Cooling effects on nitric oxide production by rabbit ear and femoral arteries during cholinergic stimulation

Transcription

1 Br. J. Pharmacol. (1994), 113, '." Macmillan Press Ltd, 1994 Cooling effects on nitric oxide production by rabbit ear and femoral arteries during cholinergic stimulation N. Fernandez, L. Monge, A.L. Garcia-Villalon, J.L. Garcia, B. Gomez & 'G. Dieguez Departamento de Fisiologia, Facultad de Medicina, Universidad Aut6noma, Arzobispo Morcillo 1, 2829 Madrid, Spain 1 Ear (cutaneous) and femoral (deep) arteries from rabbit were perfused at 37 C and 24 C (cooling) and the production of nitrite, as an index of nitric oxide production, was measured under basal conditions and cholinergic stimulation. 2 In both types of arteries under control conditions, the basal production of nitrite was similar at 24 C and 37 C. Compared with the control conditions, the basal production of nitrite was significantly lower in ear and femoral arteries without endothelium or treated with N-nitro-L-arginine methyl ester (L-NAME, 1-4 M) but it was similar in those treated with atropine (1-6 M). 3 At 37 C, methacholine (1-'-1- M) increased the production of nitrite in ear and femoral arteries; this increase persisted during 3-6 min and was practically abolished by L-NAME (1-4M), atropine (1-6 M), or removal of the endothelium. In ear arteries the total nitrite production to activation with methacholine was higher at 24 C than at 37 C due to this production persisted increased for a longer period (>15 min), whereas in femoral arteries it was lower at 24 C than at 37C. 4 It is suggested that: (a) the endothelium of rabbit ear and femoral arteries produce nitric oxide under basal conditions, which is increased by cholinergic stimulation, and (b) cooling potentiates endothelial nitric oxide production to cholinergic stimulation in cutaneous arteries, whereas it inhibits this production in deep arteries. Keywords: Skin arteries; temperature; endothelium; nitrite; femoral arteries; cholinergic stimulation; NO Introduction The main function of the cutaneous circulation is body thermoregulatory function, and it appears that cooling affects reactivity of cutaneous vessels to vasoactive stimuli (Vanhoutte & Flavahan, 1986). Also it becomes apparent that nitric oxide is a relaxant factor that produces a basal vasodilator tone and is involved in the response of different vascular beds to vasoactive stimuli (Moncada et al., 1991). Nitric oxide is synthesized in the endothelium from L- arginine and this synthesis can be stimulated by several types of stimuli, including cholinoceptor agonists, and is inhibited by L-arginine analogues (Moncada et al., 1991). In rabbit ear artery, considered as a cutaneous blood vessel (Patton & Wallace, 1978; Roberts & Zygmunt, 1984; Harker & Vanhoutte, 1988), we have recently observed that cooling increases the relaxation to cholinergic stimulation (Monge et al., 1993) and reduces the contraction to endothelin-l (Monge et al., 1991) and to adrenergic activation (Garcia- Villalon et al., 1992) probably by endothelium-dependent mechanisms. In these three studies (Monge et al., 1991; 1993; Garcia-Villalon et al., 1992) we suggested that these effects of cooling are specific for cutaneous vessels since they were not present in the femoral artery (a deep vessel), and that they might be related to an increased release of endothelial nitric oxide. The present study was performed to determine the effects of cooling on nitric oxide production by cutaneous and non cutaneous arteries measuring the production of nitrite in rabbit central ear and femoral arteries at 37 C and 24 C (cooling) under basal conditions and after cholinergic stimulation. Currently, there is not a completely satisfactory technique to measure the production of nitric oxide by vessels, mainly due to the very short life of this radical. Nitric oxide is quickly oxidized to form nitrite (Marletta et al., 1988) and determination of nitrite production has been used as an indirect measurement of nitric oxide production (Ignarro et al., 199; Bereta et al., 1992). Author for correspondence. Methods Forty-eight New Zealand White rabbits either sex, weighing kg, were killed by i.v. injection of sodium pentobarbitone (Sigma, 1 mg kg-'). The two central ear and the two femoral arteries were dissected free and each artery was cut to a length of 6 cm (ear arteries) or 4 cm (femoral arteries). The two ear and the two femoral arteries were then cannulated with a polyethylene tubing and each pair of arteries was placed inside a small chamber and perfused in series with Krebs-Henseleit solution (composition mm: NaCl 115, KCl 4.6, KH2PO4 1.2, MgSO4 1.2 CaCI2 2.5, NaHCO3 25, glucose 11.1 equilibrated with 95% oxygen and 5% CO2 to give a ph of ). The total volume of the perfusion system and perfusion chamber was.2 ml. The perfusion was carried out with a peristaltic pump (Minipuls 3, Gilson Medical), and the chamber containing each pair of arteries was placed inside an organ bath at 37C or 24 C (cooling). First, the arteries were washed with the Krebs-Henseleit solution during 15 min at a rate of.5 ml min-' and then they were continuously perfused at a rate of 2 ml h-'. After an equilibration period of 3 min, 1 ml-samples of the perfusate