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Endocrinology Vol. 145, No. 10 4532-4539
Copyright © 2004 by The Endocrine Society

Acetylcholine-Induced Relaxation and Hyperpolarization in Small Bovine Adrenal Cortical Arteries: Role of Cytochrome P450 Metabolites

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 present study characterizes the vascular responses of isolated small bovine adrenal cortical arteries to acetylcholine, an endogenous neurotransmitter in the adrenal gland. Acetylcholine (10–10 to 10–6 M) elicited a concentration-dependent relaxation, with a maximal relaxation of 96 ± 1% and EC50 of 4.2 nM. The relaxation was abolished by endothelial removal and attenuated by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 µM) but not by the cyclooxygenase inhibitor indomethacin (10 µM). The maximal relaxation and EC50 of acetylcholine in the presence of L-NA were 87 ± 4% and 22 nM, respectively. The acetylcholine-induced, indomethacin- and L-NA-resistant relaxation was eliminated by high K+ and markedly inhibited by the cytochrome P450 inhibitors SKF 525A (10 µM) and miconazole (10 µM). The maximal relaxations and EC50s with SKF 525A and miconazole were 56 ± 8 and 72 ± 2% and 0.8 and 0.5 µM, respectively. In indomethacin- and L-NA-treated arteries, acetylcholine induced a smooth muscle hyperpolarization, which was blocked by SKF 525A (3 ± 1 mV vs. 15 ± 2 mV of control). Arachidonic acid (10–9 to 10–5 M) and 14,15-epoxyeicosatrienoic acid (14,15-EET, 10–9 to 10–5 M), a cytochrome P450 metabolite of arachidonic acid, also evoked relaxations in small adrenal arteries, with maximal relaxations of 56 ± 4 and 90 ± 5%, respectively. The arachidonic acid-induced relaxation was blocked by SKF 525A. Using high-pressure liquid chromatography and gas chromatography/mass spectrometry analysis, EETs were identified in small adrenal arteries. These results demonstrate that acetylcholine is a potent vasodilator of small adrenal cortical arteries. The acetylcholine-induced relaxation is largely mediated by an endothelium-dependent hyperpolarization mechanism, presumably through cytochrome P450 metabolites of arachidonic acid.


   Introduction
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 adrenal steroidogenesis (1, 2, 3). Changes in the rate of blood flow through the adrenal cortex regulate both the delivery of major stimulants (i.e. angiotensin II and ACTH) to steroidogenic cells and the removal of the end products (i.e. aldosterone and corticosterone) from the gland. In addition, changes in adrenal blood flow can be a direct stimulant of steroid hormone release (4). Despite the important role of adrenal blood flow in adrenal steroid secretion, mechanisms involved in the regulation of adrenal vascular tone and blood flow are largely unknown.

Previous studies of adrenal vascular tone relied entirely on the perfused adrenal gland or adrenal blood flow in vivo. Studies in the intact adrenal gland indicate global changes in vascular tone but give no insight into the mechanisms regulating vascular tone due to the complexity of the gland. In the intact adrenal gland, vascular responses to an agonist could be due to a direct effect on the artery and/or an indirect effect secondary to hormones and metabolites released by adrenal cells. In this regard, isolated arterial preparations provide a useful alternative to study the factors regulating adrenal vascular tone and blood flow independent of steroidogenic cells.

Recently we reported an in vitro model of isolated small cortical arteries from the bovine adrenal gland and found that these small cortical arteries are highly responsive to various vasoconstrictor agents such as endothelin-1, 5-hydroxytryptamine, and the thromboxane mimetic U46619 (5). The contractile profiles of these isolated arterioles are consistent with their proposed important role in the regulation of adrenal vascular tone and blood flow and validate the use of these arterioles to examine the regulatory mechanisms of the adrenal circulation. As a further characterization of isolated small cortical arteries, the present study examined the vasodilator responses of these arteries to acetylcholine, a potent vasodilator in a number of vascular beds and an endogenous neurotransmitter in the adrenal cortex. Morphological and immunochemical evidence showed that the adrenal cortex is innervated with cholinergic neurons, primarily in the capsule and zona glomerulosa, and some nerve fibers terminate around blood vessels (6, 7, 8, 9, 10). We also explored the mechanisms of acetylcholine-induced relaxation with respect to the endothelium-derived relaxing factors such as nitric oxide (NO) and cytochrome P450 metabolites of arachidonic acid.


