Modulation of Endothelium-Dependent Hyperpolarization and Relaxation to Acetylcholine in Rat Mesenteric Artery by Cytochrome P450 Enzyme Activity
Acetylcholine (ACh) induced hyperpolarization and relaxation in rat mesenteric arteries contracted with norepinephrine, as indicated from studies with simultaneous microelectrode and tension recordings. We tested whether the hyperpolarization to ACh was modified by induction and depletion of cytochrome P450 enzymes. Enzyme induction by treating the animals with 3-methylcholanthrene and β-naphthoflavone for 3 days resulted in a significant increase in the endothelium-dependent hyperpolarization to a maximum of 22.7±1.0 mV from 13.9±0.4 mV in arteries from untreated animals. Enzyme depletion by treating the animals with CoCl2 for 2 days resulted in a significant reduction in the maximum hyperpolarization to 9.9±0.7 mV. When NO synthesis was inhibited by Nω-nitro-l-arginine, the relaxation was correlated to hyperpolarization. The Nω-nitro-l-arginine–resistant responses were significantly inhibited by clotrimazole. The relaxation to ACh was not altered by enzyme induction but was significantly reduced by enzyme depletion. In KCl-contracted arteries, modification of cytochrome P450 enzyme activity had no significant effect on the relaxation to ACh. Similarly, hyperpolarization and relaxation to pinacidil were not significantly affected. These results suggest that the hyperpolarization response to ACh is closely regulated by cytochrome P450–dependent enzymes.
Acetylcholine induces vasodilatation by releasing relaxing factors from the endothelium. The best known of these factors is EDRF, which has now been identified as NO.1 However, NO may not be the only relaxing factor released from the endothelium, because endothelium-dependent relaxations could still be induced in the presence of NO synthase inhibitors,2 3 methylene blue, or hemoglobin.4
ACh also induces endothelium-dependent hyperpolarization in vascular tissues. This hyperpolarization is little affected by NO synthase inhibitors,2 3 hemoglobin, or methylene blue.4 Thus, the release of an EDHF has been proposed.4 The hyperpolarization is due to opening of K+ channels and could be inhibited by certain K+ channel blockers or by raising the external K+ concentrations.4 5 6 EDHF may contribute to the component of the relaxation not mediated by NO. In the presence of NO synthase inhibitors, the remaining relaxation could be abolished by K+ channel inhibitors or by raising K+ concentration.2
There are suggestions that EDHF may be a cytochrome P450–derived epoxide such as EET.7 8 EET is synthesized in endothelial cells and has a potent vasodilatory effect.8 9 It also causes hyperpolarization of vascular cells by activating K+ channels.8 10 Cytochrome P450 enzyme inhibitors such as clotrimazole and proadifen are effective in attenuating the component of the endothelium-dependent relaxation attributable to EDHF.3 Modulation of cytochrome P450–dependent enzyme activity alters endothelium-dependent relaxation to arachidonic acid, being potentiated by induction and attenuated by depletion of the enzymes.11 12 13 In the present study, we tested whether similar induction or depletion of cytochrome P450 enzymes could modify the endothelium-dependent hyperpolarization and relaxation responses to ACh. If EDHF is a metabolic product of cytochrome P450 enzymes, then the hyperpolarization response should reflect the changes in enzyme activities.
Materials and Methods
The main superior mesenteric arteries were isolated from male Wistar rats (10 to 12 weeks old, Charles River, Wilmington, Mass) and immersed in physiological solution. Ring segments 4 mm in length were used for simultaneous recording of membrane potential and tension. Two tungsten wires were passed through the lumen and attached to an F60 transducer (Narco) for tension measurement. To facilitate recording of membrane potential, a small incision was made at one end of the segment to create a strip (1 mm in width and 2 mm in length) that could be pinned down to the bottom of the organ bath. Glass micropipettes filled with 3.0 mol/L KCl and with tip resistances of 40 to 60 MΩ were used for membrane potential recordings. The microelectrodes were inserted into the smooth muscle cells through the exposed intimal surface of the artery.
The arteries were constantly superfused with physiological solution and allowed to equilibrate for at least 2 hours before the actual experiment. During this period, the arteries were challenged with norepinephrine (10.0 μmol/L) for three to four times until the contractions had stabilized. All drugs were equilibrated with the perfusate before entering the recording chamber. For induction of cytochrome P450 enzymes, the rats were treated for 3 days with a combination of 3-methylcholanthrene (80 mg/kg per day SC) and β-naphthoflavone (80 mg/kg per day SC).11 12 13 To deplete cytochrome P450 enzymes, the rats were treated with CoCl2 (24 mg/kg per day SC) for 2 days.11 12 13
Solutions and Drugs
The physiological solution had the following composition (mmol/L): NaCl 120, NaHCO3 25, KCl 5, CaCl2 2.5, NaH2PO4 1, MgSO4 1, and glucose 11. The K+-rich solutions were prepared by replacing NaCl with KCl. The solutions were constantly bubbled with 5% CO2/95% O2 and maintained at 36°C.
