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Endocrinology Vol. 145, No. 4 1571-1578
Copyright © 2004 by The Endocrine Society

Characterization of Vasoconstrictor Responses in Small Bovine Adrenal Cortical Arteries in Vitro

David X. Zhang, Kathryn M. Gauthier and William B. Campbell

Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Address all correspondence and requests for reprints to: William B. Campbell, Ph.D., Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail: wbcamp{at}mcw.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The adrenal gland is highly vascularized with tightly regulated blood flow that is closely correlated with steroidogenesis. Mechanisms involved in the regulation of adrenal blood flow and vascular tone are largely unknown. The present study characterizes the contractile responses of isolated small cortical arteries from bovine adrenal glands. In endothelium-intact arteries, K+, the thromboxane mimetic U46619, 5-hydroxytryptamine (5-HT), and endothelin-1 (ET-1) induced concentration-dependent contractions, whereas phenylephrine, norepinephrine, and ACTH were without effect. The EC50s for K+, U46619, 5-HT, and ET-1 were 45 ± 3 mM, 150 ± 24 nM, 370 ± 38 nM, and 2.8 ± 0.8 nM, respectively. Contractions induced by U46619, 5-HT, and ET-1 were blocked by the thromboxane receptor antagonist SQ 29,548, the 5-HT2A receptor antagonist ketanserin, and the ETA receptor antagonist BQ 123, respectively. Removal of the endothelium caused a marked leftward shift of concentration responses to high K+, U46619, 5-HT, and ET-1, and revealed contractile responses to phenylephrine and norepinephrine. In U46619-preconstricted arteries, BQ 123 converted ET-1-induced contractions to relaxations (maximal relaxation of 57 ± 8%), which were subsequently blocked by the ETB receptor antagonist BQ 788. The ETB-mediated relaxations were endothelium dependent and inhibited by the nitric oxide synthase inhibitor N-nitro-L-arginine, the cytochrome P450 inhibitor SKF 525A, and high extracellular K+, but not by the cyclooxygenase inhibitor indomethacin. These results demonstrate that small adrenal cortical arteries are highly responsive to various vasoconstrictor agents. The forceful contractile responses of these arterioles are consistent with their potential role in the regulation of adrenal blood flow.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADRENAL GLAND is a highly vascularized organ with a well-regulated blood supply that is maintained even in severe hemorrhage. The mechanisms regulating adrenal vascular tone and blood flow are largely unknown but probably involve a range of neural, humoral, and local mediators (1, 2, 3, 4). For example, stimulation of the splanchnic nerve increases adrenal blood flow in both dogs and calves, an effect that may be mediated by the release of neuropeptides such as vasoactive intestinal peptide, Met-enkephalin, and calcitonin gene-related peptide (5, 6). Hormones such as ACTH and CRH also increase adrenal cortical blood flow (7, 8, 9). With regard to regional regulation, adrenal blood flow responds to changes in O2 tension (10) and to a number of endogenous vasoactive substances such as endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), catecholamines, and nitric oxide (NO) (2, 8, 11, 12).

Accumulating evidence indicates that changes in blood flow through the adrenal cortex is an important local regulatory mechanism of adrenal steroidogenesis (1, 2, 3, 4). According to Vinson et al. (1), there is a direct correlation between adrenal blood flow and steroid secretion. Changes in adrenal blood flow carries obvious implications for both the delivery of stimulants (i.e. ACTH) to steroidogenic cells and the export of secretory products (i.e. corticosterone and aldosterone) into the systemic circulation. Additionally, changes in adrenal blood flow can be a direct steroidogenic stimulant. In the isolated perfused rat adrenal gland, an increase in the flow rate of stimulant-free perfusate induces a significant increase in corticosterone secretion (13), thus further supporting the role of adrenal blood flow in the regulation of steroid hormone release.

