Pflügers Arch - Eur J Physiol (2000) 440:548–555 Digital Object Identifier (DOI) 10.1007/s004240000323
O R I G I N A L A RT I C L E
A.L. García-Villalón · J. Padilla · N. Fernández L. Monge · M.A. Martínez · B. Gómez · G. Diéguez
Effect of neuropeptide Y on the sympathetic contraction of the rabbit central ear artery during cooling Received: 22 February 2000 / Accepted: 29 March 2000 / Published online: 20 May 2000 © Springer-Verlag 2000
Abstract In order to analyse the effect of neuropeptide Y (NPY) on the cutaneous vascular response to sympathetic nerve stimulation during cooling, the isometric response of isolated 2-mm segments of the rabbit central ear (cutaneous) artery was recorded at 37°C and during cooling (30°C). Electrical field stimulation (4–16 Hz) at 37°C produced a frequency-dependent contraction, which was reduced during cooling (45% for 16 Hz) and potentiated by NPY (10–8, 3×10–8 and 10–7 M), this potentiation being greater at 30°C than at 37°C. The NPY-induced potentiation of the contraction elicited by electrical field stimulation (8 Hz) was abolished by an antagonist of Y1 subtype NPY receptors, BIBP3226 (10–6 M), at 37°C and 30°C, reduced by phentolamine (10–6 M) at 30°C but not at 37°C, was not modified by the purinoceptor antagonist PPADS (3×10–5 M) and was reduced by application of both phentolamine and PPADS at both temperatures. Both NiCl2 (10–3 M) and verapamil (10–5 M) abolished the potentiating effect of NPY at 37°C and reduced it at 30°C. Neither application of an inhibitor of nitric oxide synthesis, L-Nω-nitro-arginine (L-NOARG, 10–4 M), nor endothelium removal modified the potentiating effect of NPY at 37°C or 30°C. NPY (10–8, 3×10–8 and 10–7 M) potentiated in a concentration-dependent way the arterial contraction in response to exogenous noradrenaline (10–8–10–4 M) at 30°C but not at 37°C, and it increased the response to ATP (10–4–10–2 M) at both temperatures. Therefore, in cutaneous (ear) arteries: (1) NPY potentiates the sympathetic response at 37°C and at 30°C, (2) this potentiating effect of NPY was more marked at 30°C than at 37°C, probably because of greater potentiation of A.L. García-Villalón · N. Fernández · L. Monge · M.A. Martínez B. Gómez · G. Diéguez (✉) Departamento de Fisiología, Facultad de Medicina, Universidad Autónoma, Arzobispo Morcillo 2, 28029 Madrid, Spain e-mail:
[email protected] Tel.: +34-1-3975490, Fax: +34-1-3975324 J. Padilla Departamento de Biología, Universidad del Atlántico, Carrera 43, 50–53, Barranquilla, Colombia
the α-adrenoceptor response during cooling, and (3) the potentiating effect of NPY at both temperatures is mediated by NPY receptors of the Y1 subtype, is dependent of Ca2+ channels and is independent of the release of endothelial nitric oxide. Key words Alpha-adrenoceptor vasoconstriction · Cutaneous arteries · Nitric oxide · Purinoceptor vasoconstriction · Y1 receptors
Introduction Neuropeptide Y (NPY) is a 36-amino-acid peptide that is stored in perivascular sympathetic nerve endings [10, 23], and is released on nerve stimulation [21]. This peptide may be involved in vascular regulatory mechanisms: it may constrict some types of arteries [34], and may modulate the sympathetic response, by potentiating the contractile response to exogenous noradrenaline [10] and by inhibiting noradrenaline release from perivascular nerve terminals after nerve stimulation [22, 26]. Its direct vasoconstrictor action seems to be mediated by NPY receptors of the Y1 subtype, and its inhibitory effect on noradrenaline release may be mediated by NPY receptors of the Y2 subtype [36]. In addition, the potentiating effect of NPY on adrenergic contraction may be mediated, at least in part, by the entry of extracellular Ca2+ through dihydropyridine-sensitive [2] or through both dihydropyridine-sensitive and -insensitive [5] Ca2+ channels. Conflicting results have been reported [3, 6] regarding the role of the endothelium in the potentiating effect of NPY on adrenergic contraction. To our knowledge, the role of NPY in the adrenoceptor mechanisms involved in the regulation of the cutaneous circulation during cooling has been not explored. This issue is interesting because the cutaneous circulation is mainly regulated by sympathetic adrenoceptor mechanisms and is involved in body temperature regulation [19]. In a previous study from our laboratory, using the rabbit central ear artery as a model of cutaneous arte-