Norepinephrine, ACh, L-NA, 3-methylcholanthrene, β-naphthoflavone, CoCl2, clotrimazole, and pinacidil were obtained from Sigma Chemical Co. Preliminary experiments indicate that 10 to 30 μmol/L L-NA was effective in blocking NO synthesis, because there was no further inhibition either in higher concentrations or in combination with S-methyl-l-thiocitrulline (10 μmol/L) (Calbiochem). L-NA potentiated the contraction to norepinephrine (by 80.2±6.5%, n=8) without significantly altering the membrane depolarization (control, 13.2±0.6 mV; L-NA, 13.7±0.7 mV). For comparison purposes, the contraction level in L-NA tissues was matched to that of control by slightly reducing the norepinephrine concentration. To eliminate any nonspecific or other unrelated membrane effects, the concentration of clotrimazole used was the minimum required to effectively inhibit the endothelium-dependent hyperpolarization. Preliminary experiments indicate that the responses to ACh were not affected by indomethacin in this preparation.
Results are expressed as mean±SEM. Statistical significance was evaluated using Student's t test. Values of P<.05 were considered to indicate significant differences between means.
Hyperpolarization Induced by ACh
The mesenteric artery of untreated animals had a resting membrane potential of −51.1±0.7 mV (n=8). This was not significantly different from those treated with 3-methylcholanthrene and β-naphthoflavone (−52.0±0.5 mV, n=10) or CoCl2 (−51.7±0.4 mV, n=10). Norepinephrine (1.0 μmol/L) induced sustained contraction of the mesenteric arteries and caused similar depolarization of the smooth muscle cells in the three groups of animals (13.5±0.5 mV depolarization in untreated arteries, 12.6±0.5 mV with enzyme induction, and 13.3±0.5 mV with enzyme depletion). ACh induced membrane hyperpolarization in a concentration-dependent manner (Fig 1A⇓). A maximum hyperpolarization of 13.9±0.4 mV (n=8) was observed in the untreated arteries (Fig 2A⇓). There was a significant increase in the hyperpolarization to high concentrations of ACh with enzyme induction, reaching a maximum of 22.7±1.0 mV (n=6) (Figs 2A and 3A⇓⇓). However, the hyperpolarizations were not significantly different from those of the untreated arteries at concentrations of ACh below 1.0 μmol/L. In contrast, enzyme depletion with CoCl2 significantly attenuated the hyperpolarization response at all concentrations of ACh higher than 0.1 μmol/L (Fig 4⇓). At 3.0 μmol/L of ACh, the maximal hyperpolarization was only 9.9±0.7 mV (n=6) (Fig 2A⇓).
Relaxation Induced by ACh
ACh induced relaxation in mesenteric arteries from the three groups of animals in a concentration-dependent manner. The responses of enzyme-induced arteries were not significantly different from those of untreated arteries, and total relaxation of the norepinephrine-induced tone was attained at concentrations of ACh higher than 1.0 μmol/L (Fig 2B⇑). In CoCl2-treated arteries, relaxations to ACh were significantly attenuated except at the highest concentration of 3.0 μmol/L ACh (Fig 2B⇑).
Effects of L-NA and Clotrimazole
L-NA (30.0 μmol/L for >20 minutes) caused no significant changes in the maximum hyperpolarization to ACh in all three groups of animals (Fig 2C⇑). However, there was a small but significant reduction in the hyperpolarization at lower concentrations of ACh. At 0.1 μmol/L of ACh, the hyperpolarization was reduced from 8.1±1.0 to 5.4±0.5 mV (n=4) in untreated arteries. Similarly, the hyperpolarization was reduced from 10.7±1.1 to 6.6±0.6 mV (n=4) in enzyme-induced arteries. In CoCl2-treated arteries, the reduction from 2.8±0.8 to 1.6±0.2 mV (n=4) did not reach a statistically significant level. The differences in the hyperpolarization to ACh among the three groups were not altered by L-NA (Fig 2C⇑).
The relaxation to ACh was significantly inhibited by L-NA in all three preparations, and the dose-dependent curves were shifted significantly to the right (Fig 2D⇑). The maximum relaxation at 3.0 μmol/L ACh was reduced to 72.3±2.1% of control in the untreated arteries. Similarly, maximum relaxations to 70.7±7.0% and 59.2±7.7% of the norepinephrine-induced tone were observed in enzyme-induced and CoCl2-treated arteries, respectively. The differences in the relaxation responses among the three groups were also not altered by L-NA (Fig 2D⇑).