Until now, much of our understanding of the regulation of adrenal blood flow comes from studies on the perfused adrenal gland. Studies have not directly assessed the vascular responsiveness of the adrenal arteries in vitro. Although adrenal perfusion studies approach physiological conditions, it is often difficult to determine whether hormone- or drug-induced changes in blood flow are due to direct vascular actions or occur secondary to alterations of steroid hormone secretion or the release of other vasoactive agents from the adrenal tissue. In this respect, isolated arterial preparations provide a useful alternative to study the regulatory mechanisms of the adrenal circulation. The small arteries in the capsule and on the cortical surface of the adrenal gland have the size characteristics of resistance arteries (<500 µm internal diameter) found in a range of vascular beds (14) and are the only adrenal arteries with a thick vascular wall (1). Accordingly, these small arteries have been suggested to be a primary site of control of blood flow in the adrenal cortex (1). Despite their clear importance in the regulation of adrenal blood flow, vascular reactivity of these small arteries has received little attention. As an initial approach, this study presents a model for the study of isolated small adrenal cortical arteries. We examined the responses of isolated small adrenal arteries to several endogenous and exogenous vasoconstrictors and ACTH, and the specific receptor subtypes mediating the actions of selected agonists and their mechanisms of actions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Wire myograph
Fresh bovine adrenals were obtained from a local abattoir, placed in ice-cold HEPES solution, and transported immediately to the laboratory. Small adrenal cortical arteries closely attached to the adrenal surface (200–300 µm) were carefully dissected and cleaned of connective tissues. Isolated arterial segments were threaded on two stainless steel wires (40 µm diameter) and mounted on a four-chamber wire myograph (model 610M, Danish Myo Technology A/S) (15). One wire was attached to a micromanipulator and the other to a force transducer, which allows for the determination of the wall force when the arteries were stretched. Arteries were set to an initial luminal diameter at which passive tension was first measurable, and equilibrated in physiological saline solution (PSS), bubbled with 95% O2/5% CO2, at 37 C for 30 min. Then arteries were gradually stretched to a resting tension of 1 millinewton (mN), which was the optimal preload for active tension development as determined by length-tension relation using the classic Laplace relationship approach (15) and the length-tension curve method (16). After a further 30-min equilibration, the potassium-substituted PSS (K-PSS; 145 mM K+) was added to the bath for 3–5 min and rinsed (for two times) to activate the contractile properties of arteries and to test the vessel responsiveness. After K-PSS stimulation, arteries were allowed to equilibrate for another 30 min before the initiation of experimental protocols.

Experimental protocol
Cumulative concentration-responses of small adrenal arteries to the following agonists were assessed on the basal tension: potassium chloride (KCl, 20–100 mM), the thromboxane A2 mimetic U46619 (10-9–10-6 M), 5-HT (10-8–3 x 10-6 M), ACTH (10-12–10-8 M), phenylephrine (10-9–10-5 M), norepinephrine (10-9–10-5 M), ET-1 (10-11–5 x 10-8 M), and the selective ETB agonist IRL 1620 (10-11–10-7 M). To evaluate relaxation responses to ET-1 or IRL1620, a submaximal concentration of U46619 (100–300 nM) was added to the bath to preconstrict the arteries to 50–75% of maximal K-PSS contraction. Cumulative concentrations of ET-1 (10-11–5 x 10-8 M), or IRL 1620 (10-11–10-7 M) were then added. To identify specific receptors involved in contractile or relaxation responses to an agonist, arteries were rinsed and pretreated for 15–30 min with a corresponding receptor antagonist, and concentration responses were repeated. Receptor antagonists used in this study included SQ 29,548 (10 µM), a thromboxane receptor antagonist, ketanserin (10 µM), a selective 5-HT2A receptor antagonist, phentolamine (10 µM), a selective {alpha}-adrenergic receptor blocker, BQ 123 (5 µM), a selective ETA receptor antagonist, and BQ 788 (5 µM), a selective ETB receptor antagonist. To examine the possible role of NO, prostacyclin and cytochrome P450 metabolites of arachidonic acid in vascular responses, arteries were pretreated for 15 min with N-nitro-L-arginine (L-NA) (30 µM), an endothelial NO synthase (NOS) inhibitor, indomethacin (10 µM), a cyclooxygenase inhibitor, and/or SKF 525A (10 µM), a cytochrome P450 inhibitor (17, 18, 19). To examine the contribution of potassium channels to the action of IRL 1620, arteries were preconstricted with K-PSS and the concentration response to IRL 1620 was then determined.

