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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1999 Feb;126(3):785–793. doi: 10.1038/sj.bjp.0702345

Effects of vasopressin on the sympathetic contraction of rabbit ear artery during cooling

A L García-Villalón 1, J Padilla 2, L Monge 1, N Fernández 1, M A Sánchez 1, B Gómez 1, G Diéguez 1,*
PMCID: PMC1565852  PMID: 10188992

Abstract

  1. In order to analyse the effects of arginine-vasopressin on the vascular contraction to sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of the rabbit central ear (cutaneous) artery to electrical field stimulation (1–8 Hz) was recorded at 37 and 30°C.

  2. Electrical stimulation (37°C) produced frequency-dependent arterial contraction, which was reduced at 30°C and potentiated by vasopressin (10 pM, 100 pM and 1 nM). This potentiation was greater at 30 than at 37°C and was abolished at both temperatures by the antagonist of vasopressin V1 receptors d(CH2)5 Tyr(Me)AVP (100 nM). Desmopressin (1 μM) did not affect the response to electrical stimulation.

  3. At 37°C, the vasopressin-induced potentiation was abolished by the purinoceptor antagonist PPADS (30 μM), increased by phentolamine (1 μM) or prazosin (1 μM) and not modified by yohimbine (1 μM), whilst at 30°C, the potentiation was reduced by phentolamine, yohimbine or PPADS, and was not modified by prazosin.

  4. The Ca2+-channel blockers, verapamil (10 μM) and NiCl2 (1 mM), abolished the potentiating effects of vasopressin at 37°C whilst verapamil reduced and NiCl2 abolished this potentiation at 30°C. The inhibitor of nitric oxide synthesis, L-NOARG (100 μM), or endothelium removal did not modify the potentiation by vasopressin at 37 and 30°C.

  5. Vasopressin also increased the arterial contraction to the α2-adrenoceptor agonist BHT-920 (10 μM) and to ATP (2 mM) at 30 and 37°C, but it did not modify the contraction to noradrenaline (1 μM) at either temperature.

  6. These results suggest that in cutaneous (ear) arteries, vasopressin potentiaties sympathetic vasoconstriction to a greater extent at 30 than at 37°C by activating vasopressin V1 receptors and Ca2+ channels at both temperatures. At 37°C, the potentiation appears related to activation of the purinoceptor component and, at 30°C, to activation of both purinoceptor and α2-adrenoceptor components of the sympathetic response.

Keywords: Cutaneous arteries, temperature, vasopressin V1 receptors, alpha-adrenoceptor vasoconstriction, purinoceptor vasoconstriction, nitric oxide, endothelium, Ca2+-channels, cooling

Introduction

Vasopressin is known to exert powerful vasoconstrictor activity in a variety of vascular beds (Altura & Altura, 1977). However, a key issue is whether the vasoconstrictor action of vasopressin can be achieved with vasopressin concentrations that are physiologically or pathophysiologically achievable, as many studies of the vascular actions of vasopressin have employed large, unphysiological doses of this hormone (Share, 1988). It has been shown that vasopressin, in addition to its direct vasoconstrictor effect, may enhance the vascular response to exogenous catecholamines at low, subthreshold, doses of this peptide (Bartlestone et al., 1967), although others have not confirmed this phenomenon (Altura & Altura, 1977). Although this subject may be of interest for understanding the role of vasopressin in controlling vascular function, it has received little attention from investigators.

