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(Hypertension. 1996;27:1245-1253.)
© 1996 American Heart Association, Inc.

Articles

Differential Contribution of Endothelial Function to Vascular Reactivity in Conduit and Resistance Arteries From Deoxycorticosterone-Salt Hypertensive Rats

Richard M. White; Carlos O. Rivera; Cathy Bruner Davison

From the Department of Pharmacology and Neuroscience, Albany (NY) Medical College.

Correspondence to Cathy Bruner Davison, PhD, Department of Pharmacology and Neuoroscience, A-136, Albany Medical College, 47 New Scotland Ave, Albany, NY 12208. E-mail cdavison@ccgateway.amc.edu.

	Abstract

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Abstract The purpose of these studies was to compare changes in conduit and resistance artery function in deoxycorticosterone-salt hypertensive rats. We hypothesized that if there was a common mechanism producing changes in vascular function in hypertension, then there would be similar alterations in reactivity of conduit and resistance arteries. Helically cut strips of common carotid artery were prepared for measurement of isometric force generation, and segments of small mesenteric arteries were pressurized for video dimension analysis. Sensitivity of arteries to phenylephrine and acetylcholine was determined. Carotid arteries from deoxycorticosterone-salt hypertensive rats were more sensitive to phenylephrine than arteries from control rats, whereas mesenteric resistance arteries from hypertensive rats were less sensitive to phenylephrine. In carotid arteries, endothelial denudation or incubation with N-nitro-L-arginine increased phenylephrine sensitivity in control rats to the level seen in deoxycorticosterone-salt rats. These manipulations had no effect on phenylephrine sensitivity in arteries from deoxycorticosterone-salt rats. In mesenteric resistance arteries, endothelium denudation normalized the depressed phenylephrine sensitivity in arteries from hypertensive rats but had no effect on arteries from normotensive rats. This depressed phenylephrine sensitivity in deoxycorticosterone-salt mesenteric arteries was not reversed by incubation with N-nitro-L-arginine. Acetylcholine-induced relaxation was depressed in carotid arteries from deoxycorticosterone-salt hypertensive rats, and N-nitro-L-arginine blocked these relaxations. In contrast, acetylcholine relaxation in the mesenteric arteries from normotensive and hypertensive rats did not differ. N-nitro-L-arginine slightly but significantly attenuated acetylcholine dilation only in mesenteric resistance arteries from the hypertensive rats. We conclude that qualitatively different changes in vasoconstrictor sensitivity to phenylephrine occur in carotid arteries and mesenteric resistance arteries of deoxycorticosterone-salt hypertensive rats. The increased phenylephrine sensitivity in carotid arteries in this model of hypertension is due to the loss of endothelium-derived nitric oxide production. In contrast, the decreased phenylephrine sensitivity in mesenteric resistance arteries from deoxycorticosterone-salt rats is due to a non–nitric oxide–mediated influence of the endothelium that is absent in arteries from normotensive rats.

Key Words: acetylcholine • endothelium • carotid arteries • adrenergic agonists • hypertension, experimental • mineralocorticoids • resistance arteries

	Introduction

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Alterations in the structure and function of the vasculature have been implicated in the pathogenesis and maintenance of chronic hypertension. In particular, two types of functional changes have been widely observed and are thought to contribute to increases in blood pressure. First, increased vasoconstrictor sensitivity to agents such as norepinephrine and serotonin has been observed in numerous forms of experimental hypertension, including mineralocorticoid-salt hypertension,^{1 2 3} the spontaneously hypertensive rat,^{4 5} renovascular hypertension,⁶ and the Dahl salt-sensitive rat.⁷ Second, many groups have established that there is a defect in endothelium-dependent dilation (typically assessed by measurement of acetylcholine-induced relaxation) in these same forms of experimental hypertension.^{8 9 10 11} Both of these changes have been postulated to contribute to the elevated peripheral vascular resistance characteristic of established hypertension. In addition, there may be an important interaction between changes in vasoconstrictor and vasodilator functions, because the endothelium can alter the ability of smooth muscle to respond to vasoconstrictor stimuli in both normotension and hypertension.^{8 12 13}

Many of the above studies were performed with preparations of isolated conduit arteries such as the carotid artery and aorta. Although these observations have been informative, it has been well established that peripheral vascular resistance is primarily determined by the caliber of the small arteries and arterioles less than 300 µm.¹⁴ It is a reduction in diameter in these arteries that produces the elevation of blood pressure in hypertension.¹⁵ A critical question that has not been fully addressed is whether changes in constrictor and dilator functions observed in the conduit arteries in hypertension are also seen in the resistance vasculature. Some studies have suggested that minimal, if any, changes in contractile sensitivity occur in resistance arteries from hypertensive rats.^{16 17 18 19 20} Similarly, others have found either a small depression,^{16 17} no change,²¹ or enhanced²² endothelium-dependent vasodilation in resistance arteries from hypertensive rats. In studies of arteries from mineralocorticoid-salt hypertensive rats, both increases^{22 23} and decreases^{9 24} in endothelium-dependent vasodilation have been observed. Reasons for this discrepancy may be related to the variety of artery types and agonists used in these studies. The purpose of the current study was to systematically identify changes in vascular reactivity and endothelial function that occur in conduit and resistance arteries from DOC-salt hypertensive rats. We hypothesized that if there was a common mechanism for changes in vascular function in DOC-salt hypertension, then similar alterations in vascular reactivity would be observed in conduit and resistance arteries. To address this hypothesis, we performed the following experiments. First, we conducted studies to compare conduit and resistance artery function in DOC-salt hypertensive rats. We examined contractile sensitivity to phenylephrine and dilator sensitivity to endothelium-dependent and -independent agents in carotid arteries and mesenteric resistance arteries. Second, we studied a potential mechanism for differences in conduit and resistance artery function by examining the ability of the endothelium to modulate phenylephrine contractions. We conducted further studies using inhibitors of NO synthase to characterize the mechanism of endothelium-dependent effects in the two artery types.