Experiments were performed on arteries with intact endothelium. Where indicated, the endothelium was removed by gently rubbing the intimal surface of the vessel with the human hair while a small passive tension (0.2 mN) was applied (20). The endothelium was considered intact if acetylcholine (1 µM) caused more than 80% relaxation of arteries precontracted with U46619 and effectively removed (denuded) if acetylcholine induced less than 10% relaxation.

Data analysis
Contractile responses to an agonist are expressed as the percentage of the maximal contraction induced by the corresponding agonist or U46619 as indicated. Relaxations are expressed as a percentage relaxation relative to U46619-precontraction, with 100% relaxation representing basal tension. Where appropriate, the concentration of the drug required to produce 50% of the maximal response (EC50) was calculated from the concentration-response curves by fitting data to a logistic sigmoid equation using the GraphPad Prism program (GraphPad, San Diego, CA). Data are presented as mean ± SEM. Significance of mean values between and within multiple groups was evaluated by ANOVA followed by the Student-Newman-Keuls multiple comparison test. P < 0.05 was considered statistically significant.

Drugs and solutions
5-HT, ACTH, phenylephrine, norepinephrine, ketanserin and phentolamine, L-NA, indomethacin, and SKF 525A were purchased from Sigma (St. Louis, MO). ET-1, IRL 1620, BQ 123, and BQ 788 were obtained from American Peptide Co. (Sunnyvale, CA) and U46619 from Cayman Chemical Co. (Ann Arbor, MI) SQ 29,548 was a gift from the Squibb Institute (Princeton, NJ) for investigational use. Ketanserin was prepared as 50 mM stock, and BQ 123 and BQ 788 were prepared as 5 mM stock in dimethylsulfoxide. Indomethacin was prepared as 10 mM stock in 0.2 M Na2CO3. U46619 was prepared as 2 mM stock, and SKF 525A was prepared as 20 mM stock in 95% ethanol. All other drugs were prepared to their appropriate stock concentrations in distilled water. Subsequent dilutions were made with the HEPES solution. We used PSS of the following composition (in mM): NaCl 119, KCl 4.7, CaCl2 2.5, MgSO4 1.17, NaHCO3 24, KH2PO4 1.18, EDTA 0.026, and glucose 5.5. K-PSS was prepared by equimolar substitution of NaCl by KCl. The HEPES solution consisted of (in mM): NaCl 135, KCl 5.6, CaCl2 1.6, MgCl2 1.0, HEPES 10, and glucose 5.5 (pH 7.4).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Length-tension characteristics
To obtain the maximal contractile response, we determined the length-tension relationship of isolated small adrenal cortical arteries. Two methods for determining length-tension relation were assessed: the classic Laplace relationship approach, and the length-tension curve method using the thromboxane A2 mimetic U46619 (300 nM) as the vasoconstrictor. The two methods produced similar normalized length or basal tension for the arteries studied. The active tension reached a peak as the internal circumference of the artery approached 0.8–0.9 of L100 (the internal circumference of the vessel when exposed to a luminal pressure of 100 mm Hg) or as the passive tension reached an average of 1 mN (data not shown). Because the optimal tension was reproducible among different preparations, the initial basal tension of approximately 1 mN was used in subsequent studies.

