INTRODUCTION

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VASCULAR RESPONSIVENESS to sympathetic neural outflow and neurohumoral agents can be modulated by a number of physical and chemical factors. One of the more well documented of these factors is temperature (27). For example, the effect of decreasing temperature on vascular smooth muscle responses to adrenergic agonists and neural activation has been clearly demonstrated. In 1968, Webb-Peploe and Shepherd (31, 32) reported that the vasoconstrictor response to local cooling in dog cutaneous veins was dependent on an intact sympathetic nervous system and was potentiated by a change in vascular responsiveness to incoming neural impulses. Subsequently, Vanhoutte (27) confirmed that local cooling increases vascular reactivity to electrical stimulation and exogenous norepinephrine in canine cutaneous veins, while depressing responses in deep vessels.

In addition to agonist-mediated constriction, limited evidence suggests that vascular smooth muscle responsiveness to nitric oxide and related factors can be influenced by temperature (7, 11, 17). Relaxation responses to cholinergic agents in rabbit femoral arteries are attenuated by cooling to 24°C (7, 17). Conversely, responses to these agents are augmented by cooling in rabbit cutaneous vessels (7, 17) and rat thoracic aorta (11). The responses to nitrovasodilators are also augmented by cooling (7, 11, 17); however, this effect does not appear to be vessel specific. Taken together, these data suggest that the stimulated release of and sensitivity to nitric oxide are augmented by cooling in vascular smooth muscle.

In contrast to the effect of cooling on smooth muscle reactivity to vasoactive agents, the influence of physiologically relevant increases in temperature above 37°C on vascular reactivity to vasoactive agents is unclear because of limited information and disparate results. Therefore, the purpose of this study was to determine the effect of heating on vascular smooth muscle responses to vasoactive agents in rat mesenteric arteries. The mesenteric artery was chosen for this study because one of the primary adjustments to heating in rats is a sympathetically mediated increase in mesenteric vascular resistance (12, 14) and the concomitant redistribution of cardiac output from the viscera to cutaneous regions for heat dissipation (15). Despite a continuous increase in splanchnic sympathetic nerve activity (14), mesenteric resistance shows a triphasic response to hyperthermia (15). Resistance increases during the early stages of heating, reaches a plateau, and then declines during the latter stages of heating as body temperature approaches 41-42°C. This pattern suggests that vascular responsiveness to neural outflow in this region changes with increasing temperature, especially temperatures above 40°C. In addition to reduced vascular responsiveness to adrenergic agonists, the decrease in resistance during the latter stages of heating could also be due to an increase in the release of (9) and sensitivity to nitric oxide. Thus the hemodynamic responses to hyperthermia in the mesenteric artery imply that the vascular reactivity to vasoconstrictor and vasodilator agents are altered during heating.

	METHODS

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Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN), weighing 300-350 g, were used in all experiments. Animals were housed in individual cages on a 12:12-h light-dark schedule and allowed standard rat chow and water ad libitum before experimentation. Experiments were performed in accordance with guidelines approved by the Institutional Animal Use and Care Committee.

Vessel preparation. Rats were anesthetized with an injection of pentobarbital sodium (50 mg/kg ip) and exsanguinated. The proximal segment of the superior mesenteric artery was excised and placed in ice-cold (4°C) physiological saline solution (PSS). Fat and connective tissue were removed, and vessels were cut into rings 2.5 mm in length.

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Concentration-response curves. In separate groups of rings, cumulative concentration-response curves to norepinephrine (10¹⁰ to 10⁵ M), phenylephrine (10⁹ to 10⁴ M), acetylcholine (10⁹ to 10⁴ M), sodium nitroprusside (SNP; 10¹² to 10⁴ M), and KCl (5-120 mM) were generated at 37 and 41 or 42°C. The higher temperatures represent the point during hyperthermia when mesenteric vascular resistance begins to decline (15), suggesting that vascular responsiveness to adrenergic agonists has decreased (15). For norepinephrine, KCl, acetylcholine, and SNP, two concentration-response curves were generated in each ring, one at 37 and one at 41°C. After the first concentration-response curve was completed, rings were washed and allowed to reestablish resting tension before being heated. When rings stabilized (15-20 min), bath temperature was increased to 41°C. Rings were allowed to equilibrate at this temperature for 1 h before a second concentration-response curve was determined. Parallel experiments were conducted in separate groups of rings maintained at 37°C for the duration of the experiment to control for changes in reactivity due to time and the very small increase in tension that occurred in some rings during heating. A second protocol, which consisted of a single concentration-response curve for phenylephrine or acetylcholine generated at either 37 or 42°C, was also used to control for changes in reactivity with time or repeated exposures to an agent. In experiments utilizing acetylcholine or SNP, rings were preconstricted with 10⁶ M phenylephrine. Rings used for KCl concentration-response curves were pretreated with the -adrenergic-receptor antagonist phentolamine (10⁵ M) 10 min before KCl administration (29). Only one agent was tested in each ring.