ACh-induced hyperpolarization is inhibited by the P450 enzyme inhibitor clotrimazole (10.0 μmol/L), and a combination of L-NA and clotrimazole is most effective in abolishing the responses to ACh.3 In the present study, the hyperpolarization to ACh was significantly reduced in all three preparations by a combination of L-NA and clotrimazole. Even in enzyme-induced arteries, only a small hyperpolarization of 4.0±0.4 mV (n=4) could be evoked by 3.0 μmol/L ACh (Fig 2C⇑). A combination of clotrimazole and L-NA was also very effective in reducing the relaxation responses to ACh. The relaxations to 3.0 μmol/L ACh were 19.4±3.7%, 36.8±2.3%, and 29.6±5.1% for the untreated, enzyme-induced, and CoCl2-treated arteries, respectively (Fig 2D⇑).
Correlation of Hyperpolarization to Relaxation
The relaxation to ACh was not correlated to hyperpolarization in all three cases under control conditions (Fig 5⇓). However, when NO synthase was inhibited by L-NA (30.0 μmol/L), a significant correlation between relaxation and hyperpolarization was observed at concentrations of ACh below 1.0 μmol/L (r=.95, P<.001 for untreated arteries; r=.98, P<.001 for enzyme-induced arteries; and r=.97, P<.001 for CoCl2-treated arteries).
Relaxation in KCl-Depolarized Arteries
To test whether modulation of cytochrome P450 enzyme activities could affect responses not associated with membrane hyperpolarization, relaxation to ACh was studied in arteries contracted by high KCl (40.0 mmol/L). In high KCl solutions, ACh induced relaxation in the absence of hyperpolarization. ACh was equally effective in inducing relaxation in all three groups of animals (Fig 6⇓). A small but significant increase in relaxation was observed in the enzyme-induced arteries only at the highest concentration of ACh tested (3.0 μmol/L). In all cases, the relaxation to lower concentrations of ACh was virtually abolished by L-NA (30.0 μmol/L) in KCl-contracted arteries (Fig 6⇓), indicating that NO was probably the sole mediator of the response in the absence of membrane hyperpolarization. With maximal ACh stimulation (>1.0 μmol/L), a small relaxation of 4% to 9% persisted (Fig 6⇓).
Hyperpolarization and Relaxation to Pinacidil
Pinacidil, an endothelium-independent vasodilator, was used to test whether enzyme induction or depletion could affect hyperpolarization and relaxation of vascular smooth muscle directly. There were no significant differences in the hyperpolarization (Fig 7A⇓) and relaxation (Fig 7B⇓) to pinacidil among the three groups of arteries contracted with norepinephrine (1.0 μmol/L). The responses to pinacidil were inhibited by glibenclamide (3.0 μmol/L) (Fig 7⇓).
Many studies have implicated the involvement of cytochrome P450 enzymes in endothelium-dependent relaxation. Cytochrome P450 enzymes are located mainly in the endothelium of blood vessels.14 15 These enzymes are known to generate vasorelaxant products such as EETs from arachidonic acid.8 Modulation of cytochrome P450 enzyme activities results in corresponding changes in endothelium-dependent relaxation. Thus, cytochrome P450 inhibitors such as proadifen and clotrimazole are effective in attenuating endothelium-dependent relaxation.3 16 Induction of cytochrome P450 enzymes with 3-methylcholanthrene and β-naphthoflavone resulted in an increase in relaxation to arachidonic acid that could be related to a twofold increase in aryl hydrocarbon hydroxylase activity in vascular tissues.12 Conversely, depletion of cytochrome P450 enzymes with CoCl2 resulted in a decrease in the relaxation response and a reduction in the aryl hydrocarbon hydroxylase activity to 10% to 20% of that observed in the control.12
Cytochrome P450 and Endothelium-Dependent Hyperpolarization
It has been suggested that EDHF is a cytochrome P450 metabolite.7 8 We have previously shown that endothelium-dependent hyperpolarization to ACh is significantly diminished by cytochrome P450 inhibitors such as clotrimazole and proadifen.3 Results from the present study further support the involvement of cytochrome P450 enzymes in the hyperpolarization response to ACh. With enzyme induction, the maximal hyperpolarization to ACh increased dramatically, by ≈63%. However, the hyperpolarization was not significantly increased at lower levels of ACh stimulation. Synthesis and release of EDHF are initiated by ACh, and the level of stimulation is dependent primarily on the number of receptors activated to produce the graded concentration-dependent response.17 Cytochrome P450 enzymes are not the limiting factors for the production of EDHF and are activated only at the end stage of the signal transduction pathway. Enzyme induction would not amplify the response, as there were no increases in the preceding signals upstream of the cytochrome P450 enzyme. It is only upon the strongest ACh stimulations that the production of EDHF is limited by the availability of cytochrome P450 enzymes. Induction of cytochrome P450 enzymes would help to meet the demand for increased production of EDHF at high ACh concentrations. This may explain why increased hyperpolarization was observed only at high ACh stimulation in the enzyme-induced arteries. In CoCl2-treated arteries, the scarcity of cytochrome P450 enzymes becomes an important factor in the production of EDHF. Thus, the hyperpolarization was attenuated at all levels of ACh stimulation. Obviously, the cytochrome P450 enzymes were not totally depleted by such treatments,12 as the hyperpolarizations were not abolished.