Responses to KCl, U46619, 5-HT, and ACTH
KCl (20–100 mM), U46619 (10-9–10-6 M), and 5-HT (10-8-3 x 10-6 M) produced concentration-dependent contractions in the small adrenal cortical arteries with EC50’s of 45 ± 3 mM, 150 ± 24 nM and 370 ± 38 nM, respectively (Figs. 1Go, A–C, and 2Go, A–C). The active tension to 100 mM KCl (7.6 ± 1.1 mN) was approximately 70% of that to K-PSS (10.9 ± 0.6 mN) (n = 8). The maximal U46619-induced contraction of 9.6 ± 1.5 mN at 10-6 M was approximately 90% that of the K-PSS (10.8 ± 1.9 mN) (n = 11). Pretreatment of arteries with SQ 29,548 (10 µM), a thromboxane receptor antagonist, abolished the U46619-induced contraction. The maximal 5-HT-induced contraction of 11.1 ± 1.7 mN at 3 x 10-6 M was approximately 80% that of the K-PSS (13.6 ± 2.8 mN) (n = 12). The 5-HT-induced contraction was markedly inhibited by ketanserin (10 µM), a 5-HT2A receptor antagonist. Removal of the endothelium caused a marked leftward shift of concentration-responses to KCl, U46619 and 5-HT, as well as an increase in maximal contractions. In endothelium-denuded arteries, the maximal contractions to KCl, U46619, and 5-HT were increased to 11.9 ± 2.3 mN (n = 8), 12.0 ± 1.4 mN (n = 16) and 16.0 ± 2.3 mN (n = 8), respectively. In contrast to these potent vasoconstrictors, ACTH had no effect on vascular tone in either endothelium-intact or -denuded arteries (n = 8; Fig. 2DGo).



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  		FIG. 1. Typical tracings of the contractile effects of KCl, U46619, 5-HT and ET-1 in small bovine adrenal cortical arteries.

 


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  		FIG. 2. Concentration-response curves for (A) KCl, (B) U46619, (C) 5-HT, and (D) ACTH in small bovine adrenal cortical arteries. The thromboxane receptor antagonist SQ29,548 (10 µM) and 5-HT2A receptor antagonist ketanserin (10 µM) blocked the contractions induced by U46619 and 5-HT, whereas removal of the endothelium enhanced the contractions induced by KCl, U46619 and 5-HT. *, P < 0.05 vs. control. Results are expressed as percentage of the maximal contraction induced by the corresponding agonist or U46619 (for ACTH). All values are mean ± SEM (n = 8–16).

 
Responses to phenylephrine and norepinephrine
As shown in Fig. 3Go, A and B, phenylephrine (10-9–10-5 M) and norepinephrine (10-9–10-5 M) did not induce contractions in small adrenal cortical arteries under basal conditions. Removal of the endothelium unmasked contractile responses to phenylephrine at 10-5 M and to norepinephrine at 10-6–10-5 M. The maximal contraction to phenylephrine and norepinephrine were 17% and 53% that of U46619. The contractile responses to phenylephrine and norepinephrine in endothelium-denuded arteries were eliminated by the selective {alpha}-adrenergic receptor antagonist phentolamine (10 µM). L-NA (30 µM) and indomethacin (10 µM) alone or in combination also significantly enhanced the contractile response to norepinephrine (Fig. 3CGo).



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  		FIG. 3. Concentration-response curves for (A) phenylephrine and (B and C) norepinephrine in small bovine adrenal cortical arteries. Removal of the endothelium unmasked the contractile responses to phenylephrine and norepinephrine, which was blocked by the {alpha}-receptor antagonist phentolamine (10 µM). L-NA (30 µM) and indomethacin (Indo, 10 µM) alone or in combination also significantly enhanced the contractile response to norepinephrine. *, P < 0.05 vs. control; #, P < 0.05 vs. denuded. Results are expressed as percentage of the maximal contraction induced by U46619. All values are mean ± SEM (n = 6–8).