were collected every 3 min in separate assay tubes kept on ice. Three samples were collected under basal conditions and another five samples after adding methacholine ( M) to the perfusion solution; the first sample after methwholine was taken 3 min after adding this drug. The effects of methacholine ( s M) on nitrite production were determined in intact ear and femoral arteries at 37 C and 24 C. Also, the effects of methacholine (1-5 M) were determined at 37 C in ear and femoral arteries without endothelium, or treated with the inhibitor of nitric oxide synthase N-nitro-L-arginine methyl ester (L-NAME, 1-4 M) (Moore et al., 199; Rees et al., 199) or with atropine (1-6 M). Endothelium removal was accomplished by rubbing the vessel lumen with a roughened steel rod. L-NAME or atropine were added to the medium at the beginning of perfusion, and maintained throughout the experiment. To test if basal nitrite release changed throughout the experimental period, three preparations at 37 C and 24 C were

2 COOLING AND NITRIC OXIDE 551 used to measure nitrite production during 4 h in intact, nontreated ear and femoral arteries. Nitrite content of each sample was assayed in duplicate by the Griess reaction (Rider & Melon, 1946). Briefly, two.45 ml-aliquots were taken from the perfusate sample and each of them was incubated with 5 jtl of Griess reagent at room temperature for 15 min (final concentration:.5% sulphanilamide/.5% naphthylenediamine dihydrochloride (Merck) in 2% H3P4). The peak absorbance at 54 nm was measured with a Schimadzu u.v.-16 spectrophotometer using a reference cuvette containing Griess reagent and identical Krebs-Henseleit solution to that used for the arterial perfusion. Nitrite concentration was determined with dilutions of sodium nitrite in Krebs-Henseleit solution. Background nitrite levels were determined in Krebs-Henseleit solution passing through a polyethylene tube exposed to identical experimental conditions except contact with arteries, and subtracted from the values determined in the experimental preparation. To determine whether our experimental procedure alters the functional capability of the arteries, we performed a few experiments where we measured the level of oxygenation in the perfusate during 4 h of perfusion and recorded the in vitro reactivity of arteries after this perfusion. After ear and femoral arteries were mounted in the perfusion system we collected samples of the perfusate after passing through the arteries every 3 min during 4 h and measured gases content and ph by standard electrometric methods (Radiometer ABL 3, Copenhagen, Denmark). This was made in four preparations of ear and femoral arteries at 37 C (two preparations under control and two preparations treated with 1-6 M methacholine), and in another two preparations of ear and femoral arteries at 24 C. After this test, these same arteries were removed from the perfusion system, cut in segments 2 mm long and mounted in a set up for isometric tension recording at 37C as described elsewhere (Monge et al., 1993). In this preparation, the contraction of the arteries under basal conditions to potassium choride (5 mm), and the relaxation to methacholine ( M) and to sodium nitroprusside (I-6I- I-M) after precontraction with endothelin-l ( M) were recorded. Data analysis The production of nitrite by each pair of arteries (two ear and two femoral arteries) obtained in each sample was expressed in pmol 3 min-'. The effects of methacholine on nitrite production under the different conditions were evaluated as the peak increment (maximal level obtained after stimulation over the basal production) and as the total increment (the total amount of nitrite over the basal production obtained in the five samples recorded after stimulation). The basal production was considered as the average of the nitrite content in the three samples obtained before applying methacholine. To compare the effects of methacholine in arteries at 37TC and 24TC, and in arteries with and without endothelium or treated with L-NAME or atropine at 37C, an analysis of variance followed by the Dunnet's test was applied to the data. In each case, P<.5 was considered statistically significant. Drugs used were: methacholine chloride, N-nitro-L-arginine methyl ester hydrochloride (L-NAME) and atropine sulphate, all from Sigma. All drugs were dissolved in isotonic saline (NaCl.9%) with ascorbic acid 1% M to prevent oxidation of the drugs. Results At 37C, the amount of nitrite released under basal conditions (three samples during 9 min) averaged 46 ± 2 pmol 3 min-i (ear arteries, n = 16) and 52 ± 2 pmol 3 min-' (femoral arteries, n = 16). In three control preparations of ear and femoral arteries the spontaneous release of nitrite diminished slowly throughout a period of 4 h (these results are not shown, but the time course of this production was a 151 Methacholine 1-7 M Methacholine 1-6 M Methacholine 1-5 M 11 C E Ẹ _ C 6). z 5 i b 15] 1l 5- I Methacholine 1O7 M OJ Time (min of perfusion) Figure 1 Production of nitrite by rabbit central ear (a) and femoral (b) arteries before and during stimulation with methacholine (1-7-1O- M) at 37 C (@) and at 24 C (). Values are means ± s.e. Statistically significant difference between 37 C and 24 C; *P<.5; **P<..