	Methods

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Animal Preparation
Male Wistar rats weighing 200 to 250 g (Harlan Sprague Dawley, Indianapolis, Ind) were used in these studies. Rats were housed in groups of three in temperature-controlled, light-cycled (6 AM to 6 PM) quarters with ad libitum access to food and water. All procedures involving the use of animals were approved by the Institutional Animal Care and Use Committee and conformed to institutional, state, and federal guidelines.

All rats underwent a right uninephrectomy under pentobarbital anesthesia (50 mg/kg IP). After 1 week of recovery, rats were placed on a 1% NaCl, 0.2% KCl drinking solution. After rats had been on this regimen for a week, control systolic pressure was measured with a tail-cuff technique and photoelectric transducer (IITC). At this time, rats were divided into two groups. The first group was designated as a control group and remained on the saline drinking solution. The experimental group received DOC (200 mg/kg SC) embedded in a silicone elastomer rubber matrix and continued on the saline drinking solution. Blood pressure measurements were obtained weekly during a 3-week treatment period. At the end of the treatment period, rats were killed with an overdose of sodium pentobarbital (120 mg/kg IP), and arteries were removed for study of vascular function.

Isolated Artery Preparation
Carotid Artery
Common carotid arteries were removed bilaterally and placed immediately into cold PSS. The PSS composition for all studies was (mmol/L) NaCl 130, KCl 4.7, MgSO₄ · 7H₂O 1.17, KH₂PO₄ 1.18, NaHCO₃ 14.9, dextrose 5.5, NaCa₂EDTA 0.03, and CaCl₂ 1.6. Arteries were cleaned of excess fat and connective tissue and cut into helical strips (one strip from each artery). One strip from each rat was denuded of endothelium by rubbing the intimal surface with a moistened cotton swab. The endothelium was left intact in the other strip. Strips were mounted on metal tissue holders and placed in 50-mL tissue baths filled with warmed (37°C) PSS bubbled with 95% O₂/5% CO₂, pH 7.35. Each strip was connected to a force transducer (FT.03, Grass Instrument Co) for measurement of isometric force, which was recorded on a Grass polygraph. Vascular strips were allowed to equilibrate for 120 minutes at a passive force of 7.84 mN.

Mesenteric Resistance Artery
A segment of the small intestine was removed and placed in cold PSS. Under a dissecting microscope, two third- to fourth-order branches of the superior mesenteric artery were dissected free of adhering fat and connective tissues. In one vessel from each rat, the endothelium was removed by a modification of the procedure of Osol et al.²⁵ Briefly, an individual vessel was placed in a Petri dish containing several human hairs of various diameters. The vessel was placed over a hair approximating the lumen diameter and rubbed several times over the surface of the hair. At this point, the vessel was transferred to one chamber of a dual-chamber pressure arteriograph (Living Systems Instrumentation) and cannulated at one end with glass microcannulas. A small volume of PSS (between 1 and 2 mL) containing several small air bubbles was then flushed through the lumen. PSS was then flushed through the lumen for 5 minutes, at which time the distal end of the artery was cannulated. The other artery was left with endothelium intact; after cannulation at the proximal end, blood was gently flushed from the lumen with PSS and the distal end was cannulated. All arteries were secured to the cannulas with silk sutures. Pressure was monitored by a flow-through pressure transducer and maintained at 60 mm Hg with a servo-controlled device (Living Systems Instrumentation). There was no luminal flow through the arteries. Vessels were superfused with warmed (37°C), gassed (95% O₂/5% CO₂) PSS at a flow rate of 20 mL/min and allowed to equilibrate for 60 minutes. The arteriograph chamber was placed on the stage of an inverted microscope, and an image of the vessel was sent to a computer equipped with a Frame-Grabber board (PCVision Plus, Microsciences Inc). Vessel outer diameter was continuously monitored with image-analysis software (Microsciences Inc). Resting vessel diameters were as follows: control intact, 331±10 µm (n=17); control denuded, 368±10 µm (n=17); DOC intact, 359±13 µm (n=14); and DOC denuded, 361±7 µm (n=14).

Vascular Reactivity Protocols
Vasoconstrictor Responses
For all vessels, cumulative concentration-response curves to the -adrenergic agonist phenylephrine were performed. The arteries were then rinsed with fresh PSS and allowed to reequilibrate for 20 to 30 minutes. In some experiments, arteries were then incubated with the NO synthase inhibitor LNA (10^-4 mol/L) for 20 minutes, and the phenylephrine concentration-response curves were repeated.

Vasodilator Responses
Arteries were precontracted with a phenylephrine concentration sufficient to cause 80% of the maximal response for each vessel as previously determined. In carotid arteries, cumulative concentration-response curves to the endothelium-dependent dilator acetylcholine were performed in the presence or absence of LNA (10^-4 mol/L). In addition, concentration-response curves to the endothelium-independent dilator sodium nitroprusside were also performed in carotid arteries. In mesenteric resistance arteries, concentration-response curves to acetylcholine were performed with or without LNA. In the mesenteric resistance arteries, endothelium-independent dilation was assessed by performing concentration-response curves to adenosine. In all arteries, effective removal of the endothelium was functionally assessed by the absence of a dilator response to acetylcholine.