   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, Aarhus, N, Denmark) as we described previously (5). Briefly, 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. Arteries were then gradually stretched to a resting tension of 1 millinewton (mN) during an additional 30-min equilibration period. Thereafter, the preparation was stimulated two times with the potassium-substituted PSS (K-PSS; 145 mM K+) for 3–5 min at 10-min intervals. Arteries were then allowed to equilibrate for another 30 min before the initiation of experimental protocols.

To examine relaxation responses, submaximal concentrations of U46619 (100–300 nM) were added to the bath to precontract the arteries to 50–75% of maximal KPSS contraction. After the contraction reached steady state, cumulative concentration response curves to the following vasodilators were determined: acetylcholine (10–10 to 10–6 M), sodium nitroprusside (10–8 to 10–4 M), iloprost (10–10 to 10–5 M), 14,15-epoxyeicosatrienoic acid (14,15-EET, 10–9 to 10–5 M), or arachidonic acid (10–9 to 10–5 M). To examine the possible role of NO, prostacyclin and cytochrome P450 metabolites of arachidonic acid in relaxation responses, arteries were pretreated for 15–30 min with N-nitro-L-arginine (L-NA, 30 or 100 µM), an endothelial NO synthase (NOS) inhibitor, indomethacin (10 µM), a cyclooxygenase inhibitor, and/or SKF 525A (10 µM) or miconazole (10 µM), cytochrome P450 inhibitors (11, 12, 13). To examine the contribution of K+ channels to relaxation responses, arteries were pretreated for 20 min with iberiotoxin (100 nM), a blocker of large-conductance Ca2+-activated K+ channels or preconstricted with high K+, and the concentration responses to a vasodilator 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, with a small passive tension (0.2 mN) applied. The endothelium was considered intact if acetylcholine (1 µM) caused greater than 80% relaxation of arteries precontracted with U46619 and effectively removed (denuded) if acetylcholine induced less than 10% relaxation.

Metabolism of arachidonic acid
Small adrenal cortical arteries were isolated, cleaned of adhering connective tissue and fat, and cut into 3-mm-long segments, and incubated for 15 min at 37 C in HEPES buffer. Vessels were preincubated with vehicle (0.095% ethanol), SKF 525A (10 µM), or indomethacin (10 µM) for 10 min, followed by sequential addition of [14C] arachidonic acid (0.05 µCi, 100 nM) for 10 min and A23187 (10 µM) for another 10 min. Reactions were stopped by adding ethanol to a final concentration of 15%. The solutions were extracted on octadecasilyl solid-phase extraction column and dried under a stream of nitrogen gas. The extracts were redissolved in acetonitrile and chromatographed by reverse-phase HPLC with a Nucleosil C-18 reverse phase column (5 µm, 4.6 x 250 mm) (Phenomenex, Torrance, CA) as previously described (14). Solvent A was distilled water and solvent B contained acetonitrile: glacial acetic acid (999:1). A linear gradient from 50% solvent B in A to 100% B over 40 min was used with a flow rate of 1 ml/min. Column effluent was collected in 0.2-ml fractions and analyzed for radioactivity by liquid scintillation spectrometry. An additional set of samples was analyzed by HPLC/mass spectrometry (LC/MS) using an 1100 LC/MSD, SL model (Agilent Technologies, Berks, UK) as previously described (15). Arachidonic acid metabolites were resolved using a C18 (Kromasil, 250x 2 mm) column using water/acetonitrile with 0.005% acetic acid at a flow rate of 0.2 ml/min. The separation used a 60–80% acetonitrile in water linear gradient over 30 min followed by an increase to 100% over 5 min and held at 100% for 5 min. Drying gas flow was 12 liters/min at a temperature of 350 C, nebulizer pressure was 35 psig, vaporizer temperature was 325 C, and capillary voltage was 3000 V. The detection was made in the negative mode.