In a previous study (Padilla et al., 1997), we found that vasopressin potentiated the vasoconstrictor response of the rabbit central ear artery, a cutaneous blood vessel, to sympathetic stimulation at 30 but not at 37°C. We therefore suggested that vasopressin may play a role in the regulation of the cutaneous circulation during changes in temperature, by enhancing the sympathetic nerve-induced contraction that occurs during cooling in cutaneous blood vessels. The objective of the present study was to analyse further this potentiating action of vasopressin. Vasopressin receptors have been classified into V1 and V2 subtypes, and in a previous study we have found that the constriction in several types of arteries from the rabbit is mediated by the V1 subtype and may be modulated by endothelial nitric oxide (García-Villalón, 1996). Also, the vasoconstriction to vasopressin may be produced by mobilization of intracellular Ca2+ (McDonald et al., 1994) and/or by facilitating the entry of extracellular Ca2+ through membrane channels (Altura & Altura, 1977). Therefore, in the present study we have analysed the subtype of vasopressin receptor mediating the potentiating effects of vasopressin on sympathetic contraction, and have determined whether this potentiating effect is dependent on Ca2+ channels or is modulated by the endothelium and nitric oxide.

It has been previously reported that sympathetic vasoconstriction in rabbit ear arteries may be mediated by release of noradrenaline and ATP from perivascular nerve terminals (Kennedy et al., 1986). These transmitters then produce constriction by acting on postjunctional α-adrenoceptors and purinoceptors, respectively. In the present study we have also analysed whether the potentiation by vasopressin of the sympathetic contraction is mediated by α-adrenoceptors and purinoceptors.

The present studies have been performed at 37 and 30°C in the rabbit central ear artery, a superficial, cutaneous blood vessel which is involved in thermoregulation (Harker & Vanhoutte, 1988; Patton & Wallace, 1978; Roberts & Zygmunt, 1984).

Methods

Thirty-eight New Zealand White rabbits, weighing 2–2.5 kg, were killed by intravenous injection of sodium pentobarbital (100 mg kg−1). Central ear arteries were dissected free and cut into cylindrical segments 2 mm in length. Each segment was prepared for isometric tension recording in a 6-ml organ bath containing modified Krebs-Henseleit solution with the following composition (millimolar): NaCl, 115; KCl, 4.6; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 11.1. The solution was equilibrated with 95% oxygen and 5% carbon dioxide to give a pH of 7.3–7.4, which was measured with a pH-meter micropH 2001 (Crison Instruments). Briefly, the method consists of passing two fine, stainless steel pins, 150 μm in diameter, through the lumen of the vascular segment. One pin is fixed to the organ bath wall, while the other is connected to a strain gauge for isometric tension recording, thus permitting the application of passive tension in a plane perpendicular to the long axis of the vascular cylinder. The recording system included a Universal Transducing Cell UC3 (Statham Instruments, Inc.), a Statham Microscale Accessory UL5 (Statham Instruments, Inc.) and a Beckman Type RS Recorder (model R-411, Beckman Instruments, Inc.). A previously determined resting passive tension of 0.5 g was applied to the vascular segments, and then they were allowed to equilibrate for 60–90 min before any drug was added. The temperature of the bath was adjusted from the beginning of the experiment at 37 and 30°C, and the arteries remained at the chosen temperature throughout the duration of the experiment.

Electrical field stimulation (1, 2, 4 and 8 Hz, 0.2 ms pulse duration, at a supramaximal voltage of 70 V, during 5 s) was applied to the arteries with two platinum electrodes placed on either side of the artery and connected to a CS-14 stimulator (Cibertec). An interval of at least 5 min was imposed between stimulation periods to allow recovery of the response, and the stimulation trains were repeated until the responses were reproducible over at least 40 min under control conditions. Thereafter, the effect of vasopressin (10 pM, 100 pM and 1 nM) on the arterial response to electrical stimulation was studied by cumulative addition of this peptide to the organ bath. The segments were incubated with each vasopressin concentration for 5 min before electrical stimulation. One stimulation series (1–8 Hz) was applied to the arteries after each dose of the peptide. Each arterial segment was treated with every dose of vasopressin. The effect of vasopressin on the response to electrical stimulation was studied in arterial segments at 37 or 30°C. Each arterial segment was tested at one temperature only.