Statistical Analysis
All values are expressed as mean±SE. For each artery, the phenylephrine response at each concentration was expressed as a percentage of maximal effect, and the EC₅₀ (concentration causing 50% of the maximal response) was calculated with probit analysis.²⁶ Statistical analysis was performed on the pD₂ (-log EC₅₀) values for each artery. The maximal response of the carotid artery was expressed as millinewtons of force. Maximal responses for resistance arteries were calculated as the percent reduction in baseline diameter by the formula Maximal Response=(D_i-D_m)/D_i, where D_i is initial diameter, and D_m is the diameter at maximal constriction. Differences in pD₂ and maximal response were assessed with a two-way between-groups ANOVA (factor 1: normotensive versus hypertensive; factor 2: endothelium present or absent or presence of LNA). Relaxation responses were calculated as a percentage of the phenylephrine-induced contraction. Differences between groups were assessed at each dilator dose with a between-within ANOVA. Blood pressures were compared at each week of treatment with a between-within ANOVA. When the F value was significant, individual comparisons were made with the Newman-Keuls post hoc test for significance. In all cases, a value of P<.05 was considered statistically significant.

	Results

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Blood Pressure Response
In the DOC-salt group, subcutaneous implantation of DOC resulted in a significant increase in blood pressure after 4 weeks (control, 120±2 mm Hg, to 167±6 mm Hg, n=15, P<.05). Blood pressure in the control group did not change over the period of the study (baseline, 120±2 mm Hg; 4 weeks, 121±2 mm Hg, n=18, P=NS).

Vasoconstrictor Responses
Phenylephrine Sensitivity
Endothelium-intact carotid artery strips from DOC-salt hypertensive rats were significantly more sensitive to phenylephrine than those from control rats (Fig 1). The magnitude of this increase in sensitivity was approximately a threefold shift in the EC₅₀ value (see Table 1 for pD₂ values). In contrast, mesenteric resistance arteries from the hypertensive rats were markedly less sensitive to phenylephrine than those from control rats (Fig 2); arteries from the hypertensive rats were approximately 10-fold less sensitive to phenylephrine (see Table 2 for pD₂ values). The maximal response elicited by phenylephrine in the mesenteric resistance artery was significantly lower in arteries from DOC-salt hypertensive rats compared with arteries from control rats (Table 2); the maximal response in carotid arteries did not differ (Table 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Phenylephrine concentration-response curves in carotid arteries from control and DOC-salt hypertensive rats (n=6 to 7 in each group). Data are plotted as percentage of maximal contractile response to phenylephrine. Intact and denuded refer to presence or absence of endothelial cells, respectively. Refer to Table 1 for pD2 and maximal response values.

View this table: [in this window] [in a new window]   Table 1. Sensitivity and Maximal Response of Carotid Arteries From Deoxycorticosterone-Salt and Control Rats to Phenylephrine

View larger version (21K): [in this window] [in a new window]   Figure 2. Phenylephrine concentration-response curves in mesenteric resistance arteries from control (n=17) and DOC-salt hypertensive (n=14) rats. Data are plotted as percentage of maximal contractile response to phenylephrine. Intact and denuded refer to presence or absence of endothelial cells, respectively. Refer to Table 2 for pD2 and maximal response values.

View this table: [in this window] [in a new window]   Table 2. Sensitivity and Maximal Response of Mesenteric Resistance Arteries From Deoxycorticosterone-Salt and Control Rats to Phenylephrine

Effect of Endothelium Denudation on Phenylephrine Responses
Endothelium-denuded carotid artery strips from control rats were more sensitive to phenylephrine than endothelium-intact arteries. In contrast, phenylephrine sensitivity of endothelium-denuded strips from hypertensive rats was not significantly different from that in endothelium-intact strips (Fig 1 and Table 1). As a result, phenylephrine sensitivity in endothelium-denuded carotid arteries from control and hypertensive rats did not differ. In the mesenteric resistance arteries, endothelium removal increased phenylephrine sensitivity in arteries from the hypertensive rats but not in those from control rats (Fig 2 and Table 2). Thus, in the mesenteric resistance arteries, endothelium removal also eliminated differences in phenylephrine sensitivity between control and DOC-salt rats; however, this occurred by increasing sensitivity of arteries only from hypertensive rats.

In carotid artery strips, endothelium denudation increased maximal response only in arteries from control rats (Table 1). In the mesenteric resistance arteries, endothelium removal increased maximal response in arteries from both control and DOC-salt hypertensive rats; however, maximal response was greater in endothelium-denuded mesenteric resistance arteries from control rats than in those from hypertensive rats (Table 2).

Effect of NO Synthase Inhibition on Phenylephrine Responses
Inhibition of NO synthase with LNA replicated the effect of endothelium removal in carotid arteries. Thus, LNA increased phenylephrine sensitivity in carotid arteries from control but not hypertensive rats (Fig 3 and Table 1). LNA eliminated the difference in phenylephrine sensitivity in endothelium-intact carotid arteries from control and DOC-salt hypertensive rats. In the mesenteric resistance arteries, LNA slightly but significantly increased phenylephrine sensitivity of endothelium-intact arteries from both control and hypertensive rats (Fig 4 and Table 2). However, phenylephrine sensitivity of mesenteric resistance arteries from DOC-salt hypertensive rats was still significantly decreased compared with arteries from normotensive rats. LNA did not affect phenylephrine sensitivity in endothelium-denuded carotid or mesenteric resistance arteries (Tables 1 and 2).

View larger version (19K): [in this window] [in a new window]   Figure 3. Phenylephrine concentration-response curves in endothelium-intact carotid arteries from control and DOC-salt hypertensive rats in the presence or absence of 100 µmol/L LNA. Data are plotted as percentage of maximal contractile response to phenylephrine. Refer to Table 1 for pD2 and maximal response values.