Ca²⁺ sensitivity. Ca²⁺ sensitivity or the contractile response to a given concentration of Ca²⁺ can change in the presence of pharmacological agents or under pathophysiological conditions (18, 19). Therefore, experiments were conducted to determine whether heating increases Ca²⁺ sensitivity in vascular rings. Rings were incubated in a Ca²⁺-free PSS containing 2.0 mM EGTA (see Drugs and solutions) for 15 min at 37°C, then treated with 2-3 maximal doses of phenylephrine (10⁵ M) to deplete intracellular Ca²⁺ stores (25). Rings were then washed with Ca²⁺-free PSS containing 0.1 mM EGTA (see Drugs and solutions) and incubated for 10 min. After 5 min, phenylephrine (10⁶ M) was added to the bath and a cumulative concentration-response curve to CaCl₂ (10⁵ to 6 × 10³ M) was generated. When maximal constriction was attained, rings were washed with normal PSS for 15 min and the protocol was repeated after a 45-min equilibration period at 41°C. The Ca²⁺-free PSS was warmed to 41°C before the protocol was repeated.

Role of the endothelium. To address the role of endothelial factors in changes in vascular responsiveness during heating, concentration-response curves to norepinephrine, KCl, SNP, and CaCl₂ were generated at 37 and 41°C in vascular rings without an endothelium. The influence of nitric oxide on constrictor responses to norepinephrine during heating was specifically addressed by pretreating a subset of endothelium-intact rings with the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME; 10 mM). L-NAME was added to the organ chamber bath 20 min before norepinephrine concentration-response curves were started. Relaxation responses to acetylcholine (10⁶ M) were used to verify blockade by L-NAME and determine endothelial cell integrity.

Drugs and solutions. The normal PSS contained (in mM) 118.3 NaCl, 24 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.9 CaCl₂, and 11.1 glucose. Two Ca²⁺-free solutions were used in these experiments. In one Ca²⁺-free solution, EGTA (2 mM) was substituted for CaCl₂ and all other components remained the same as in normal PSS. In the other Ca²⁺-free PSS, 0.1 mM EGTA and excess NaCl (1.8 mM) were used as a substitute for CaCl₂.

Data analysis and statistics. Data are expressed as means ± SE. Drug concentrations eliciting EC₅₀ or IC₅₀ were calculated from plotted data by using a four-parameter logistic equation (Sigma Plot, Jandel Scientific). Concentration-response curves were compared by using repeated-measures analysis of variance followed by a modified Student's t-test with a Bonferroni correction for multiple comparisons. Maximal responses and EC₅₀ and IC₅₀ values for curves obtained before and during heating were compared by using paired and unpaired (acetylcholine and phenylephrine) Student's t-tests. Statistical significance was set at P < 0.05.