To investigate the nature of the vasopressin receptor subtypes involved, the effect of vasopressin on the arterial response to electrical stimulation was recorded in the presence of the V1 vasopressin antagonist, d(CH2)5 Tyr(Me)AVP (100 nM). In addition, the effect of the V2 agonist, desmopressin (1 μM) on the response to electrical stimulation was studied.

The relative contribution of the sympathetic neurotransmitters noradrenaline and ATP in the effect of vasopressin on the arterial response to electrical stimulation was studied by performing a series of experiments in the presence of the α1- and α2-adrenoceptor antagonist, phentolamine (1 μM), of the α1-adrenoceptor antagonist, prazosin (1 μM), of the α2-adrenoceptor antagonist, yohimbine (1 μM), of the purinoceptor antagonist, pyridoxalphosphate-6-azophenyl-2,4′-disulphonic acid (PPADS, 30 μM), and of phentolamine (1 μM) plus PPADS (30 μM).

The effect of vasopressin on the response to electrical stimulation was also recorded in the presence of NiCl2 (1 mM) and verapamil (10 μM), which are non-specific (Narahashi et al., 1987) and L-type-specific (McDonald et al., 1994) Ca2+-channel blockers, respectively.

To analyse the role of nitric oxide and the vascular endothelium, the effects of vasopressin on the response of the artery to electrical stimulation at 37 and at 30°C were studied in arteries without endothelium or in arteries with endothelium pretreated with the inhibitor of nitric oxide synthesis L-NG-nitro-arginine (L-NOARG, 100 μM). Endothelium removal was accomplished by gentle rubbing of the vascular lumen with a steel rod, and tested, after finishing the experiment with vasopressin and electrical stimulation, by the abolition of the relaxing response to acetylcholine (10 μM) after precontraction with endothelin-1 (100 nM). This peptide was used to precontract the arteries as in our experimental conditions it produces more stable contractions than other vasoactive drugs such as noradrenaline or serotonin.

Reproducible responses to electrical stimulation (1–8 Hz) were obtained over a period of 40 min. Thereafter, an antagonist was added to the organ bath, and two series of electrical stimulation (1–8 Hz) were applied in the presence of the antagonist. After these series of electrical stimulations, vasopressin (10 pM, 100 pM and 1 nM) was added to the organ bath cumulatively and the response to electrical stimulation was recorded in the arteries in the presence of each concentration of vasopressin plus the antagonist previously applied. Each of the antagonists used was added to the bath 40 min before applying vasopressin. As a control, one vascular segment (each temperature) was treated with vasopressin but not antagonist.

To examine the site of the effect of vasopressin (i.e., pre- or post-junctional) on the arterial response to electrical stimulation, the response of ear arteries to exogenous noradrenaline, ATP or to the α2-adrenoceptor agonist BHT-920 were studied at 37 or 30°C, in the absence (control) and in the presence of vasopressin (10 pM, 100 pM and 1 nM). The response to BHT-920 was always studied in the presence of prazosin (1 μM) to block a possible α1 agonist effect of this agonist. A single submaximal concentration (García-Villalón et al., 1997b) of noradrenaline (1 μM), BHT-920 (10 μM) or ATP (2 mM) was used, and it was applied to the arteries every 15 min, each time followed by washing, until a consistent response was obtained over at least 40 min. After this period, vasopressin, in increasing concentrations (10 pM, 100 pM and 1 nM), was added to the bath, and 15 min thereafter noradrenaline, BHT-920 or ATP were again applied to the arteries, followed by washout and addition of the next vasopressin concentration.

Data are expressed as means±s.e.mean, and were evaluated by analysis of variance (ANOVA) applied to each group of data. To compare the response in the presence and the absence of vasopressin, paired Student's t-test after analysis of variance was applied to the absolute contraction values at each temperature. Then, to compare the effects of vasopressin found at 37 and 30°C, the increments or decrements in the contraction of arteries to sympathetic stimulation were calculated (i.e., the difference between the control response and the response in the presence of vasopressin) and three-way analysis of variance was applied to these data: in this case one factor was stimulation frequency, another factor was vasopressin concentration and another was temperature. To analyse the effects of the antagonists on the potentiation by vasopressin, the increments or decrements in the contraction produced by electrical stimulation were also calculated and analysed by three-way analysis of variance, in which one factor was stimulation frequency, another factor was vasopressin concentration and another presence or absence of the antagonist. A probability value of less than 0.05 was considered significant.