View larger version (23K): [in this window] [in a new window]   Figure 4. Phenylephrine concentration-response curves in endothelium-intact mesenteric resistance arteries from control (n=9) and DOC-salt hypertensive (n=10) rats in the presence or absence of 100 µmol/L LNA. Data are plotted as percentage of maximal contractile response to phenylephrine. Refer to Table 2 for pD2 and maximal response values.

In endothelium-intact carotid arteries, LNA increased the maximal force generation only in arteries from control rats. LNA had no effect on maximal force in endothelium-denuded carotid arteries from DOC-salt or control rats (Table 1). In the mesenteric resistance arteries, LNA increased maximal response only in endothelium-intact arteries from DOC-salt hypertensive rats (Table 2).

Addition of LNA to the tissue bath caused a contraction of endothelium-intact carotid arteries from hypertensive rats (0.47±0.14 mN, n=14) that was approximately 20% of the phenylephrine-induced maximal contraction. The magnitude of this contraction was significantly less in endothelium-denuded carotid arteries (0.16±0.04 mN, n=14, P<.05). LNA had no effect on baseline diameter in mesenteric resistance arteries.

Endothelium-Dependent Relaxation
Relaxation responses to acetylcholine were depressed in carotid arteries from the hypertensive compared with normotensive rats at almost all acetylcholine concentrations tested (Fig 5). Incubation with LNA fully eliminated the response to acetylcholine in endothelium-intact carotid arteries from both normotensive and hypertensive rats (Fig 5). In contrast to the results in carotid arteries, relaxation responses to acetylcholine were not different in mesenteric resistance arteries from control and DOC-salt hypertensive rats (Fig 6). LNA failed to alter the acetylcholine-induced dilation in mesenteric resistance arteries from control rats. However, LNA slightly depressed acetylcholine relaxation in the mesenteric resistance arteries from DOC-salt hypertensive rats at acetylcholine concentrations of 3x10^-8 to 3x10^-7 mol/L.

View larger version (17K): [in this window] [in a new window]   Figure 5. Acetylcholine concentration-response curves in carotid arteries from control and DOC-salt hypertensive rats (n=6 to 8 in each group) in the presence or absence of 100 µmol/L LNA. Arteries were precontracted with an EC80 concentration of phenylephrine. *Significant difference between control and DOC.

View larger version (20K): [in this window] [in a new window]   Figure 6. Acetylcholine concentration-response curves in mesenteric resistance arteries from control and DOC-salt hypertensive rats in the presence (n=6 to 9) or absence (n=8 to 13) of 100 µmol/L LNA. Arteries were preconstricted with an EC80 concentration of phenylephrine. *Significant difference between DOC and DOC+LNA groups.

Endothelium-Independent Relaxation
Maximal relaxation to the endothelium-independent dilator sodium nitroprusside was greater in carotid arteries from the hypertensive rats compared with those from normotensive rats (Fig 7). Arteries from the hypertensive rats relaxed to a tension less than that of resting baseline tension, whereas arteries from control rats relaxed just to baseline. The ability of nitroprusside to relax arteries from DOC-salt rats below resting tension indicates that they had intrinsic tone that was absent in carotid arteries from control rats. Arteries from DOC-salt rats also were less sensitive to nitroprusside than those from control rats (Fig 7).

View larger version (19K): [in this window] [in a new window]   Figure 7. Sodium nitroprusside concentration-response curves in carotid arteries from control and DOC-salt hypertensive rats (n=7 to 8 in each group). Arteries were precontracted with an EC80 concentration of phenylephrine. Intact and denuded refer to the presence or absence of endothelium, respectively. *Significant difference between control intact and DOC intact groups.

In preliminary experiments, we found that sodium nitroprusside did not consistently relax mesenteric resistance arteries. As a result, we assessed the relaxation response to the endothelium-independent dilator adenosine in these arteries. The response to adenosine in mesenteric arteries from normotensive and hypertensive rats did not differ significantly. However, endothelium removal significantly enhanced adenosine-induced dilation equally in the mesenteric resistance arteries from these two rat groups (Fig 8).

View larger version (20K): [in this window] [in a new window]   Figure 8. Adenosine concentration-response curves in phenylephrine-preconstricted mesenteric resistance arteries from control (n=10) and DOC-salt hypertensive (n=5) rats. Arteries were preconstricted with an EC80 concentration of phenylephrine. Intact and denuded refer to the presence or absence of endothelial cells, respectively. *Significant difference between control intact and control denuded groups; significant difference between DOC intact and DOC denuded groups.

	Discussion

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We designed the current studies to assess the effect of DOC-salt hypertension on contractile and dilator functions in conduit and resistance arteries. We also examined the role of the vascular endothelium in the regulation of contractile sensitivity and how this may be altered in hypertension. There were five major findings of these experiments. First, the enhanced phenylephrine sensitivity observed in carotid arteries from hypertensive rats was not observed in mesenteric resistance arteries. Instead, a marked decrease in phenylephrine sensitivity was seen in the mesenteric resistance arteries. Second, the increased phenylephrine sensitivity in carotid arteries from DOC-salt hypertensive rats was solely due to lack of endothelium-derived NO that normally suppresses phenylephrine contraction. Third, the marked depression of phenylephrine sensitivity in mesenteric resistance arteries of DOC-salt hypertensive rats was eliminated by removal of the endothelial cell layer. The mediator of this endothelial depression of phenylephrine sensitivity in hypertension does not appear to be NO. Fourth, the depression of endothelium-dependent dilation in response to acetylcholine in the carotid arteries of hypertensive rats was not present in the mesenteric resistance arteries. Fifth, the major mediator of acetylcholine-induced dilation was different between the conduit and resistance vessels.