	RESULTS

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Concentration-response curves. The contractile responses to norepinephrine in mesenteric arterial rings are presented in Fig. 1. Norepinephrine elicited concentration-dependent increases in tension in all rings at 37 and 41°C. Maximal contractions in response to norepinephrine were comparable in rings with and without endothelium at both temperatures. Heating had no effect on the responses to norepinephrine in endothelium-intact or -denuded rings (Fig. 1, Table 1). Pretreatment with L-NAME significantly enhanced the maximal responses to norepinephrine in all rings at 37°C, but there was no significant difference between maximal tension generated at 37 and 41°C. The sensitivity to norepinephrine in mesenteric rings was also comparable across treatments and temperatures (Table 1). In the time control experiments, maximal responses to norepinephrine in endothelium-intact rings were slightly higher for the second concentration-response curve (2.0 ± 0.1 g) compared with the first (1.8 ± 0.1 g).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Contractions in response to norepinephrine in endothelium-intact (A; n = 7 rats), endothelium-denuded (B; n = 7 rats), and 10 mM NG-nitro-L-arginine methyl ester (L-NAME)-treated (C; n = 5 rats) mesenteric arterial rings at 37 and 41°C. Endothelium-intact rings were treated with L-NAME 20 min before addition of norepinephrine. Concentration-response curves were generated at 37 and 41°C in each ring. Heating did not alter tension generation in any of the rings. n, No. of rats. * P < 0.05 compared with 37°C.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Contractions in response to phenylephrine in mesenteric arterial rings at 37 and 42°C (n = 8/temperature). Rings were incubated at either 37 or 42°C for 1 h before testing. Only 1 concentration-response curve was generated in each ring.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Contractions in response to potassium chloride in endothelium-intact (top) and endothelium-denuded (bottom) mesenteric arterial rings at 37 and 41°C. Concentration-response curves were generated at both temperatures in each ring. Heating significantly enhanced maximal tension generated in endothelium-intact rings but had no effect on endothelium-denuded rings. n, No. of rats. * P < 0.05 compared with 37°C.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Acetylcholine-induced relaxations in mesenteric arterial rings at 37 and 41°C. Concentration-response curves were generated at 37 and 41°C in each ring after constriction with phenylephrine. Heating significantly attenuated relaxant response to acetylcholine. n = 9 rats. * P < 0.05 compared with 37°C.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Relaxation responses to sodium nitroprusside (SNP) in endothelium-intact (top) and -denuded (bottom) mesenteric arterial rings at 37 and 41°C. Concentration-response curves were generated at 37 and 41°C in each ring after constriction with phenylephrine. Heating significantly attenuated relaxant responses in endothelium-intact and -denuded rings. n, No. of rats. * P < 0.05 compared with 37°C.

Ca²⁺ sensitivity. Ca²⁺ sensitivity was assessed in mesenteric arterial rings by comparing concentration-response curves to CaCl₂ at 37 and 41°C (Fig. 6). At 37°C, tension increased in response to CaCl₂; however, the highest concentrations caused the rings to relax (Fig. 6). As a result, the maximal responses to Ca²⁺ were significantly higher at 41 than at 37°C (Table 1). The EC₅₀ value at 41°C was also significantly different from the value at 37°C. When comparing endothelium-intact and -denuded rings, maximal responses at 37°C in endothelium-denuded rings were higher than responses in intact rings at the same temperature but comparable to those from endothelium-intact rings at 41°C (Table 1). At 37°C, the EC₅₀ value for denuded rings was also shifted to a higher concentration compared with intact rings (Table 1). In contrast, responses were similar in endothelium-intact and -denuded rings at 41°C. Furthermore, heating did not alter the responses in endothelium-denuded rings (Fig. 6, Table 1).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Responses to calcium (CaCl2) in endothelium-intact (top) and -denuded (bottom) mesenteric arterial rings at 37 and 41°C. Intracellular Ca2+ stores were depleted by repeated administration of phenylephrine (105 M) to rings incubated in Ca2+-free PSS containing 2.0 mM EGTA. Rings were then incubated in Ca2+-free PSS containing 0.1 mM EGTA and activated with phenylephrine (106 M) before addition of CaCl2. Concentration-response curves were generated at 37 and 41°C in each ring. Heating significantly enhanced contractile responses to CaCl2 in endothelium-intact rings. n, No. of rats. * P < 0.05 compared with 37°C.