Drugs used were: [Arg8]-vasopressin acetate; [deamino-Cys1, D-Arg8]-vasopressin (desmopressin) acetate; the V1 antagonist (b-Mercapto-b,b-cyclopenta-methylenepropionyl1, O-Me-Tyr2,Arg8)-vasopressin [d(CH2)5Tyr(Me)AVP]; adenosine 5′-triphosphate, disodium salt (ATP); (−)-arterenol, bitartrate salt (noradrenaline); L-NG-nitro-arginine (L-NOARG); nickel chloride hexahydrate (NiCl2); phentolamine hydrochloride; prazosin hydrochloride; verapamil hydrochloride; and yohimbine hydrochloride; all from Sigma; pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS tetrasodium salt) from Tocris Cookson Ltd.; and 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5]-dazepin hydrochloride (BHT-920) was a gift from Europharma S.A.

Results

Response to electrical field stimulation

Electrical stimulation (1–8 Hz) produced frequency-dependent contraction of the vascular segments at 37 and 30°C, but at 30°C the response was significantly lower (P<0.001) than at 37°C, at every frequency of stimulation (Figure 1). For example, the arterial contraction (8 Hz) at 30°C was 0.59±0.06 g (n=32) and at 37°C was 1.6±0.12 g (n=30: P<0.01).

Figure 1.

Figure 1

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in arteries: (a) in the absence (control) and in the presence of vasopressin (10 pM–1 nM); (b) pretreated with the antagonist of vasopressin V1 receptors d(CH2)5 Tyr(Me)AVP (100 nM) in the absence (control) and in the presence of vasopressin (10 pM–1 nM); and (c) in the absence (control) and in the presence of desmopressin (1 μM). Points are means±s.e.mean. * Significantly different (P<0.01) from the control. n=number of animals.

Effects of vasopressin on the response to electrical field stimulation

Control

Vasopressin, at the concentrations used, did not produce contraction, or in some cases produced a contraction that markedly diminished after applying electrical stimulation. If the contraction induced by vasopressin did not return to less than 25% of the maximal response to electrical stimulation, these data were discarded following the protocol used in a previous study (Padilla et al., 1997).

In the presence of vasopressin (10 pM, 100 pM and 1 nM) the contraction to electrical stimulation was greater than that in the absence of the peptide (Figure 1a). At 37°C, the potentiating effect of vasopressin was significant (P<0.01) only at 1 Hz for every vasopressin concentration, at 2 Hz for vasopressin at 10 and 100 pM, and at 4 Hz only for vasopressin at 10 pM. At 30°C, vasopressin increased the contraction to electrical stimulation in a concentration-dependent manner, and this increase was significant (P<0.01) at every stimulation frequency and vasopressin concentration. The increments induced by vasopressin at 30°C were significantly higher (P<0.01) than at 37°C for 4 and 8 Hz frequencies and 100 pM and 1 nM vasopressin concentrations; for 1 Hz it was higher (P<0.05) at 37°C for all vasopressin concentrations (Table 1).

Table 1.

Vasopressin-induced increments in g, of the contraction of rabbit central ear arteries to electrical field stimulation (1 – 8 Hz,0.2 ms pulse duration, 70 V for 5 s) at 37 and 30°C

graphic file with name 126-0702345t1.jpg

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Vasopressin receptor subtypes involved

The antagonist of V1 vasopressin receptors d(CH2)5Tyr(Me)AVP (100 nM) by itself did not modify the contraction to electrical stimulation (data not shown). In the presence of this antagonist, the potentiation of the response to electrical stimulation produced by vasopressin in control conditions was not present either at 37 or 30°C (Figure 1b) (n=7).