3 552 N. FERNANDEZ et al. similar to that exhibited by the arteries that were treated with 1- M methacholine, Figure 1). Methacholine (l-7- I-I M), added to the perfusion solution, provoked a concentration-dependent, transient increase of nitrite production over the basal production that was comparable in ear and femoral arteries at 37C; this increment for 1 6 and I-5 M methacholine was evident during 3-6 min (Figure 1 and Table 1). In the presence of atropine (1-6 M) the basal production of nitrite in ear (n = 3) and femoral (n = 3) arteries was not significantly different from that obtained in non-treated arteries, but the increase in nitrite production induced by 1-i M methacholine was reduced by 9% (Figure 2). In the presence of L-NAME (1-4 M) the levels of basal production of nitrite in both types of arteries were about 4% of those found in non-treated arteries, and the increase in nitrite production caused by 1-5 M methacholine in ear (n = 5) and femoral (n = 5) arteries was also reduced by about 9% (Figure 2). Both ear (n = 3) and femoral (n = 3) arteries without endothelium released an amount of nitrite that was not significantly different from background levels, both under basal conditions and after addition of 1-5 M methacholine. At 24 C (cooling) the basal production of nitrite was comparable to that at 37C, both in ear (46 ± 2 vs 46 ± 2 pmol 3min-', n= 12, P>.5) and femoral (48± 3 vs 52±2 pmol 3 min-', n = 12, P>.5) arteries. In ear arteries during cooling, methacholine (lo M) also increased nitrite production over the basal release in a concentrationdependent way, but the total increment was consistently higher than at 37 C (Figure 1 and Table 1). Although in these arteries the peak increment of nitrite production at 24'C was similar to that at 37 C, the increment was more prolonged (> 15 min) (Figure 1) so that the total increment of nitrite production induced by methacholine (1-'- 1' M) was higher at 24 C. On the contrary, in femoral arteries at 24 C, methacholine ( M) did not modify significantly the release of nitrite with regard to basal production, so that the effect of this drug in these particular arteries was lower at 24 C than at 37 C (Figure 1 and Table 1). In our experimental conditions the values for P2, Pco2 and ph measured in the perfusate collected after passing through ear and femoral arteries were similar at 37 and 24 C, both in the arteries treated or non treated with 1-6 M methacholine. These values for P2, Pco2 and ph in ear arteries averaged 188 ± 7 mmhg, 38 ± 4 mmhg and 7.32 ±.1 mmhg, respectively, and in femoral arteries they averaged 199 ± 7 mmhg, 39 ± 4 and 7.3 ±.9, respectively, throughout the perfusion period. After this test, the arteries were removed from the perfusion system and cut in segments 2 mm in length for isometric tension recording at 37C. In these arteries (eight segments) under basal conditions, potassium chloride (5 mm) produced contraction of ear (1.86 ±.16 g) and of femoral arteries (3.43 ±.43 g). After contraction with endothelin-l the arteries (eight segments) exhibited concentration-dependent relaxation to methacholine ( M, the maximal relaxation with regard to the active tone was 71 ± 5% for ear arteries and 64 ± 7% for femoral arteries) and to sodium nitroprusside ( M, the maximal relaxation was 74 ± 4% for ear arteries and 73 ± 6% for femoral arteries). No major differences were observed between the contractcion or relaxation responses of ear and femoral arteries that had been perfused at 37 C or 24 C, or that had been treated or not treated with 1-6 M methacholine during perfusion. Table 1 Total increases in nitrite production (pmol) in rabbit methacholine at 37 C and 24C (cooling) Methacholine 1-' (M) 37C 5 ± 3 (6) 72 ± 14 (5) 13 ± 21 (5) Ear artery ear and femoral arteries during 15 min of stimulation with 24eC 2± 2 (3) 147 ± 2* (4) 35 ± 27** (5) 37 C 13±6 (6) 79 ± 16 (5) 118± 19 (5) Femoral artery 24 C 3 ± 4 (3) 21 ± 12* (4) 25 ± 19** (5) In parentheses, number of experiments. Values are means ± s.e. Statistically significant difference between 37'C and 24'C; *P<.5; **P<.1. a 151 Methacholine 1-5 M b 1 Methacholine 1-5 M E cż -c r-.. z 1-5- * +s**f f p p + :q* Time (min of perfusion) Figure 2 Production of nitrite by rabbit central ear (a) and femoral (b) arteries before and during stimulation with 1-5 M methacholine at 37 C in control conditions (-) and in the presence of 1-4 M Nly-nitro-L-arginine methyl ester () or 1-' M atropine(o). Values are means ± s.e. Statistically significant difference between the control and treatment with NG-nitro-L-arginine methyl ester or atropine; *P<.5; **P<.1.