 
Responses to ET-1 and IRL 1620
ET-1 (10-11–5 x 10-8 M) produced concentration-dependent contractions in small adrenal cortical arteries under basal conditions, with an EC50 of 2.8 ± 0.8 nM (Figs. 1DGo and 4AGo). The maximal contraction of 9.7 ± 1.2 mN at 5 x 10-8 M was approximately 100% that of K-PSS (9.8 ± 1.3 mN) (n = 10). BQ 123 (5 µM), a selective ETA antagonist, blocked ET-1-induced contraction. BQ 788 (5 µM), a selective ETB antagonist, had no effect on ET-1 responses. Removal of the endothelium induced a marked leftward shift of concentration-responses to ET-1. The maximal contraction to ET-1 in denuded arteries was 8.4 ± 1.1 mN at 5 x 10-8 M. Figure 4BGo shows ET-1 responses in U46619-preconstricted arteries. ET-1 induced concentration-dependent contractions, which converted to relaxation responses by BQ 123 (5 µM). Maximal relaxations of 57 ± 8% were observed at 10-8 M ET-1. Treatment of arteries with BQ 123 plus BQ 788 (5 µM) abolished any effect of ET-1. As in resting arteries, BQ 788 alone had no significant effect on ET-1-induced contractions.



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  		FIG. 4. A, Concentration-response curves for ET-1 in small bovine adrenal cortical arteries. The ETA receptor antagonist BQ 123 (5 µM) blocked the contractions induced by ET-1, whereas removal of the endothelium enhanced the contractions induced by ET-1. *, P < 0.05 vs. control. Results are expressed as percentage of the maximal contraction induced by ET-1. B, Concentration-response curves for ET-1 in arteries precontracted with a submaximal concentration of U46619. BQ 123 (5 µM) reversed ET-1-induced contractions to relaxations, which was subsequently blocked by the ETB receptor antagonist BQ 788 (5 µM). *, P < 0.05 vs. control; #, P < 0.05 vs. BQ 123. Results are expressed as percentage change in tension relative to U46619 preconstriction. All values are mean ± SEM (n = 8–11).

 
To further characterize the role of ETB receptor in ET-1-induced relaxations in small adrenal arteries, the responses to IRL 1620, a selective ETB agonist, were determined. As shown in Fig. 5AGo, IRL 1620 did not alter vascular tension under basal conditions. However, in U46619-precontracted arteries, IRL 1620 induced concentration-related relaxations, which were blocked by BQ 788 (5 µM). Maximal relaxations to IRL 1620 averaged 60 ± 7%. (Fig. 5BGo). This did not differ significantly from ET-1-induced relaxations in the presence of BQ 123 (57 ± 8%).



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  		FIG. 5. A, Concentration-response curve for IRL 1620 in small bovine adrenal cortical arteries. Results are expressed as the percentage of the maximal contraction induced by U46619. B, Concentration-response curve for IRL 1620 in arteries precontracted with a submaximal concentration of U46619. BQ 788 (5 µM) significantly inhibited the relaxation responses to IRL 1620. *, P < 0.05 vs. control. Results are expressed as percentage relaxation relative to U46619 preconstriction. All values are mean ± SEM (n = 8–12).

 
Mechanisms of IRL 1620-induced relaxation
We examined the roles of three major endothelium-derived relaxing factors, i.e. NO, prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs), in IRL 1620-induced relaxations. Small adrenal cortical arteries were pretreated with either L-NA (30 µM), an endothelial NOS inhibitor, indomethacin (10 µM), a cyclooxygenase inhibitor, or SKF 525A (10 µM), a cytochrome P450 and EDHF inhibitor (17, 18, 19), and responses to IRL 1620 were determined (Fig. 6AGo). IRL 1620-induced relaxations were significantly inhibited by L-NA, as indicated by a rightward shift of the concentration-responses to IRL 1620. In contrast, indomethacin had no effect. SKF 525A in the presence of L-NA and indomethacin caused a further rightward shift of concentration-responses to IRL 1620 as compared with L-NA alone, as well as a decrease in the maximal response to IRL 1620. Removal of the endothelium abolished IRL 1620-induced relaxations (Fig. 6BGo). We also determined the role of K+ channels in IRL 1620 responses using high extracellular K+ (K-PSS). In arteries precontracted with K-PSS, IRL 1620 did not induce relaxations (Fig. 6BGo).