The agonist of vasopressin V2 receptors desmopressin did not produce any contraction of rabbit ear artery and did not modify the contraction to electrical stimulation at 37 and 30°C (Figure 1c) (n=7).

Adrenoceptor and purinoceptor blockade

The contraction of the ear artery to electrical stimulation was significantly reduced by application of phentolamine (1 μM, Figure 2a, P<0.01, n=6) or prazosin (1 μM, Figure 2b, P<0.05, n=6), and was significantly increased (P<0.01) by yohimbine (1 μM, Figure 2c, n=6), at both 37 and 30°C. However, application of PPADS (30 μM, n=7) slightly increased the response at 37°C (P<0.01) and did not affect the response at 30°C (Figure 3a). Application of PPADS plus phentolamine markedly reduced (P<0.01, n=7) the contraction to electrical stimulation at both temperatures (Figure 3b).

Figure 2.

Figure 2

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in the absence (control) and in the presence of: (a) phentolamine (1 μM); (b) prazosin (1 μM); and (c) yohimbine (1 μM). Points are means±s.e.mean. *, **, Significantly different from the control (*P<0.05, **P<0.01). n=number of animals.

Figure 3.

Figure 3

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in the absence (control) and in the presence of: (a) PPADS (30 μM); and (b) PPADS (30 μM) plus phentolamine (1 μM). Points are means±s.e.mean. *, **, Significantly different from the control (*P<0.05, **P<0.01). n=number of animals.

At 37°C, the potentiation of the arterial contraction to electrical stimulation by vasopressin was increased (P<0.001) by phentolamine (Figure 4a, n=6) or prazosin (Figure 4b, n=6) but was not modified by yohimbine (Figure 4c, n=6). At this temperature PPADS (P<0.001, Figure 4d, n=7) or PPADS plus phentolamine (P<0.001, Figure 4e, n=7) changed the effect of vasopressin on the contraction to electrical stimulation, as during these two treatments vasopressin reduced the response to electrical stimulation. At 30°C, the potentiation produced by vasopressin was reduced by phentolamine (P<0.05, Figure 4a, n=6), yohimbine (P<0.05, Figure 4c, n=6) or PPADS (P<0.001, Figure 4d, n=7), was abolished by phentolamine plus PPADS (P<0.001, Figure 4e, n=7) and was not modified by prazosin (Figure 4c, n=6).

Figure 4.

Figure 4

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in the absence (control) and in the presence of vasopressin (10 pM–1 nM) in arteries pretreated with: (a) phentolamine (1 μM); (b) prazosin (1 μM); (c) yohimbine (1 μM); (d) PPADS (30 μM); and (e) PPADS (30 μM) plus phentolamine (1 μM). Points are means ±s.e.mean. *, **, Significantly different from the control (*P<0.05, **P<0.01). n=number of animals.

Role Ca2+ channels

The specific blocker of L-type Ca2+ channels verapamil (1 μM) did not modify the response of vascular segments to electrical stimulation at 37 or 30°C (Figure 5a, n=6), whereas the non-specific Ca2+ channel blocker, NiCl2 (1 mM) reduced the arterial response at 37°C (P<0.01, n=6) but not at 30°C (Figure 5b, n=6). At 30°C, the potentiating effect of vasopressin on the contraction of ear arteries to electrical stimulation was reduced by verapamil (P<0.001, n=6) and abolished by NiCl2 (P<0.001, n=6). At 37°C, in the arteries pretreated with verapamil (n=6) or NiCl2 (n=6) vasopressin did not potentiate but reduced (P<0.001) the contraction to electrical stimulation (Figure 6a and b).

Figure 5.