Enhanced vascular sensitivity to -adrenergic agonists such as norepinephrine has previously been observed in the conduit vasculature of mineralocorticoid-salt hypertensive rats.^{24 27 28} Changes in vascular reactivity have been postulated to contribute to the elevations of total peripheral resistance characteristic of established hypertension.²⁹ The mechanism by which alterations in -adrenergic sensitivity occur in mineralocorticoid-salt hypertension is not well understood. Several investigators have examined -adrenergic receptors in conduit arteries of mineralocorticoid-salt hypertensive rats and found that there is no alteration in receptor number, affinity, or major subtype.^{27 28} Similarly, the EC₅₀ for intracellular calcium stimulation of contraction is not altered in mineralocorticoid-salt hypertension, indicating no intrinsic alteration in contractile machinery sensitivity to calcium.^{28 30} It has been suggested that at least part of the increase in vascular sensitivity to -adrenergic agonists may be due to enhanced phosphoinositide production and/or metabolism in arteries from hypertensive rats.³¹ The results of the current study suggest an additional mechanism that may be responsible for increased vascular sensitivity to -adrenergic agonists in conduit arteries of DOC-salt hypertensive rats: the loss of endothelial production of NO that normally acts to suppress phenylephrine-induced contraction.

Endothelium-derived relaxation factors such as NO have been shown to oppose vasoconstriction induced by agents such as norepinephrine and vasopressin in conduit arteries.^{8 12 13 32 33} We found that endothelium-intact carotid arteries from DOC-salt hypertensive rats are approximately threefold more sensitive to phenylephrine than those from normotensive rats. However, in endothelium-denuded arteries, this difference in sensitivity was eliminated because of the increase in sensitivity of carotid arteries from control rats. Since LNA mimicked the effect of endothelium removal in carotid arteries, our data are consistent with the postulate that the endothelium of carotid arteries from normotensive rats produces NO, which suppresses the phenylephrine-induced contraction. It is the absence of this endothelial production of NO in carotid arteries from DOC-salt hypertensive rats that renders these arteries more sensitive to phenylephrine. Thus, in contrast to results obtained by other investigators,²⁸ we found no evidence for alterations in smooth muscle sensitivity to phenylephrine in conduit arteries in this model of hypertension. Reasons for this discrepancy are not clear, although one possibility is the use of different conduit vessels in these studies (carotid artery versus superior mesenteric artery). It is interesting to note, however, that the finding of a contractile response to LNA itself in the hypertensive carotid arteries may indicate an enhanced basal release of NO in these arteries. The mechanism for differences in basal versus agonist-stimulated NO release in the hypertensive carotid arteries is not clear from these experiments, although these data highlight the fact that NO release from hypertensive carotid artery endothelium may not be impaired under all conditions. One possibility not addressed in these studies is that contraction per se may be responsible for the phenylephrine-induced release of NO and that this mechanism is defective in the hypertensive carotid artery. In preliminary experiments, however, we found that concentration-response curves to potassium chloride were not significantly different in endothelium-intact and -denuded carotid arteries from normotensive rats, suggesting that contraction per se is not a mechanism for NO release in these arteries.

A primary aim of these studies was to determine whether changes in vascular reactivity commonly observed in the conduit arteries of hypertensive rats would also be seen in the resistance vasculature, where they may be proposed to directly contribute to elevations of peripheral vascular resistance. In our studies, we found that the increased phenylephrine sensitivity seen in the carotid artery of DOC-salt hypertensive rats is absent in the mesenteric small arteries; instead, phenylephrine sensitivity in the resistance arteries from hypertensive rats is markedly depressed. This suggests that intrinsic increases in adrenergic constrictor sensitivity may not contribute to elevations of mesenteric vascular resistance in this form of hypertension. Moreover, these data highlight the fact that changes in vascular reactivity observed in conduit vessels may not be "models" of changes occurring in the resistance vasculature. Our results are consistent with other reports in the literature demonstrating that few, if any, changes in contractile sensitivity occur in resistance arteries in hypertension,^{16 17 18 19 20} once again suggesting that increases in reactivity per se may not be a pathophysiological factor in the elevation of peripheral vascular resistance.

In contrast to our result of an apparent depression of endothelial function in conduit arteries of DOC-salt hypertensive rats, our results and those of others^{22 23} suggest that in some arterial preparations, there may be greater endothelial suppression of -adrenergic contractile sensitivity in arteries from hypertensive rats compared with those from normotensive rats. There also is evidence that NO is the endothelial factor that mediates this effect.²³ Our findings in the mesenteric resistance arteries in DOC-salt hypertension differ from these earlier studies in two important ways. First, we found that the enhanced endothelial function in the resistance arteries from the hypertensive rats does not mask increases in smooth muscle sensitivity; rather, we found no change in smooth muscle sensitivity in hypertension. Our studies demonstrate that the endothelium in these arteries causes a marked depression of contractile sensitivity to phenylephrine to a level below that of control arteries. Second, we also found that the mediator of this enhanced endothelial function in the hypertensive resistance arteries does not appear to be NO. Although NO synthase inhibition slightly increased the sensitivity of the mesenteric resistance arteries to phenylephrine, this was the case for arteries from both control and hypertensive rats. Thus, there appears to be production of NO from both control and hypertensive mesenteric resistance arteries sufficient to oppose phenylephrine-induced constriction. However, the endothelial depression of phenylephrine sensitivity in arteries from DOC-salt rats cannot be fully explained by NO production because the magnitude of the effect of LNA was less than that of endothelium denudation. The fact that NO does not appear to be the major mediator of the endothelial effect may not be surprising in light of studies suggesting that an endothelium-derived hyperpolarizing factor may be an important mediator of endothelium-dependent relaxation in the mesenteric resistance arteries.³⁴ Our data suggest that there may be another factor, perhaps endothelium-derived hyperpolarizing factor, that may mediate the endothelial depression of phenylephrine sensitivity in mesenteric resistance arteries from DOC-salt hypertensive rats.