	DISCUSSION

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Most of the previous studies examining the effect of temperature on smooth muscle responses to vasoactive agents have focused on the effect of cooling. Cooling reduces the responsiveness of deep vessels to constrictor agents and increases constrictor responses in cutaneous vessels (27). In contrast, the effect on vascular smooth muscle of heating to physiologically relevant temperatures above 37°C has not been as well documented. Therefore, the purpose of this study was to determine the effect of heating to 41 or 42°C on vascular reactivity to vasoactive agents in rat mesenteric arterial rings. At temperatures above 40°C, mesenteric resistance in rats begins to decline despite high levels of sympathetic neural outflow to the region (14, 15). The physiological changes that occur at or above this temperature are important because this decline in vascular resistance generally marks the onset of circulatory collapse (15). Previous in vivo and in vitro data indicate that heating to 39°C has little or no effect on the responses to vasoactive agents (13, 21). However, the systemic hemodynamic responses to adrenegeric agonists are blunted at temperatures above 40°C (13), whereas the microvascular responses to vasoconstrictor agents are augmented or unchanged by warming (26). Data from in vitro preparations heated to 40°C or higher are equivocal (2, 11, 20, 23, 27, 28). In the present study, heating to 41°C had no effect on the maximal contractions in response to -adrenergic agonists in the mesenteric artery. In contrast, the contractile responses to KCl and CaCl₂ were augmented at 41°C in endothelium-intact, but not -denuded rings. Furthermore, relaxation responses to endothelium-dependent (acetylcholine) and endothelium-independent (SNP) agonists were blunted at 41°C. Collectively, these data indicate that heating alters vascular responsiveness to constrictor and dilator agents. Moreover, the results imply that the changes in vascular reactivity observed in the present study are likely due to a change in vascular smooth muscle responsiveness to nitric oxide-containing compounds. This decrease in responsiveness can limit the ability of vascular smooth muscle to relax and possibly decreases the capacity of the endothelium to buffer vasocontrictor responses.

Heating has been reported to increase (2, 11, 26, 28), decrease (20), or have no effect (21, 23, 26) on the vascular responses to vasoconstrictor agents in normotensive rats. In the present study, increasing temperature did not alter the responses to adrenergic agonists in mesenteric arterial rings. This observation is consistent with the data of Ryan and Gisolfi (23). They demonstrated that heating to 42 or 43°C did not alter the concentration-dependent responses to norepinephrine in isolated mesenteric arteries. Stojanov and Proctor (26) also reported that increasing skin temperature did not alter the vasoconstrictor responses to norepinephrine and angiotensin II in hamster skin arterioles in vivo. Conversely, heating to temperatures above 37°C has variable effects on the responses to norepinephrine in rat thoracic aorta (11, 20, 21), suggesting that responses may be vessel specific.

Although heating did not change the responses to adrenergic agonists, the contractile responses to KCl were enhanced at 41°C in endothelium-intact rings. These findings contradict those of Peiper et al. (20) but support observations made in rat thoracic aorta (21) and canine saphenous veins (2, 28). In contrast, heating did not alter the contractile responses to KCl in endothelium-denuded rings. Although the mechanism for these disparate responses is unclear, the data imply that KCl may stimulate the release of an endothelium-derived contracting factor at 41°C. However, further experiments are necessary to confirm this postulate.

Heating also significantly increased the maximal contractions in response to CaCl₂ in endothelium-intact rings, but not in endothelium-denuded rings. Endothelium removal or heating intact rings to 41°C also inhibited the relaxation response to high concentrations of Ca²⁺. Ca²⁺ is known to have a dual effect on vascular smooth muscle, eliciting both contraction and relaxation (1, 33). Relaxation at high concentrations of extracellular Ca²⁺ involves a membrane-stabilizing effect of Ca²⁺ that is dependent, in part, on the endothelium (33). This dual effect of Ca²⁺ was evident in the present study during the cumulative concentration-response curves to CaCl₂. High concentrations of Ca²⁺ in the presence of phenylephrine elicited relaxation at 37°C, but not at 41°C. Furthermore, this relaxation to Ca²⁺ was not observed in endothelium-denuded rings at either temperature, supporting the role of the endothelium in this response. Inhibition of the membrane-stabilizing effect of Ca²⁺ during heating may be related to a change in Ca²⁺ binding to the lipid bilayer of vascular smooth muscle cells (membrane destabilization) (3), a sufficient degree of membrane depolarization to prevent relaxation by Ca²⁺ (8), an increase in vascular smooth muscle sensitivity to Ca²⁺ (18, 19), or a decrease in the release of endothelial factors (33).