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ries, we found that NPY potentiates the in vitro response to electrical stimulation to a greater extent during moderate cooling (30°C) than at normal temperatures [24]. Some cutaneous arteries, such as the rabbit central ear artery [20, 28] and the rat tail artery [32], have a dense sympathetic innervation and these nerves contain noradrenaline and ATP as neurotransmitters, together with a relatively large quantity of NPY. Endogenous NPY may contribute to the contraction of the rabbit ear artery to electrical field stimulation [7]. Also, plasma levels of NPY in the rat may be increased by exposure to the cold [37]. These observations suggest that this peptide may be significant in the regulation of the cutaneous circulation during cooling. The wall of the ear artery may be exposed physiologically to temperatures lower than 37°C, because of countercurrent transfer of heat between the arterial and venous blood [29], as may also occur in the arteries of human [27] or sloth [30] limbs. The aim of this study was to analyse the mechanisms involved in the potentiating action of NPY on the response of cutaneous arteries to sympathetic stimulation, specially during cooling. We used the central ear artery from rabbits as an example of a cutaneous artery, and we examined the role of the subtypes of NPY receptors, the α-adrenoceptors, purinoceptors, endothelial nitric oxide, and Ca2+ channels in the response of this artery during cooling to sympathetic nervous stimulation.
Materials and methods Forty-five male New Zealand White rabbits, weighing 2–2.5 kg, were killed by intravenous injection of sodium pentobarbital, 100 mg/kg. 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), a Statham Microscale Accessory UL5 (Statham Instruments) and a Beckman Type RS Recorder (model R-411, Beckman Instruments). 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 to 37°C or 30°C (cooling), and the arteries remained at the chosen temperature for the duration of the experiment. This temperature of 30°C was chosen to study the effects of cooling, because previous studies have shown that it produces clear differences in the vascular response compared with 37°C [24], and it is a temperature that may occur in the skin of the rabbit ear in physiological conditions [11]. Electrical field stimulation (4–16 Hz, 0.2 ms pulse duration, at a supramaximal voltage of 70 V, for 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 in every case until the responses were reproducible over at least 40 min under control conditions. Thereafter, the effects of NPY (10–8, 3×10–8 and 10–7 M) on the arterial response to electrical stimulation were studied by cumulative addition of this peptide to the organ bath. Electrical field stimulation (4–16 Hz) was applied 20 min after adding each concentration of the peptide, and each arterial segment was treated with the three concentrations of NPY. The effect of this peptide on the response to electrical stimulation was studied in the arterial segments at 37°C or 30°C, each arterial segment being tested at one temperature only. In each experiment, a segment that was electrically stimulated but not treated with NPY was used as a time control. To analyse whether there was desensitization of NPY receptors when cumulative concentration–response curves to this peptide were constructed, in a group of experiments the effects of non-cumulative concentrations of NPY were studied. In this protocol, only one NPY concentration (10–8, 3×10–8 or 10–7 M) was applied to each vascular segment, and the response to electrical stimulation (4–16 Hz) was studied before and after addition of this concentration to the bath. To analyse the mechanisms underlying NPY’s effects on the response to electrical stimulation, the rest of the experiments were performed using the intermediate stimulation frequency of 8 Hz, which showed both a clear contraction to electrical stimulation in control conditions, and a marked potentiation by NPY. To investigate the nature of the NPY receptor subtypes involved, the effects of NPY on the contractions evoked by electrical stimulation (8 Hz) were studied in the absence and in the presence of the Y1 receptor subtype antagonist BIBP3226 (10–6 M). This antagonist concentration may be specific for this subtype of receptors, as it produces a marked blockade of Y1 receptors, without affecting Y2, Y3 or Y4 subtypes [8]. The relative contributions of the sympathetic neurotransmitters noradrenaline and ATP to the