Our results indicate that qualitatively different changes in endothelial function occur in conduit and resistance arteries in DOC-salt hypertension: a decrease in endothelial suppression of contraction in carotid arteries and an increase in endothelial suppression of contraction in resistance arteries. The mechanism by which conduit and resistance arteries may demonstrate differential alterations in endothelial function in hypertension is not clear. At least two mechanisms may be operative in producing these alterations. First, the carotid artery and mesenteric resistance arteries may be exposed to different shear stresses in vivo. Shear stress across the vascular endothelium can be markedly altered by changes in several factors, including flow velocity and vessel diameter.³⁵ In hypertension, significant vasoconstriction (as may be found in the mesenteric circulation) could produce elevations in shear stress in the mesenteric arteries³⁶ relative to that found in the carotid arteries. Shear stress is known to be one of the major stimuli for endothelial release of relaxation factors such as NO³⁷ and could potentially be involved in the production of other endothelium-dependent relaxation factors that act to suppress phenylephrine-induced contraction. Second, enhanced activity of the sympathetic nervous system in DOC-salt hypertension may differentially affect the mesenteric resistance arteries and carotid artery. In the carotid arteries, synaptic terminations are sparse near smooth muscle cells. In contrast, the smaller, more muscular mesenteric arteries have rich numbers of these synaptic terminals.³⁸ Because there is evidence of increased activity of the sympathetic nervous system in mineralocorticoid-salt hypertension,^{39 40} the mesenteric arteries could be exposed to higher levels of products of the sympathetic nerve terminals such as norepinephrine. Prolonged exposure to high levels of norepinephrine has been shown to induce vascular tachyphylaxis to -adrenergic agonists via stimulation of NO release from the vascular endothelium.^{41 42 43} Thus, if the mesenteric resistance arteries from the hypertensive rats are exposed to high levels of norepinephrine from the sympathetic nerve terminals, there may be stimulation of endothelium-derived factors that oppose vasoconstriction and result in a decrease in phenylephrine sensitivity in this form of hypertension.

Our studies confirm earlier findings of a depression of acetylcholine-induced relaxation in conduit arteries of hypertensive rats.⁹ Because LNA fully eliminated the response to acetylcholine in these arteries, it is likely that NO is the major mediator of acetylcholine-induced dilation. There are at least two possible mechanisms for the depressed response to acetylcholine in conduit arteries from DOC-salt hypertensive rats. First, given our result that the carotid arteries from hypertensive rats demonstrated a decreased sensitivity to sodium nitroprusside (an NO donor), smooth muscle sensitivity to NO may be decreased. Second, NO release from the endothelium may be diminished when stimulated with acetylcholine. On the other hand, because LNA caused an endothelium-dependent contraction of carotid arteries only from the hypertensive rats, basal NO release from these arteries may be greater. It is possible, however, that this effect is due to the presence of intrinsic tone in carotid arteries from DOC-salt rats that was not present in arteries from normotensive rats.

The finding of depressed endothelium-dependent dilation in the carotid artery of hypertensive rats was not replicated in the mesenteric resistance arteries; rather, we found no change in acetylcholine-induced relaxation in the mesenteric resistance arteries of the hypertensive rats. This result suggests that changes in endothelial function in the resistance arteries from hypertensive rats are agonist specific; endothelium-dependent dilation in response to acetylcholine does not change, yet endothelial suppression of phenylephrine-induced contraction is marked. Interestingly, we found that although NO appears to fully mediate acetylcholine-induced relaxation in the carotid arteries from both control and hypertensive rats, this was not the case for the mesenteric vasculature. In the mesenteric resistance arteries, NO synthase inhibition failed to block acetylcholine-induced relaxation, although there was a slight but significant effect in the hypertensive arteries. This finding is in agreement with studies^{35 44} which demonstrate that an endothelium-derived hyperpolarizing factor, rather than NO, may be the major mediator of endothelium-dependent relaxation in the mesenteric vasculature. In preliminary studies, we also found that the relaxation response to sodium nitroprusside, an NO donor, was highly variable in the mesenteric resistance arteries, ranging from no relaxation to mild relaxation and in some cases full relaxation. This finding may also indicate that NO may not be a major relaxation factor in this segment of the vasculature. As a result, we chose adenosine as an endothelium-independent dilator in the mesenteric resistance arteries and found no alteration in the ability of smooth muscle to relax in these arteries. The mechanism by which endothelium removal augmented adenosine-induced dilation is not clear; one possibility is the removal of adenosine deaminase in the denuded vessels.^{45 46}

In summary, enhanced phenylephrine contractile sensitivity is seen in the carotid artery of DOC-salt hypertensive rats, whereas depressed phenylephrine sensitivity is observed in mesenteric resistance arteries. The primary factor responsible for changes in vascular sensitivity to phenylephrine in hypertension in these two artery types appears to be the function of the endothelium. Carotid arteries from DOC-salt hypertensive rats exhibit an enhanced sensitivity to phenylephrine that is fully explained by the loss of endothelial production of NO which normally acts to suppress contraction. In contrast, mesenteric resistance arteries from hypertensive rats demonstrate a marked reduction in phenylephrine sensitivity fully due to endothelial suppression of phenylephrine-induced contraction that is absent in arteries from normotensive rats. This endothelial suppression of contraction in mesenteric resistance arteries cannot be explained by enhanced release of endothelium-derived NO. The alterations in endothelial function in resistance arteries from DOC-salt hypertensive rats are agonist specific because acetylcholine-induced dilation in these arteries did not change. Although it has previously been suggested that increases in -adrenergic vasoconstrictor sensitivity in hypertension may at least partially contribute to elevations in peripheral vascular resistance, our data do not support a role for such an alteration. Instead, this study demonstrates that the vascular endothelium of mesenteric resistance arteries from DOC-salt hypertensive rats suppresses phenylephrine-induced contraction to a greater degree than that in control rats. This function of the vascular endothelium may represent a mechanism that acts to limit increases in adrenergic vasoconstrictor tone in this form of hypertension.