Limited data suggest that changing temperature may also alter the ability of the endothelium to generate or release nitric oxide (11, 16). Decreased release of nitric oxide from endothelial cells at 41°C could explain the endothelium-dependent potentiating effect of heating on the contractile responses to CaCl₂. However, several lines of evidence suggest that nitric oxide release in vivo and in vitro increases at high temperatures (4, 9). Therefore, decreased spontaneous release of nitric oxide with heating appears unlikely. In contrast, temperature may have a specific effect on the stimulated release of nitric oxide. Karaki and Nagase (11) reported that the relaxation responses to carbachol were enhanced with cooling and decreased with heating. A similar effect of cooling on the relaxant responses to acetylcholine and methacholine was reported by Monge et al. (17) for rabbit ear arteries. Conversely, Lubbe (16) reported that the responses to acetylcholine were attenuated in normothermic rat cremaster arterioles after an acute heating period (30 min at 41°C). In the present study, the relaxation responses to acetylcholine were attenuated at 41°C compared with responses at 37°C. The blunted responses to cholinergic agonists (11, 17) could be due to decreased release of a relaxing factor, as suggested by the data of Fernández et al. (5). They reported that methacholine-induced nitrite production was potentiated by cooling in cutaneous arteries. Heating may have the opposite effect on nitric oxide release in response to acetylcholine, but this has yet to be determined by direct measurement of nitric oxide release.

In addition to possibly decreasing the release of endothelium-derived relaxing factors, heating may also reduce the sensitivity to nitric oxide, as suggested by the blunted responses to SNP at 41°C. However, the effect of temperature on vascular smooth muscle sensitivity to nitric oxide is unclear. Monge et al. (17) reported that the relaxant responses to SNP were attenuated by cooling in rabbit ear and femoral arteries. Their findings imply that heating might augment the responses to nitrovasodilators. However, Lubbe (16) reported that after an acute bout of heating (30 min at 41°C) the responses to SNP in vivo were not altered under normothermic conditions. In contrast, our data support the findings of Karaki and Nagase (11), who also observed decreased relaxation responses to SNP during heating. One potential mechanism for the blunted responses to SNP is a desensitization of soluble guanylate cyclase to nitrovasodilators (30). The relaxation induced by SNP and related compounds is mediated by the release of nitric oxide, which activates soluble guanylate cyclase and stimulates cGMP production and relaxation of vascular smooth muscle (22). After a single, prolonged (1-h) exposure to nitroglycerin, the ability of nitrovasodilators to stimulate guanylate cyclase activity during a second exposure can be reduced (30), possibly limiting their ability to elicit relaxation in vascular smooth muscle. Other vasorelaxant agents such as carbachol and acetylcholine also activate soluble guanylate cyclase (24) after stimulating the release of an endothelium-derived relaxing factor (6, 22). Thus desensitization of guanylate cyclase, a decrease in cGMP production, or a change in cGMP-mediated relaxation of vascular smooth muscle could also explain the attenuated responses to cholinergic agonists and the augmented responses to CaCl₂ in endothelium-intact rings during heating. However, the responses to SNP and acetylcholine were not altered in time control experiments. Therefore, in the present study, the loss of responsiveness to SNP and acetylcholine, possibly due to desensitization of soluble guanylate cyclase, appears to be a specific effect of heating.

The ability of vascular smooth muscle to maintain vascular tone during moderate heating despite the presence of vasorelaxant stimuli may have a functional role in vivo. During the early stages of hyperthermia in rats, vasoconstriction in the viscera is necessary to maintain blood pressure and redistribute blood flow to cutaneous regions. This visceral vasoconstriction may stimulate regional nitric oxide production and release (9) due to increased shear stress (10). This increase in nitric oxide release, which under normothermic conditions would elicit vasodilation, may have little effect on vascular tone during hyperthermia due to a heat-induced decrease in vascular responsiveness. Therefore, small increases in circulating relaxing factors would not dramatically impact normal physiological responses to mild stressors.

In summary, the results of this study demonstrate that heating alters both the contractile and relaxant responses to vasoactive agents in ring segments from rat mesenteric arteries. The contractile responses to KCl and CaCl₂ were augmented during heating in an endothelium-dependent manner. In contrast, heating attenuated the relaxant responses to acetylcholine and SNP. Taken together, these data suggest that heating decreases the ability of nitric oxide to relax vascular smooth muscle. Although the mechanism for this attenuated response is unclear, it may be related to changes in guanylate cyclase and/or cGMP-mediated relaxation. However, further studies are necessary to determine the effect of heating on guanylate cyclase activity and to determine whether this loss of sensitivity contributes to the endothelium-dependent changes in vasoconstriction observed in this study.