effects of NPY on the arterial response to electrical stimulation (8 Hz) were examined by performing a series of experiments in the presence of the α-adrenoceptor antagonist phentolamine (10–6 M), of the purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2,4′-disulphonic acid (PPADS, 3×10–5 M), and of phentolamine (10–6 M) plus PPADS (3×10–5 M). The effects of each of these antagonists in the arterial segments at 37°C and 30°C were recorded. To examine the role of Ca2+ channels, the effect of NPY on the response to electrical stimulation (8 Hz) at 37°C and 30°C was also recorded in the presence of verapamil (10–5 M) and NiCl2 (10–3 M), which are L-type-specific and non-specific Ca2+ channel blockers, respectively. To analyse the role of nitric oxide and the vascular endothelium, we studied the effect of NPY on the response to electrical stimulation (8 Hz) at 37°C and 30°C in arteries pretreated with the inhibitor of nitric oxide synthesis L-Nω-nitro-arginine (L-NOARG, 10–4 M), and in arteries deprived of endothelium. Endothelium removal was accomplished by gently rubbing the vascular lumen with a steel rod, and functionally tested at the end of the experiments by checking the ability of acetylcholine (10–5 M) to induce a relaxation after precontraction with endothelin-1 (10–8 M). Reproducible responses to electrical stimulation (8 Hz) were obtained over a period of 40 min. Thereafter, an antagonist was added to the organ bath, and 20 min after this electrical stimulation was again applied for 40 min. After this electrical stimulation in the presence of each antagonist, NPY (10–8–10–7 M) was added to the organ bath cumulatively, and the response to electrical stimulation in the arteries was again recorded in the presence of each concentration of NPY plus the antagonist previously applied. As a control, one vascular segment (at each temperature) was treated with NPY but not antagonists. To examine the level at which NPY acts in the perivascular sympathetic terminals, i.e. pre- or post-junctionally, the responses of ear arteries to exogenous noradrenaline and to ATP were studied at 37°C and 30°C, in the absence (control) and in the presence of NPY (10–8–10–7 M). Cumulative concentration–response curves to noradrenaline (10–8–10–4 M) and ATP (10–4–10–2 M) were recorded from the segments treated with 10–8, 3×10–8 or 10–7 M
550 NPY. The peptide was added to the bath 20 min before beginning the test with noradrenaline or ATP, each segment was treated with only one concentration of NPY, and one segment which was not treated with NPY was used as control. Data are expressed as mean ±SEM, 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 NPY, paired Student’s t-test after ANOVA was applied to the absolute contraction values at each temperature. Then, to compare the effects of NPY found at 37°C and 30°C, the increments 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 NPY) and two-way ANOVA was applied to these data: in this case one factor was NPY concentration and the other was temperature. To analyse the effects of each antagonist on the potentiation by NPY, the increments or decrements in the contraction produced by electrical stimulation were also calculated and analysed by two-way ANOVA, in which one factor was NPY concentration and the other was the presence or absence of the antagonist. A probability value of less than 0.05 was considered significant. To analyse the concentration–response curves for noradrenaline and ATP, EC50 values were calculated as the concentration producing 50% of the maximal effect by geometric interpolation, and the results are expressed as the pD2 (–log EC50). Drugs used were: NPY (porcine); adenosine 5′-triphosphate, disodium salt (ATP); (–)arterenol, bitartrate salt (noradrenaline); L-Nω-nitro-arginine (L-NOARG); nickel chloride hexahydrate (NiCl2); phentolamine hydrochloride; and verapamil hydrochloride; all from Sigma; and pyridoxalphosphate-6-azophenyl-2′,4′disulphonic acid (PPADS tetrasodium salt) from Tocris Cookson; {(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl) methyl]-D-arginine amide} (BIBP3226) courtesy of Dr. Karl Thomae, Biberach, Germany.
Fig. 1 Contraction of rabbit central ear arteries evoked by electrical field stimulation (4–16 Hz, 0.2 ms pulse duration, 70 V, for 5 s) in the absence (control) and in the presence of NPY (10–8, 3×10–8, 10–7 M) at 37°C (a) and at 30°C (b). Points are means ±SEM. Statistically significant difference compared to control (*P