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  		FIG. 6. A, Effects of L-NA, indomethacin (Indo), and SKF 525A (SKF) on IRL 1620-induced relaxations in small bovine adrenal cortical arteries. L-NA (30 µM) significantly inhibited the relaxation responses to IRL-1620. Further inhibition was obtained by the combination of L-NA, indomethacin (10 µM) and SKF 525A (10 µM). Indo alone was without effect. *, P < 0.05 vs. control; #, P < 0.05 vs. L-NA. B, Effects of high K+ and endothelial removal on IRL 1620-induced relaxations. IRL 1620 did not induce significant relaxations in K-PSS (145 mM K+)-precontracted arteries or in endothelium-denuded arteries. *, P < 0.05 vs. control. All values are mean ± SEM (n = 6–8).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study is the first to characterize the pharmacological profiles of isolated small adrenal cortical arteries from the bovine adrenal gland to a number of endogenous and exogenous vasoconstrictors and ACTH. We demonstrated that these arteries are highly responsive to high K+, U46619, 5-HT, and ET-1 but are relatively insensitive to phenylephrine, norepinephrine, and ACTH. The contractile responses to U46619, 5-HT, and ET-1 are mediated via thromboxane, 5-HT2A and ETA receptors, respectively. In addition, the contractile responses are markedly modulated by the endothelium.

The regulation of vascular tone and blood flow in the adrenal cortex is complex and involves a range of humoral, neural, and local mediators (1, 2, 3, 4). In addition to circulating hormones such as ACTH, various bioactive substances are produced in the adrenal cortex by nerve endings, endothelial cells, chromaffin cells, cells of immune system (i.e. mast cells), or adrenal steroid-secreting cells. For example, norepinephrine is released by catecholaminergic nerve fibers that are located in the capsular and subcapular region (21, 22). 5-HT-like immunoreactivity has been detected in perivascular mast cells in the rat and human adrenal glands (8, 23). Thromboxane A2, an arachidonic acid metabolite released by platelets, mast cells and vascular endothelial cells (24, 25), was also found to be synthesized by bovine microvascular endothelial cells (our unpublished observation). ET-1, a potent vasoactive peptide secreted by vascular endothelial cells (26), is released from adrenal capillary endothelial cells in response to peptide agonists (27) and from the perfused rat adrenal gland in response to changes in flow (11). In addition to endothelium-derived ET-1, adrenal steroid-secreting cells may also synthesize and release ET-1 (28). Because these local factors are released in the vicinity of steroidogenic cells and cortical arterioles, there has been considerable interest in studying their roles in the regulation or modulation of steroid production and blood flow. To date, the regulation of steroid production by these local mediators is relatively well understood, whereas the local regulation of adrenal cortical vascular tone and blood flow remains largely unknown. The present study provides the first evidence that small adrenal cortical arteries are highly reactive to factors such as ET-1, 5-HT, and thromboxane, thus supporting their potential role in the local regulation of vascular tone and blood flow in the adrenal cortex.