Figure 5

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in arteries under control conditions and in arteries: (a) pretreated with verapamil (10 μM); (b) pretreated with NiCl2 (1 mM); (c) without endothelium; and (d) pretreated with L-NOARG (100 μM). Points are means±s.e.mean. *, **, Significantly different from the control (*P<0.05, **P<0.01). n=number of animals.

Figure 6.

Figure 6

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Contraction of rabbit ear arteries at 37 and 30°C to electrical field stimulation (1–8 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in arteries in the absence (control) and in the presence of vasopressin (10 pM–1 nM): (a) pretreated with verapamil (10 μM); (b) pretreated with NiCl2 (1 mM); (c) without endothelium; and (d) pretreated with L-NOARG (100 μM). Points are means±s.e.mean. *, **, Significantly different from the control (*P<0.05, **P<0.01). n=number of animals.

Nitric oxide synthesis inhibition and endothelium removal

The contraction of rabbit ear artery to electrical stimulation was increased by endothelium removal at 30°C (P<0.01, n=6) but not at 37°C (Figure 5c, n=6), and was increased by pretreatment of the arteries with L-NOARG (100 μM) at both temperatures (P<0.01, Figure 5d, n=6). However, the potentiation by vasopressin was not significantly modified by endothelium removal (n=6) or L-NOARG (n=6) (Figure 6c and d) compared to intact, non treated arteries, at both temperatures.

Effect of vasopressin on the contraction to noradrenaline, BHT-920 and ATP

A submaximal concentration of noradrenaline (1 μM) produced contraction of ear arteries, which was not significantly different (P>0.05) at 37°C (2.78±0.3 g, n=6) and 30°C (2.23±0.19 g, n=6), and this contraction was not modified in the presence of vasopressin, either at 37 or 30°C (Figure 7a, n=6).

Figure 7.

Figure 7

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Contraction of rabbit central ear arteries at 37 and 30°C, in the absence (control) and in the presence of vasopressin (10 pM–1 nM), in response to: (a) noradrenaline (1 μM); (b) BHT-920 (10 μM) in the presence of prazosin (1 μM); and (c) ATP (2 mM). Values are means±s.e.mean. *Significantly different from its control (*P<0.01). n=number of animals.

The selective α2-adrenoceptor agonist, BHT-920 (10 μM) after treatment with prazosin (1 μM), produced a small contraction at 37°C (0.09±0.03 g, n=6) and no observable response at 30°C in control arteries (Figure 7b, n=6). However, after pretreatment with both prazosin (1 μM) and vasopressin, the response to BHT-920 was markedly increased (P<0.01, n=6) at 37°C, and this adrenoceptor agonist produced a clear contraction at 30°C (n=6).

A submaximal concentration of ATP (2 mM) contracted arterial segments at 37°C (0.69±0.07 g, n=6) and 30°C (0.47±0.07 g, n=6). There was not statistically significant difference in the response at these temperatures (P>0.05). The presence of vasopressin in the organ bath increased (P<0.01) the arterial contraction to ATP. The magnitude of this increment was similar at both 37 and 30°C (Figure 7c, n=6).

Discussion

The results of this study indicate that the contractile response of rabbit ear arteries to sympathetic nerve stimulation was reduced at 30°C compared to 37°C and that vasopressin potentiated this response to a greater extent at 30 than at 37°C, for most of the frequencies of electrical stimulation used. These results thus confirm a previous study from our laboratory (Padilla et al., 1997). The main aim of this study was to analyse the mechanisms underlying the potentiating effect of vasopressin on sympathetic vasoconstriction.