4 COOLING AND NITRIC OXIDE 553 Discussion In this study we have evaluated the effects of cooling on the production of nitric oxide in cutaneous and non-cutaneous arteries under basal conditions and when they were stimulated with methacholine. Nitric oxide production was evaluated indirectly by measuring nitrite content in the perfusate of rabbit ear (cutaneous) and femoral (non cutaneous) arteries exposed to 37 C and moderate cooling (24 C). Nitrite production seems to increase when the release of nitric oxide is augmented in perfused arteries (Ignarro et al., 1987), cultured endothelial cells (Bereta et al., 1992) and whole organs (Ignarro et al., 199). Also, nitrite production can be blocked by inhibitors of the enzyme, nitric oxide synthase (Ignarro et al., 1991; Schultz et al., 1991), suggesting that the nitrite measured in these preparations is mostly produced from nitric oxide metabolism, and therefore measurements of nitrite production can be taken as an index of the nitric oxide release from vessels. In the present study we measured the levels of P2, Pco2 and ph in the perfusate during the period of arterial perfusion, and after this period we also evaluated the reactivity of the perfused arteries. The results of these experiments suggest that the experimental procedure used in our study provides a good oxygenation of the arteries and mostly preserves the functional capability of the arteries to contract and to relax. The contraction to potassium chloride as well as the relaxation to cholinergic stimulation and to sodium nitroprusside of the arteries after perfusion was comparable to that determined for the same type of arteries in a previous study from our laboratory (Monge et al., 1993). Both ear and femoral arteries under basal conditions produced nitrite, and this production was lower in the arteries without endothelium or treated with L-NAME. The arteries treated with atropine, however, exhibited a basal nitrite production similar to that found in non treated arteries. These results suggest that the endothelium of these arteries releases nitric oxide, which might be independent of muscarinic receptors under basal conditions. The basal release of nitrite diminished slowly throughout the experimental period and this was similar in ear and femoral arteries at both 37 C and 24'C. The reason for this feature is unclear. A possible deterioration of the preparations during the perfusion could be reasonably excluded as the experimental conditions do not seem to alter the functional capability of the arteries as indicated above. The cholinoceptor agonist, methacholine, increased the production of nitrite in ear and femoral arteries over the basal release in a concentration-dependent way, and it was blocked by L-NAME, suggesting that the increase in nitrite production by methacholine is mostly due to an increment in the production of nitric oxide. In addition, the results with methacholine in arteries without endothelium or treated with atropine suggest that the increase in nitric oxide production induced by this drug takes place in the endothelium and is mostly caused by activation of endothelial muscarinic receptors. This feature agrees with the idea generally accepted that cholinoceptor stimulation increases the release of nitric oxide by the endothelium (Moncada et al., 1991). At 37 C, in ear and femoral arteries stimulated with methacholine the production of nitrite reached a maximal peak followed by a decrease, returning to basal levels 9 min after stimulation, in spite of the fact that arteries were still exposed to methacholine. Although it is accepted that cooling specifically alters the response of cutaneous arteries to some types of vasoactive stimuli (Vanhoutte & Flavahan, 1986), very little is known about the role played by the endothelium in vascular reactivity at low temperature (Karaki & Nagase, 1987; Bodelsson et al., 1989). This could be of particular interest in cutaneous vessels as they have a thermoregulatory function. We have recently reported