Our data for the constrictor effects of ET-1 in small adrenal cortical arteries are generally in agreement with their stimulatory actions in various other vascular beds. In this study, the ETA receptor selective antagonist BQ 123 converted ET-1-induced constriction to a relaxation, which was subsequently blocked by the ETB receptor selective antagonist BQ 788. The selective ETB receptor agonist IRL 1620 induced only a potent relaxation, which was abrogated by BQ 788. Removal of the endothelium abolished ETB-mediated relaxations but left intact ETA-mediated constrictions. These results indicate that ET-1 activates both smooth muscle ETA and endothelial ETB receptors to evoke contraction and relaxation, respectively, in small adrenal cortical arteries. Under our experimental conditions, ETA-mediated contraction dominates. These findings are in agreement with a study by Mozzocchi et al. (29), which showed that, in the in situ rat adrenal perfusion model, ET-1 infusion caused a decrease in flow rate via the activation of ETA receptor. ET-1 may also activate ETB receptor in this model to induce an increase in flow rate; however, this effect could be masked by the predominant effect of ETA receptor activation. Taken together, these results suggest that ET-1-induced vasoconstriction on small cortical arteries may contribute to the changes of adrenal blood flow induced by this peptide.

Endothelial ETB receptors mediate relaxation by stimulating the release of relaxing factors from the endothelium (30, 31). Three major endothelium-derived relaxing factors include NO, prostacyclin, and EDHFs that include the cytochrome P450 metabolites, the epoxyeicosatrienoic acids (17, 18, 19). In the present study, the NOS inhibitor L-NA significantly inhibited IRL 1620-induced relaxations, indicating that NO is involved in the relaxation response to ETB activation in small adrenal cortical arteries. These results are in line with those of a previous study showing that L-NA blocks ETB-mediated increases in the flow rate of perfused rat adrenal glands (29). Moreover, we found that the cytochrome P450 inhibitor SKF 525A caused a further inhibition of the L-NA-resistant relaxations to IRL. The cyclooxygenase inhibitor indomethacin was without effect. This suggests that cytochrome P450, but not cyclooxygenase metabolites, also contribute to the relaxations induced by ETB activation. The finding that IRL 1620-induced relaxations were abolished by high K+ indicates that the membrane hyperpolarization is involved in the relaxation response to IRL 1620. This supports the possible role of cytochrome P450 metabolites as EDHFs in ETB-mediated relaxations.

In contrast to the potent constrictor effect in other vascular beds such as renal and mesenteric arteries, norepinephrine did not constrict small adrenal cortical arteries under normal condition (i.e. with intact endothelium). The lack of significant vasoconstriction in response to {alpha}-adrenergic agonists has also been reported in perfused dog adrenals (2). The relative insensitivity of adrenal vessels to adrenergic stimulation has been suggested to be important in maintaining sufficient blood flow through the adrenal gland under stress conditions (i.e. hemorrhage) when a large amount of epinephrine and norepinephrine is released from the chromaffin cells in the adrenal gland (32). However, a potential role of adrenergic agonists in the regulation of vascular tone cannot be excluded, considering that cortical arterioles are densely innervated with noradrenergic neurons (21, 22). In support of this possibility, we revealed a norepinephrine-induced contraction in adrenal arteries pretreated with NOS and cyclooxygenase inhibitors or in the presence of other vasoconstrictors such as U46619 (data not shown). Therefore, norepinephrine may be involved in the fine regulation of vascular tone in small cortical arteries.