Membrane potential measurements
Membrane potential (Em) recordings were conducted as previously described (14). Briefly, arteries were mounted on the myograph and equilibrated in PSS for 60 min. Arteries were impaled from the adventitial side with glass microelectrodes (30–80 M{Omega} filled with 3 M KCl) connected to a high-impedance amplifier (Dagan Cell Explorer, Dagan Instruments). Criteria for a successful impalement included an abrupt drop in potential to a new steady-state value, which was maintained for a minimum of 5 sec, an Em value greater than –20 mV, and an abrupt return to the original baseline when the electrode was retracted from the tissue. The artery was treated with either vehicle, vehicle with U46619 (100 nM), vehicle with U46619 and acetylcholine (100 nM), SKF 525A (10 µM), SKF 525A with U46619, or SKF 525A with U46619 and acetylcholine, followed by the measurement of Em. All measurements were performed in the presence of indomethacin (10 µM) and L-NA (30 µM). Multiple successful impalements were obtained and averaged for each experimental protocol.

Data analysis
Relaxation responses are expressed as a percentage relaxation relative to U46619 or K-PSS precontraction, with 100% relaxation representing basal tension. Where appropriate, the EC50 concentration of the drug 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 differences between mean values was evaluated by Student t test or ANOVA followed by the Student-Newman-Keuls multiple comparison test. P < 0.05 was considered statistically significant.

Drugs and solutions
Acetylcholine, sodium nitroprusside, arachidonic acid (sodium salt), L-NA, indomethacin, iberiotoxin, SKF 525A, and miconazole were purchased from Sigma (St. Louis, MO). U46619 and iloprost were obtained from Cayman Chemical Co. (Ann Arbor, MI). 14,15-EET was synthesized as previously described (7). The following agents were prepared in 95% ethanol: U46619 as 2 mM stock, iloprost and 14,15-EET as 10 mM stock, and SKF 525A and miconazole as 20 mM stock. L-NA was prepared as 30 mM stock in 0.1 N HCl. Indomethacin was prepared as 10 mM stock in 0.2 M Na2CO3. Arachidonic acid was dissolved in distilled water previously sparged with nitrogen gas; stock solution and dilutions (with water) were made fresh daily for each experiment and stored on ice under nitrogen. All other drugs were prepared to their appropriate stock concentrations in distilled water. Subsequent dilutions were made with the HEPES solution. All solvents were HPLC grade and purchased from Burdick and Jackson, and [14C-U] arachidonic acid (920 mCi/mmol) was obtained from NEN Life Science Products (Boston, MA). We used PSS of the following composition (in millimoles): 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 millimoles): NaCl, 150; KCl, 5; CaCl2, 1.8; MgCl2, 1.0; HEPES, 10; and glucose, 5.5 (pH 7.4).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Relaxation responses to acetylcholine
We first examined responses to acetylcholine in small bovine adrenal cortical arteries precontracted with U46619 and the contribution of three major endothelium-derived relaxing factors, i.e. NO, prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs), to these responses. In U46619-precontracted arteries, acetylcholine (10–10 to 10–6 M) elicited concentration-dependent relaxations, with a maximal relaxation of 96 ± 1% and EC50 of 4.2 nM (Fig. 1AGo). Pretreatment of arteries with L-NA (30 µM) caused a rightward shift of the concentration-response curve to acetylcholine (maximal relaxation of 87 ± 4% and EC50 of 22 nM). High K+ (K-PSS) markedly inhibited relaxations to acetylcholine (maximal relaxation of 44 ± 5%) (Fig. 1CGo). In contrast, indomethacin (10 µM) was without effect (Fig. 1BGo). L-NA and indomethacin had no significant effect on basal tension. However, L-NA and indomethacin alone or in combination caused a leftward shift of concentration responses to U46619 (data not shown). Removal of the endothelium eliminated acetylcholine-induced relaxations (Fig. 1DGo).



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  		FIG. 1. Effect of L-NA (A), indomethacin (B), high K+ (C), and endothelial removal (D) on acetylcholine-induced relaxations of small bovine adrenal cortical arteries. Acetylcholine-induced relaxations were markedly attenuated in arteries pretreated with L-NA (30 µM for 30 min) and in K-PSS-contracted arteries. Indomethacin (10 µM for 30 min) alone was without effect. Acetylcholine did not induce relaxations in endothelium-denuded arteries. *, P < 0.05 vs. control/endothelium-intact. All values are mean ± SEM (n = 5–16).