	Selected Abbreviations and Acronyms

 

DOC = deoxycorticosterone LNA = N-nitro-L-arginine NO = nitric oxide PSS = physiological salt solution

	Acknowledgments

 
This research was supported by grant HL-45673 from the National Institutes of Health (NIH), Bethesda, Md. R.M. White is a predoctoral fellow supported by NIH HL-07194.

Received August 14, 1995; first decision October 3, 1995; accepted February 16, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Soltis EE, Field FP. Sodium pump activity and norepinephrine responsiveness of femoral arterial smooth muscle from DOCA-salt rats. Pharmacology. 1987;34:104-110. [Medline] [Order article via Infotrieve]

2. Longhurst PA, Rice PJ, Taylor DA, Fleming WW. Sensitivity of caudal arteries and the mesenteric vascular bed to norepinephrine in DOCA-salt hypertension. Hypertension. 1988;12:133-142. [Abstract/Free Full Text]

3. Bruner CA, Mangiapane ML, Fink GD, Webb RC. Area postrema ablation and vascular reactivity in deoxycorticosterone-salt–treated rats. Hypertension. 1988;11:668-673. [Abstract/Free Full Text]

4. Hermsmeyer K. Electrogenesis of increased norepinephrine sensitivity of arterial vascular muscle in hypertension. Circ Res. 1976;38:362-367. [Abstract/Free Full Text]

5. Webb RC. Increased vascular sensitivity to serotonin and methysergide in hypertension in rats. Clin Sci. 1982;63:73s-75s.

6. Chen M, Webb RC, Malvin RL. Naloxone prevents increased vascular sensitivity in Goldblatt hypertensive rats. Clin Exp Hypertens A. 1990;12:1361-1376. [Medline] [Order article via Infotrieve]

7. Soltis EE, Katovich MJ. Alterations in vascular and ß adrenergic responsiveness in the Dahl rat: a preliminary report. Res Commun Chem Pathol Pharmacol. 1986;51:3-10. [Medline] [Order article via Infotrieve]

8. Konishi M, Su C. Role of endothelium in dilator responses of spontaneously hypertensive rat arteries. Hypertension. 1983;5:881-886. [Abstract/Free Full Text]

9. Lockette W, Otsuka Y, Carretero O. The loss of endothelium-dependent vascular relaxation in hypertension. Hypertension. 1986;8(suppl II):II-61-II-66.

10. Hongo K, Nakagomi T, Kassell NF, Sasaki T, Lehman M, Vollmer DG, Tsukahara T, Ogawa H, Tomer J. Effects of aging and hypertension on endothelium-dependent vascular relaxation in rat carotid artery. Stroke. 1988;19:892-897. [Abstract/Free Full Text]

11. Van de Voorde J, Vanheel B, Leusen I. Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats. Circ Res. 1992;70:1-8. [Abstract/Free Full Text]

12. Martin W, Furchgott RF, Villani GM, Jothianandan D. Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther. 1986;237:529-538. [Abstract/Free Full Text]

13. Berkenboom G, Unger P, Fang ZY, Fontaine J. Endothelium-derived relaxing factor and protection against contraction to norepinephrine in isolated canine and human coronary arteries. J Cardiovasc Pharmacol. 1991;17(suppl 3):S127-S132.

14. Schiffrin EL. Reactivity of small blood vessels in hypertension: relation with structural changes. State-of-the-art lecture. Hypertension. 1992;19(suppl II):II-1-II-9.

15. Bohlen HG. Localization of vascular resistance changes during hypertension. Hypertension. 1986;8:181-183. [Free Full Text]

16. Dohi Y, Thiel MA, Buhler FR, Luscher TF. Activation of endothelial L-arginine pathway in resistance arteries: effect of age and hypertension. Hypertension. 1990;15:170-179. [Abstract/Free Full Text]

17. Diederich DA, Yang Z, Buhler FR, Luscher TF. Impaired endothelium-dependent relaxations in hypertensive resistance arteries involve the cyclooxygenase pathway. Am J Physiol. 1990;258:H445-H451. [Abstract/Free Full Text]

18. Dohi Y, Luscher TF. Altered intra- and extraluminal effects of 5-hydroxytryptamine in hypertensive mesenteric resistance arteries: contribution of the endothelium and smooth muscle. J Cardiovasc Pharmacol. 1991;18:278-284. [Medline] [Order article via Infotrieve]

19. Jameson M, Dai F-X, Luscher T, Skopec J, Diederich A, Diederich D. Endothelium-derived contracting factors in resistance arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993;21:280-288. [Abstract/Free Full Text]

20. Deng LY, Schiffrin EL. Morphological and functional alterations of mesenteric small resistance arteries in early renal hypertension in rats. Am J Physiol. 1991;261:H1171-H1177. [Abstract/Free Full Text]

21. Li J, Bukoski RD. Endothelium-dependent relaxation of hypertensive resistance arteries is not impaired under all conditions. Circ Res. 1993;72:290-296. [Abstract/Free Full Text]

22. King CM, Webb RC. The endothelium partially obscures enhanced microvessel reactivity in DOCA hypertensive rats. Hypertension. 1988;12:420-427. [Abstract/Free Full Text]