ACTH stimulates increases in adrenal blood flow in a number of preparations (1, 2, 3, 4, 7, 8). Although it has been suggested that ACTH acts by constricting medullary arteries and diverting blood into the cortex, subsequent studies have demonstrated that ACTH induces an overall increase in the flow in the perfused rat adrenals, suggesting a decreased vascular resistance within the adrenal gland (13). Using the isolated artery preparation, we found that ACTH has no significant effect on the vascular tone of small adrenal cortical arteries, indicating that ACTH-induced increase in adrenal blood flow is not due to its direct effect on adrenal cortical vasculature but is probably secondary to relaxing factors from other adrenal cells. This finding is in agreement with that of previous studies by Hinson et al. (7, 8), indicating that ACTH-induced decrease in vascular resistance is due to the release of histamine and 5-HT from perivascular mast cells and the subsequent vasorelaxation induced by these compounds. Similar to ACTH, a discrepancy in vascular responses to 5-HT was observed between isolated arteries and in vivo preparations. We found that 5-HT induces a constriction in isolated small adrenal cortical arteries. A 5-HT-induced vasoconstriction has also been observed in isolated small adrenal cortical arteries from fetal sheep (33). However, 5-HT seems to have a vasodilator effect in vivo. It increases flow rate in the perfused adrenal gland of rat (8). The discrepancies between these findings may be due to differences in experimental conditions, and/or species variability. Alternatively, relaxations induced by 5-HT in vivo may occur secondary to the stimulation and release of other vasoactive mediators from the adrenal gland. Taken together, these observations indicate that the regulation of cortical blood flow in the adrenal gland is complex and may involve interactions among different types of cells within the adrenal cortex.

In the present study, removal of the endothelium markedly increased the sensitivity of small adrenal cortical arteries to high K+, U46619, 5-HT, and ET-1, as indicated by a leftward shift of concentration-response curves to these vasoconstrictors. In addition, endothelial denudation unmasked contractile responses to phenylephrine and norepinephrine. These results indicate that the endothelium is an important determinant of the extent of smooth muscle contraction in these small arteries. The endothelium-mediated inhibition of contractile responses to angiotensin II, {alpha}-adrenergic agonists, endothelin, and other vasoconstrictors has been described previously in a number of other vascular beds (34, 35, 36, 37). The mechanisms responsible for this modulation by the endothelium are currently unknown but could be due to the release of relaxing factors (i.e. NO and cyclooxygenase metabolites) that decrease the sensitivity of smooth muscle cells to vasoconstrictors. Metabolism and inactivation of the vasoconstrictors seem unlikely because the vasoconstrictors have diverse chemical structures (eicosanoids, amines, and peptide). Indeed, we found that NOS inhibitor L-NA and cyclooxygenase inhibitor indomethacin caused a leftward shift of concentration responses to norepinephrine and U46619 (data not shown). Using the perfused adrenal glands, a previous study has shown that the NOS inhibitor N-nitro-L-arginine methyl ester decreases the blood flow through the adrenal cortex and L-arginine reverses this effect (12). Therefore, it is possible that endothelium-derived relaxing factors such as NO participate in the local regulation of blood flow in the adrenal cortex by exerting a tonic dilatory effect on small cortical arteries.

Adrenal steroidogenesis and hormone secretion are regulated by direct trophic hormone stimulation as well as increases in adrenal blood flow. Revealing the direct effects of vasoactive agents on adrenal arterial tone is imperative to understanding the regulation of adrenal blood flow. The present study demonstrates that small adrenal cortical arteries are highly responsive to a number of vasoconstrictors such as high K+, U46619, 5-HT, and ET-1 but relatively insensitive to phenylnephrine, norepinephrine, and ACTH. The forceful contractile responses of these arterioles are consistent with their role in the regulation of adrenal cortical blood flow.


   Acknowledgments
 
We thank Dr. Zhi-Dong Ge for advice with the wire myographs and Gretchen Barg for secretarial assistance.


   Footnotes
 
This work was supported by a grant from the National Institute of Digestive and Kidney Diseases (DK-58145). D.X.Z. is a postdoctoral fellow of the American Heart Association, Greater Midwest Affiliate, and a recipient of the Jenkins Cardiovascular Research Fellowship.

Abbreviations: EDHF, Endothelium-derived hyperpolarizing factor; ET-1, endothelin-1; 5-HT, 5-hydroxytryptamine; K-PSS, potassium-substituted PSS; L-NA, N-nitro-L-arginine; mN, millinewton; NO, nitric oxide; NOS, NO synthase; PSS, physiological saline solution.

Received October 27, 2003.

Accepted for publication January 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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