Under control conditions, the characteristics of the response of ear arteries to sympathetic stimulation at 37 and at 30°C are qualitatively similar to those reported in previous studies (García-Villalón et al., 1997a,1997b). In those studies we suggested that the reduction in the sympathetic response of these arteries at 30°C may be mainly due to a diminished participation of postjunctional α1-adrenoceptors. Also, we suggested that a purinoceptor component, in addition to the α1-adrenoceptor component, may be present in the sympathetic response of these arteries at 30°C, and that this response is modulated by endothelial nitric oxide release, to a greater extent at 30 than at 37°C. The results of the present study and of one of those studies (García-Villalón et al., 1997b) also suggest that there may be inhibitory prejunctional α2-adrenoceptors and/or purinoceptors in the nerve terminals of the rabbit ear artery since the arterial response to electrical stimulation was increased by purinoceptor inhibition at 37°C, and by yohimbine at both 30 and 37°C. According to previous studies from our and other laboratories, the concentrations of the receptor antagonists or enzyme inhibitors used in the present study are likely to be selective and/or effective: antagonists for vasopressin V1 receptors (100 nM, García-Villalón et al., 1996; Martínez et al., 1994), phentolamine (1 μM, García-Villalón et al., 1997b), prazosin (1 μM, García-Villalón et al., 1997b; Lefebvre & Smits, 1992), yohimbine (1 μM, DeMan et al., 1994; García-Villalón et al., 1997b), PPADS (30 μM, García-Villalón et al., 1997b), verapamil (10 μM, unpublished observations from our laboratory), NiCl2 (1 mM; Hirano et al., 1989) and L-NOARG (100 μM; Padilla et al., 1998).

With regard to the mechanisms underlying the potentiation of the sympathetic contraction of ear arteries by vasopressin, our results suggest that this potentiation is mediated by activation of vasopressin V1 receptors, because this potentiation was abolished by the vasopressin V1 receptor antagonist d(CH2)5 Tyr(Me)AVP, whilst desmopressin, an agonist of vasopressin V2 receptors, did not affect the response of these arteries to sympathetic stimulation. These results agree with those found in mesenteric arteries of humans (Medina et al., 1997) or rats (Noguera et al., 1997) in which vasopressin also potentiated the contraction to adrenoceptor stimulation by activation of vasopressin V1 receptors.

Another aspect of the present work is the role of adrenoceptors and purinoceptors in the potentiation of the sympathetic contraction by vasopressin. In the rabbit ear artery it has been shown previously that the constriction to sympathetic stimulation may be mediated by release of both noradrenaline and ATP from perivascular sympathetic nerve terminals (Kennedy et al., 1986), and these transmitters then act on α-adrenoceptors and purinoceptors, respectively. The present study suggests that, at 37°C, vasopressin did not potentiate the response to α1-adrenoceptor activation. This suggestion is supported by the observations that phentolamine or prazosin increased instead of reduced the potentiating effect of vasopressin on the response to nerve stimulation, and that vasopressin did not modify the arterial contraction to exogenous noradrenaline (this amine contracts rabbit ear arteries by activating mainly α1-adrenoceptors, García-Villalón et al., 1992). On the other hand, at 37°C, the potentiating effect of vasopressin on nerve stimulation was abolished by PPADS, and at the same time, vasopressin increased the contraction to exogenous ATP. This suggests that vasopressin, at this temperature, potentiates the contraction to sympathetic stimulation mainly by facilitating the response to purinoceptor activation. This might explain why the potentiation by vasopressin at 37°C is greater at the lower stimulation frequencies than at the higher ones (Table 1), as the purinergic component of the sympathetic response may be more apparent at lower frequencies of stimulation (Kennedy et al., 1986). The involvement of α2-adrenoceptors in the potentiating effect of vasopressin at 37°C is unclear, since this effect of vasopressin was not modified by yohimbine, but vasopressin did potentiate the contraction to BHT-920.