that cooling reduces the contraction to endothelin-i (Monge et al., 1991) or adrenergic activation (Garcia-Villalon et al., 1992), and at the same time it increases the relaxation to cholinergic stimulation (Monge et al., 1993) in ear (cutaneous) but not in femoral (deep) arteries from rabbits. In these studies we suggested that these effects of cooling on the contraction and relaxation are specific for cutaneous vessels and hypothesized that they could be related to an increased release of endothelial nitric oxide in the stimulated arteries during cooling (Monge et al., 1991; 1993; Garcia-Villal6n et al., 1992) The results found in the present study are consistent with the hypothesis previously proposed (Monge et al., 1993) in the sense that in ear arteries, but not in femoral arteries the release of endothelial nitric oxide to cholinoceptor stimulation is potentiated during cooling. In ear arteries methacholine produced a peak increment in nitrite production that was similar at 37C and 24 C, but the increment in this production consistently persisted for a longer period at 24 C than at 37C. Therefore, although the peak increment in nitrite production was comparable at both temperatures, the total amount of nitrite, and consequently that of nitric oxide, released by ear arteries during activation with methacholine was higher at 24 C than at 37C. In femoral arteries methacholine did not mostly affect the basal release of nitrite during cooling, thus the effects of cholinoceptor stimulation on release of nitric oxide in these particular arteries is lower at 24 C than at 37 C, which clearly contrasts with that found in ear arteries. The facilitated production of nitrite (and consequently-of nitric oxide) in cutaneous arteries to methacholine at 24 C could be related to a more prolonged effectiveness of cholinoceptor stimulation and/or to an increased function of metabolic pathway for nitric oxide synthesis in these arteries during cooling. Cooling might augment the sensitivity of endothelial cholinoceptors to cholinoceptor agonists or the postreceptorial mechanisms involved in nitric oxide synthesis in cutaneous arteries. Although these results are consistent with the increased cholinoceptor relaxation of ear arteries observed during cooling (Monge et al., 1993), the comparison of the present results with those in the arterial relaxant response must be cautious, because the effects of cooling on the nitrite production by ear arteries when they were activated with methacholine required a relatively long period to be observed, whereas the time-course of the vascular relaxant response to cholinoceptor stimulation is much shorter. With regard to femoral arteries, our study shows that the production of nitrite in these arteries after cholinoceptor stimulation was reduced by cooling. This observation apparently does not correlate well with previous experiments where we found that the in vitro relaxation of femoral arteries to cholinoceptor activation was not affected by cooling (Monge et al., 1993). This apparent discrepancy might be related to the different methods used, and it is possible that although cooling can decrease the production of nitric oxide this reduction is not sufficient to decrease the relaxation of femoral arteries to cholinoceptor stimulation during cooling. It is apparent, however, that the effects of cooling on cutaneous (ear) and deep (femoral) arteries to produce nitric oxide after cholinoceptor stimulation are different. This difference might be related to a different behaviour of endothelial cholinoceptors and of the metabolic pathway for nitric oxide synthesis in cutaneous and deep arteries during cooling. In conclusion, if the production of nitrite is a reliable index of nitric oxide production, the present results indicate that cooling potentiates the production of nitric oxide from the endothelium of cutaneous arteries, but not of deep arteries, when they are stimulated. These effects of cooling would explain, at least in part, the reduced contraction to endothelin-1 (Monge et al., 1991) and adrenergic activation (Garcia-Villalon et al., 1992) and the increased relaxation to cholinoceptor stimulation (Monge et al., 1993) of cutaneous arteries during cooling. Moreover, other endothelial factors,

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