergic transmission per se which renders resistance to endogenous nitric oxide. In the present study, blockade of purinergic transmission by {alpha},β-methylene ATP did not uncover a residual noradrenergic nerve-mediated response which could be potentiated by L-NAME.

A particular feature of sympathetic vasoconstrictor neurotransmission in the rabbit cutaneous resistance artery is its robustness. Not only is transmission resistant to local nitric oxide influence, but pharmacological blockade of either adrenergic or purinergic transmission leaves a substantial response from the other component at all but the lowest frequencies. In general this suggests that in this type of resistance vessel the sympathetic nervous system has the ability to override local factors and that the two components of co-transmission may act as a back-up to each other.


   5 Conclusions
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Conclusions
References
 
In this resistance artery, nitric oxide release has relatively little influence on nerve-induced compared with norepinephrine-induced vasoconstriction suggesting a spatial limitation to the zones of vascular smooth muscle influenced by the adrenergic nerves and by nitric oxide from the endothelium, respectively. We postulate that nitric oxide’s influence remains close to the endothelium and away from the nerves. Generalising from the results, the role of endogenous nitric oxide in modulating vascular tone may be lesser in resistance arteries than in conducting arteries or capacitance vessels and purinergic transmission may be particularly resistant.

Time for primary review 33 days.


   Acknowledgements
 
We wish to acknowledge the advice on confocal techniques provided by Dr. Silvia M. Arribas. We are grateful to Professor William Martin for criticism of the manuscript. The authors wish to thank Dr. Jillian M. Peacock for assistance in the preparation of this manuscript. This work was supported by the Medical Research Council. Our laboratory was a member of the EC Biomed Project ‘EureCa’ (BMH1-CT94-1375) during this study.


   Notes
 
1 Present address: Department of Physiology and Pharmacology, University of Strathclyde, Royal College, 204 George St., Glasgow G1 1XW. Back


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 5 Conclusions
 References
 

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