 
Effects of cytochrome P450 inhibitors on L-NA- and indomethacin-resistant relaxations to acetylcholine
To confirm the role of EDHFs in acetylcholine-induced relaxations and investigate whether cytochrome P450 metabolites may function as EDHFs in small bovine adrenal arteries, vascular responses to acetylcholine were determined in arteries pretreated with L-NA (30 µM) and indomethacin (10 µM). Consistent with the results in Fig. 1Go, a significant portion of relaxation to acetylcholine was resistant to the combination of L-NA and indomethacin (Fig. 2Go). Higher concentration of L-NA (100 µM) did not further inhibit acetylcholine-induced relaxations. The maximal relaxations in the presence of 30 and 100 µM LNA plus indomethacin were 94 ± 1 and 90 ± 2%, respectively. The L-NA- and indomethacin-resistant relaxation was abolished by high K+ (K-PSS or 60 mM KCl) and iberiotoxin (100 nM) and markedly inhibited by two structure-unrelated cytochrome P450 inhibitors, SKF 525A (10 µM) and miconazole (10 µM). Maximal relaxations and EC50s in the presence of SKF 525A and miconazole were 57 ± 8 and 72 ± 2% and 0.8 and 0.5 µM, respectively. Although SKF 525A and miconazole had no significant effect on basal tension, they decreased U46619 sensitivity, and concentrations of U46619 were adjusted to obtain the similar levels of preconstriction as controls.



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  		FIG. 2. Effect of SKF 525A, miconazole, and high K+ on acetylcholine-induced relaxations of small bovine adrenal cortical arteries. Arterial segments were pretreated with L-NA (30 µM for 30 min) and indomethacin (10 µM for 30 min) in the absence or presence of iberiotoxin (100 nM for 20 min), SKF 525A (10 µM for 15 min), or miconazole (10 µM for 15 min). Some arteries were precontracted with K-PSS. Acetylcholine-induced relaxations were significantly inhibited by SKF 525A and miconazole and abolished by iberiotoxin and K-PSS. *, P < 0.05 vs. control. All values are mean ± SEM (n = 8–16).

 
Relaxation responses to sodium nitroprusside, iloprost, and 14,15-EET
In U46619-precontracted vessels, sodium nitroprusside (10–8 to 10–4 M; a NO donor), iloprost (10–10 to 10–5 M; a prostacyclin analog), and 14,15-EET (10–9 to 10–5 M; a cytochrome P450 metabolite of arachidonic acid) relaxed small bovine adrenal cortical arteries in a concentration-dependent manner, with maximal relaxations of 92 ± 1, 95 ± 1, and 90 ± 2%, respectively (Fig. 3Go, A–C). High K+ (K-PSS) partially inhibited relaxation responses to sodium nitroprusside (maximal relaxation of 62 ± 3%) and iloprost (maximal relaxation of 44 ± 2%), whereas it abolished relaxation responses to 14,15-EET.



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  		FIG. 3. Concentration-response curves for sodium nitroprusside (A), iloprost (B), and 14,15-EET (C) of small bovine adrenal cortical arteries. Arteries were precontracted with either U46619 (control) or K-PSS. K-PSS partially inhibited relaxations to sodium nitroprusside and iloprost but abolished relaxations to 14,15-EET. *, P < 0.05 vs. control. All values are mean ± SEM (n = 8).

 
Relaxation responses to arachidonic acid
To determine whether endogenous cytochrome P450 metabolites from arachidonic acid cause relaxations in small adrenal cortical arteries, we examined vascular responses to arachidonic acid in the presence of SKF 525A. As shown in Fig. 4AGo, arachidonic acid (10–9 to 10–5 M) produced concentration-dependent relaxations, which was only slightly inhibited by indomethacin (10 µM). However, treatment with SKF 525A (10 µM) almost abolished relaxation responses to arachidonic acid (maximal relaxation of 16 ± 5 vs. 60 ± 4% of control) (Fig. 4BGo).



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  		FIG. 4. Effect of indomethacin (A) and SKF 525A (B) on arachidonic acid-induced relaxations of small bovine adrenal cortical arteries. Arterial segments were pretreated with indomethacin (10 µM for 30 min) or SKF 525A (10 µM for 15 min). Relaxation responses to arachidonic acid were slightly inhibited by indomethacin but nearly abolished by SKF 525A. *, P < 0.05 vs. control. All values are mean ± SEM (n = 7–10).