23. Bockman CS, Jeffries WB, Pettinger WA, Abel PW. Enhanced release of endothelium-derived relaxation factor in mineralocorticoid hypertension. Hypertension. 1992;20:304-313. [Abstract/Free Full Text]

24. Bruner CA. Vascular responsiveness in rats resistant to aldosterone-salt hypertension. Hypertension. 1992;20:59-66. [Abstract/Free Full Text]

25. Osol G, Cipolla M, Knutson S. A new method for mechanically denuding the endothelium of small (50-150 µm) arteries with a human hair. Blood Vessels. 1989;26:320-324. [Medline] [Order article via Infotrieve]

26. Tallarida RJ, Murray RB. Manual of Pharmacologic Calculations With Computer Programs. 2nd ed. New York, NY: Springer-Verlag; 1987.

27. Smith JM, Jones SB, Bylund DB, Jones AW. Characterization of the alpha-1 adrenergic receptors in the thoracic aorta of control and aldosterone hypertensive rats: correlation of radioligand binding with potassium efflux and contraction. J Pharmacol Exp Ther. 1987;241:882-890. [Abstract/Free Full Text]

28. Storm DS, Webb RC. -Adrenergic receptors and ⁴⁵Ca⁺⁺ efflux in arteries from deoxycorticosterone acetate hypertensive rats. Hypertension. 1992;19:734-738. [Abstract/Free Full Text]

29. Bohr DF, Dominiczak AF, Webb RC. Pathophysiology of the vasculature in hypertension. Hypertension. 1991;18(suppl III):III-69-III-75.

30. McMahon EG, Paul RJ. Calcium sensitivity of isometric force in intact and chemically skinned aortas during the development of aldosterone-salt hypertension in the rat. Circ Res. 1985;56:427-435. [Abstract/Free Full Text]

31. Jones AW, Geisbuhler BB, Shukla SD, Smith JM. Altered biochemical and functional responses in aorta from hypertensive rats. Hypertension. 1988;11:627-634. [Abstract/Free Full Text]

32. Urabe M, Kawasaki H, Takasaki K. Effect of endothelium removal on the vasoconstrictor response to neuronally released 5-hydroxytryptamine and noradrenaline in the rat isolated mesenteric and femoral arteries. Br J Pharmacol. 1991;102:85-90. [Medline] [Order article via Infotrieve]

33. Stallone JN. Role of endothelium in sexual dimorphism in vasopressin-induced contraction of rat aorta. Am J Physiol. 1993;265:H2073-H2080. [Abstract/Free Full Text]

34. Garland CJ, McPherson GA. Evidence that nitric oxide does not mediate the hyperpolarization and relaxation to acetylcholine in the rat small mesenteric artery. Br J Pharmacol. 1992;105:429-435. [Medline] [Order article via Infotrieve]

35. Stoltz JF, Zannad F. Blood rheology in hypertension. J Hypertens. 1992;10(suppl 5):S69-S78.

36. Thomas GR, Walder CE, Thiemermann C, Vane JR. Regional vascular resistance and hemodynamics in the spontaneously hypertensive rat: the effect of bradykinin. J Cardiovasc Pharmacol. 1990;15:211-217. [Medline] [Order article via Infotrieve]

37. Noris M, Morigi M, Donadelli R, Aieloo S, Foppolo M, Todeschini M, Orisio S, Remuzzi G, Remuzzi A. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res. 1995;76:536-543. [Abstract/Free Full Text]

38. Luff SE, McLachlan EM. Frequency of neuromuscular junctions on arteries of different dimensions in the rabbit, guinea pig and rat. Blood Vessels. 1989;26:95-106. [Medline] [Order article via Infotrieve]

39. Reid JL, Zivin JA, Kopin IJ. Central and peripheral adrenergic mechanisms in the development of deoxycorticosterone-saline hypertension in rats. Circ Res. 1975;37:569-579. [Abstract/Free Full Text]

40. Bouvier M, de Champlain J. Increased apparent norepinephrine release rate in anesthetized DOCA-salt hypertensive rats. Clin Exp Hypertens A. 1985;7:1629-1645. [Medline] [Order article via Infotrieve]

41. Nakaki T, Otsuka Y, Kato R. Tension-induced release of endothelium-derived relaxation factors: possible role in establishment of desensitization of norepinephrine-induced contraction in rat aorta. Jpn J Pharmacol. 1990;54:491-494.[Medline] [Order article via Infotrieve]

42. Liu SF, Crawley DE, Evans TW, Barnes PJ. Endogenous nitric oxide modulates adrenergic neural vasoconstriction in guinea-pig pulmonary artery. Br J Pharmacol. 1991;104:565-569. [Medline] [Order article via Infotrieve]

43. Kaneko K, Sunano S. Involvement of -adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta. Eur J Pharmacol. 1993;240:195-200. [Medline] [Order article via Infotrieve]

44. Hwa JJ, Ghibaudi L, Williams P, Chatterjee M. Comparison of acetylcholine-dependent relaxation in large and small arteries of rat mesenteric vascular bed. Am J Physiol. 1994;266:H952-H958. [Abstract/Free Full Text]

45. Deussen A, Bading B, Kelm M, Schrader J. Formation and salvage of adenosine by microvascular endothelial cells. Am J Physiol. 1993;264:H692-H700. [Abstract/Free Full Text]

46. Nakane T, Chiba S. Pharmacological analysis of vasodilation induced by extracellular adenosine 3',5'-cyclic monophosphate in the isolated and perfused canine coronary artery. J Pharmacol Exp Ther. 1993;264:1253-1261.[Abstract/Free Full Text]

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