At 30°C, the mechanisms underlying the vasopressin effect on sympathetic stimulation may differ from those at 37°C. Our data with PPADS and ATP suggest that at 30°C, vasopressin potentiated the purinoceptor component of the sympathetic contraction, and that this potentiation for high stimulation frequencies (4 and 8 Hz) was more marked at 30 than at 37°C. Moreover, our results at 30°C suggest that, in addition to potentiating the purinoceptor component, vasopressin may also potentiate the response to α2-adrenoceptor activation. This is suggested by the observation at 30°C that the potentiation of the sympathetic response by vasopressin was reduced by phentolamine and yohimbine but not by prazosin, and that the potentiating effect of vasopressin still present after PPADS treatment was abolished by phentolamine. This is in line with the results of Guc et al. (1992) who observed, in pithed rats, that vasopressin increased the pressor effects of noradrenaline, but not that due to selective stimulation of α1-adrenoceptors.

Regarding the role of Ca2+ channels, our results at 37 and 30°C suggest that the potentiation of the vasoconstriction to sympathetic stimulation by vasopressin is mediated by entry of extracellular Ca2+, at least in part through Ca2+ channels of the L-type, because the vasopressin effect was reduced by the Ca2+ channels blockers verapamil and NiCl2, at both temperatures. Results reported in the literature in this respect show that the potentiating effect of vasopressin on the contraction to adrenoceptor stimulation was mediated by L-type Ca2+ channels in rat (Medina et al., 1997) but not in human (Noguera et al., 1997) mesenteric arteries, suggesting that species differences may be involved. Our results also suggest that T-type Ca2+ channels might be also involved in the potentiating effect of vasopressin at 30°C, since at this temperature NiCl2 was more effective than verapamil in inhibiting the potentiation by vasopressin. To clarify this question, however, antagonists more specific for this subtype of Ca2+ channels should be used.

In relation to the role of the vascular endothelium and nitric oxide, we have found in a previous study (García-Villalón et al., 1996) that the modulatory role of nitric oxide in the effect of vasopressin on rabbit arteries varies widely between vascular beds, and that this modulatory role of nitric oxide was particularly small in ear arteries. In line with this, the present results at 37 and 30°C suggest that the role of the endothelium and nitric oxide may be of relatively little importance for the potentiating effect of vasopressin on the sympathetic contraction of ear arteries, as this potentiation was not modified by endothelium removal nor by nitric oxide synthase inhibition, at both temperatures. However, nitric oxide may modulate, at both temperatures, the response of ear arteries to sympathetic stimulation, since nitric oxide synthase inhibition in the absence of vasopressin did increase the response to electrical stimulation. Thus, it is suggested that, at 37 and 30°C, nitric oxide release may be stimulated by sympathetic stimulation to a similar extent in the absence and in the presence of vasopressin.

Therefore, it may be suggested that vasopressin potentiates the contraction of cutaneous (ear) arteries to sympathetic stimulation, to a greater extent at 30 than at 37°C. At both temperatures, this potentiating effect of vasopressin may be mediated by activation of vasopressin V1 receptors and Ca2+ channels, and it may be independent of endothelial nitric oxide. The increased potentiating effect of vasopressin at 30°C may not be due to cooling-induced changes of vasopressin receptor subtype, nor to changes of Ca2+ channels or endothelial nitric oxide effects. It may be hypothesized, instead, that cooling (30°C) changes the facilitating action of vasopressin on the receptors that mediate the sympathetic response of cutaneous arteries, so at normal temperature (37°C) vasopressin would potentiate mainly purinoceptor effects whereas during cooling (30°C) it would potentiate both purinoceptor and α2-adrenoceptor effects.

Acknowledgments

The authors are grateful to Mrs M.E. Martínez and H. Fernández-Lomana for technical assistance. This work was supported, in part, by FIS ((6/0474), DGICYT (PM 95/0032), and CAM (AE 263/95).

Abbreviations

BHT-920

5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5]-dazepin hydrochloride

d(CH2)5 Tyr(Me)AVP

b-Mercapto-b,b-cyclopenta-methylenepropionyl1,O-Me-Tyr2,Arg8)-vasopressin

L-NOARG

L-NG-nitro-arginine

PPADS

pyridoxalphosphate-6-azophenyl-2-4′-disulphonic acid

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