 
To provide further evidence that small adrenal cortical arteries produce the cytochrome P450 metabolites of arachidonic acid, isolated arteries were incubated with [14C]-labeled arachidonic acid in the presence of vehicle, SKF 525A (10 µM), or indomethacin (10 µM). The [14C] metabolites of arachidonic acid were then separated and analyzed by HPLC/scintillation spectrometry. We found that small adrenal arteries produced metabolites of arachidonic acid that comigrated with 6-keto-PGF1{alpha}, 15-hydroxyeicosatetraenoic acid (15-HETE), and 14,15-EET standards. The production of EETs was inhibited by SKF 525A (10 µM), and the production of 6-keto-PGF1{alpha} by indomethacin. 15-HETE was not altered by indomethacin or SKF 525A (Fig. 5Go). The production of HETEs and EETs was confirmed by LC/MS using different HPLC conditions (Fig. 6Go). Metabolites eluting at 21.19, 24.07, 29.89, and 32.67 min comigrated with 15-HETE, 12-HETE, 14,15-EET, and 11,12-EET, respectively (Fig. 6Go, A and C). Analysis of the mass spectra of these metabolites indicated a major M-1 ion of 319 m/z for the four metabolites (Fig. 6Go, B and D). These mass spectra are consistent with a HETE and EET structure (15).



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  		FIG. 5. Effect of SKF 525A and indomethacin on metabolism of [14C] arachidonic acid by adrenal arteries. Bovine small adrenal cortical arteries were treated with vehicle (0.095% ethanol), SKF 525A (10 µM), or indomethacin (10 µM) for 10 min, followed by sequential addition of [14C] arachidonic acid (0.05 µCi, 100 nM) for 10 min and A23187 (10 µM) for another 10 min. Metabolites were extracted and resolved by reverse-phase HPLC. Migration times of known standards are noted on the top chromatogram.

 


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  		FIG. 6. Identification of arachidonic acid metabolites of adrenal cortical arteries by LC/MS. The EETs (A) and HETEs (C) were resolved by HPLC. Migration times of known standards are shown above the chromatogram. The mass spectra for 14,15-EET (B) and 15-HETE (D) are indicated. Similar mass spectra were observed for 11,12-EET and 12-HETE. The major ion of 319 m/z represents the M-1 ion and is consistent with an EET and HETE.

 
Effect of cytochrome P450 inhibitor on smooth muscle hyperpolarization to acetylcholine
Small adrenal cortical arteries pretreated with L-NA and indomethacin had an average resting Em of –61 ± 1.7 mV (Fig. 7Go). U46619 (100 nM) caused a significant depolarization of smooth muscles (Em of –38 ± 2 mV), and subsequent addition of acetylcholine (100 nM) induced an average of 15 mV hyperpolarization (Em of –53 ± 2 mV). Preincubation of arteries with SKF 525A (10 µM) did not alter Em under resting condition and after U46619 stimulation. However, SKF 525A significantly attenuated acetylcholine-induced hyperpolarization to an average of –37 ± 1.3 mV.



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  		FIG. 7. Effect of SKF 525A on acetylcholine-induced hyperpolarization of small bovine adrenal cortical arteries. Arteries were pretreated with vehicle or SKF 525A (10 µM) for 15 min in the presence of L-NA (30 µM) and indomethacin (10 µM). Em was measured as vehicle control, vehicle with U46619 (100 nM), vehicle with U46619 and acetylcholine (100 nM), SKF 525A, SKF 525A with U46619, or SKF 525A with U46619 and acetylcholine. A, Original traces of Em responses from arteries exposed to various treatments. B, Summarized data of the Em responses. *, P < 0.05 vs. U46619; #, P < 0.05 vs. acetylcholine in control. All values are mean ± SEM (n = 7 arteries).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we characterized the vascular responses to acetylcholine in isolated small cortical arteries from bovine adrenal glands. We found that acetylcholine induces a potent endothelium-dependent relaxation, which is highly resistant to L-NA and indomethacin. The acetylcholine-induced, NO- and prostacyclin-independent relaxation is eliminated in arteries depolarized by high K+, and markedly inhibited by cytochrome P450 inhibitors. The cytochrome P450 inhibitor SKF 525A blocks the L-NA- and indomethacin-resistant acetylcholine-induced smooth muscle hyperpolarization. In addition, the cytochrome P450 metabolites EETs are identified in small adrenal arteries. This suggests that acetylcholine-induced relaxation is mediated by an endothelium-dependent hyperpolarization mechanism, presumably through cytochrome P450 metabolites of arachidonic acid.

The regulation of vascular tone and blood flow in the adrenal cortex is complex and probably involves a range of humoral, neural, and local mediators (1, 2, 3). 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 the immune system (i.e. mast cells), or adrenal steroid-secreting cells. With regard to cholinergic regulation, the presence of cholinergic nerve fibers has been shown in the adrenal cortex of various species such as rat, guinea pig, and human, primarily in the capsule and zona glomerulosa among adrenal cells and around blood vessels (6, 7, 8, 9, 10). Because this local factor is released in the vicinity of steroidogenic cells and cortical arterioles, there has been considerable interest in studying its role in the regulation or modulation of steroid production and blood flow. The stimulatory effect of acetylcholine on adrenal steroid hormone release was first reported in 1955 (16), and these findings have subsequently been confirmed and extended (10, 17, 18).

In contrast to relatively well-defined effects of acetylcholine on steroid production, the role of acetylcholine in the local regulation of adrenal vascular tone and blood flow is largely unknown. In addition, it remains elusive whether acetylcholine-induced changes in blood flow contribute to its steroidogenic effect in the adrenal cortex. The present study provides evidence that acetylcholine is a direct and potent vasodilator of small bovine adrenal cortical arteries, a proposed primary site of control of the blood flow in the adrenal cortex (1). Acetylcholine-induced vasorelaxation has also been observed in isolated adrenal arteries from fetal and newborn sheep (19). In addition, splanchnic nerve stimulation increases adrenal blood flow that is blocked by cholinergic antagonists, indicating that neurally released acetylcholine can induce vasodilation in the adrenal gland (2). Thus, these results support a potential role of acetylcholine in the local regulation of vascular tone and blood flow in the adrenal cortex.

Previous studies have shown that acetylcholine induces relaxation in a number of vascular beds by stimulating the release of relaxing factors from the endothelium. Three major endothelium-derived relaxing factors are NO or its related compounds, prostacyclin and EDHFs (11, 12, 13). It is generally agreed that acetylcholine-induced relaxation in the presence of NOS and cyclooxygenase inhibitors is mediated by EDHF, which acts by producing a hyperpolarization of smooth muscle cells (20). The contribution of EDHF to acetylcholine-induced relaxations and chemical identity of EDHFs may vary in species and vascular bed. Considerable evidence suggests that epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid, function as EDHFs in several blood vessels (11, 21, 22, 23, 24). In the present study, we found that the NOS inhibitor, L-NA, caused a right-shift of acetylcholine-induced relaxations in small adrenal cortical arteries without affecting the maximal relaxation to this agent, indicating that NO partially mediates the relaxation response to acetylcholine. These results are in agreement with those of a previous study showing that acetylcholine induces relaxations in adrenal arteries of fetal and newborn sheep precontracted with high K+ presumably via endothelial NO, although the contribution of other relaxing factors such as EDHFs to acetylcholine-induced relaxations was not examined (19).

In the current study, the acetylcholine-induced L-NA- and indomethacin-resistant relaxations were similarly abolished by high K+ and iberiotoxin, indicating that large-conductance Ca2+-activated K+ channel-mediated membrane hyperpolarization is involved in the relaxation responses to acetylcholine. The cytochrome P450 inhibitors SKF 525A and miconazole also significantly inhibited the L-NA- and indomethacin-resistant relaxations. In addition, SKF 525A blocked acetylcholine-induced smooth muscle hyperpolarization in arteries treated with L-NA and indomethacin. Arachidonic acid and 14,15-EET, a cytochrome P450 metabolite of arachidonic acid, also caused concentration-dependent relaxations in small adrenal arteries. The arachidonic acid-induced relaxation was largely blocked by SKF 525A. Moreover, we found that small adrenal arteries produced metabolites of arachidonic acid that comigrated with 14,15-EET standard and that the production of EETs was inhibited by SKF 525A. The identity of EETs was confirmed by LC/MS. Because prostacyclin synthase is a member of cytochrome P450 family (CYP8A1) (25, 26), it is not surprising that SKF 525A also inhibited the production of 6-keto-PGF1{alpha}. However, this action of SKF 525A should not interfere with the interpretation of the role of EETs in acetylcholine-induced relaxations. The acetylcholine-induced relaxation of small adrenal cortical arteries was not altered by the cyclooxygenase inhibitor indomethacin, indicating that prostacyclin is not involved in the relaxations to acetylcholine. Taken together, these results indicate that an EDHF-dependent mechanism mediates relaxations to acetylcholine in small adrenal cortical arteries and that cytochrome P450 metabolites may function as an EDHF in this vasculature.

Recently we reported that endothelin (ET)-1 causes relaxations via the ETB receptors in small adrenal cortical arteries (5). Similar to acetylcholine, ET-1-induced relaxations are also mediated by NO and cytochrome P450 metabolites but not by cyclooxygenase metabolites of arachidonic acid. These results indicate that multiple signals may converge on overlapping pathways to induce relaxation of small adrenal cortical arteries.

The specific receptor subtypes activated by acetylcholine were not explored in this study. Previous studies indicate that the muscarinic receptors that mediate the release of EDHF belong to the M1-muscarinic subtype on the endothelium, whereas those activating the synthesis of NO are M2-muscarinic subtype (27). Whether different subtypes of muscarinic receptors are also involved in acetylcholine response in small adrenal arteries requires further investigation.

Although prostacyclin seems not involved in relaxation responses to acetylcholine, the prostacyclin analog iloprost potently relaxed the small adrenal arteries, suggesting that the prostacyclin pathway is still functional in the regulation of vascular tone in these arteries. Indeed, we found that small adrenal arteries produced 6-keto-PGF1{alpha}, a stable metabolite of prostacyclin. In addition, indomethacin caused a marked leftward shift of concentration responses to U46619. Thus, it is likely that prostacyclin participates in the regulation of basal vascular tone by exerting a tonic dilatory effect on small adrenal arteries.

As discussed earlier, it remains unknown whether changes in blood flow in response to acetylcholine contribute to its steroidogenic effect in the adrenal cortex. However, recent studies indicate that a close functional association may exist between vascular cells and steroidogenic cells of the adrenal cortex. The vascular endothelial cell-derived factors including NO, prostacyclin, and EETs have been shown to regulate adrenal steroidogenesis. For example, prostacyclin and EETs stimulate steroid release (28, 29), whereas NO inhibits steroid production (30). In addition, endothelial cells release a transferable steroidogenic factor that may function in the adrenal gland as a paracrine regulator of steroid secretion (31). The data from the present study indicate that acetylcholine releases a number of relaxing factors from endothelial cells such as NO, prostacyclin, and EETs. Therefore, it is tempting to speculate that acetylcholine-induced vasorelaxation and increases in blood flow might contribute to its steroidogenic effects in the adrenal cortex through the release of these endothelial factors, although further studies are needed to address this possibility.

Blood flow through the adrenal cortex is an important local regulatory mechanism of adrenal steroidogenesis. However, mechanisms responsible for the regulation of adrenal blood flow and its interaction with steroid release are largely unknown. The present study demonstrates that small adrenal cortical arteries are highly responsive to acetylcholine, an endogenous neurotransmitter in the adrenal cortex. The potent acetylcholine-induced relaxation in these arteries is consistent with its potential role in the regulation of vascular tone and blood flow in the adrenal cortex.


   Acknowledgments
 
We thank Erik Edwards for technical assistance and Gretchen Barg for secretarial assistance.


   Footnotes
 
This work was supported by a grant from the National Institute of Diabetes and 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; 14,15-EET, 14,15-epoxyeicosatrienoic acid; Em, membrane potential; ET, endothelin; 15-HETE, 15-hydroxyeicosatetraenoic acid; K-PSS, potassium-substituted PSS; LC/MS, HPLC/mass spectrometry; L-NA, N-nitro-L-arginine; NO, nitric oxide; NOS, NO synthase; PSS, physiological saline solution.

Received April 8, 2004.

Accepted for publication June 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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