Specificity of Action of Enzyme Induction and Depletion
Changes in P450 enzyme activity could modify the hyperpolarization response to ACh by nonspecific actions on endothelial or vascular cells. For example, the synthesis or release of vasoactive substances from endothelial cells could be altered nonspecifically. In such a case, one would expect the synthesis and release of EDRF/NO to be affected in a manner similar to that for EDHF, resulting in increased EDRF/NO-mediated relaxation with enzyme induction and decreased EDRF/NO-mediated relaxation with enzyme depletion. Our studies with KCl-depolarized arteries in which the hyperpolarization component was eliminated indicate that the EDRF/NO component of the response was not altered. Thus, modulation of cytochrome P450 enzyme appears to affect only the component of the response mediated by hyperpolarization.
Changes in P450 enzyme activity could also alter nonspecifically the responses of vascular smooth muscle cells. Pinacidil, a direct-acting vasodilator,18 was used to test for specificity of action at the vascular smooth muscle level. No change in the hyperpolarization and relaxation response to pinacidil was observed with modulation of cytochrome P450 enzyme activity, and glibenclamide was equally effective in inhibiting the responses in all three cases. Furthermore, the depolarization and contraction induced by norepinephrine, as well as the resting membrane potential, were also not affected. Thus, changes observed in the present study were not likely to be due to nonspecific alteration in the properties of the vascular smooth muscle cells.
Hyperpolarization and Relaxation
Vascular tone is closely regulated by membrane potential within a certain range.19 20 21 22 Depolarization of the membrane above a threshold of ≈−45 mV elicits vascular contraction.19 20 21 The threshold for contraction in the rat mesenteric artery induced by potassium depolarization is also ≈−45 mV (D.W. Cheung and M.J. MacKay, unpublished data, 1990). Conversely, hyperpolarization of the membrane causes relaxation, but only within this membrane potential range. Hyperpolarization below −45 mV would not evoke further relaxation.19 Thus, contraction/relaxation mediated by electromechanical coupling is valid only when the membrane potential is above the threshold of ≈−45 mV. Contraction induced by norepinephrine is mediated by electromechanical and pharmacomechanical (membrane potential–independent) coupling mechanisms.22 23 Endothelium-dependent hyperpolarization induced by ACh would be expected to be more effective in inhibiting the component of the vascular tone mediated by electromechanical coupling.
When NO synthesis was blocked by L-NA, the remaining relaxation was correlated to membrane hyperpolarization at concentrations of ACh below 1.0 μmol/L. Higher concentrations of ACh were not used in the correlation studies for the following two reasons: (1) Studies with KCl-contracted arteries indicate that the inhibition by L-NA was complete only at concentrations of ACh below 1.0 μmol/L. At higher concentrations, the inhibition by L-NA was not complete, and a small relaxation (5% to 9%) persisted (see Fig 6⇑). This would introduce an error in assessing the contribution of hyperpolarization to relaxation (determined as the L-NA–resistant component) if included. (2) Hyperpolarization to ACh at concentrations higher than 1.0 μmol/L repolarized the membrane potential to ≈−52 mV in untreated arteries (see Fig 1⇑) and −60 mV in the enzyme-induced arteries (see Fig 3⇑). These membrane potentials were lower than −45 mV and outside of the effective range for electromechanical coupling and therefore would not contribute to relaxation. It is for this same reason that despite a big increase in the maximal hyperpolarization in the enzyme-induced arteries, no further increase in relaxation was induced at high ACh concentrations. At lower concentrations of ACh, hyperpolarizations in enzyme-induced arteries were not significantly different from those in untreated arteries. Therefore, the overall relaxation to ACh was not significantly altered in the enzyme-induced arteries. The relaxation response to ACh was also not altered by enzyme induction in canine coronary arteries.12
The hyperpolarization and relaxation to ACh were significantly attenuated in CoCl2-treated arteries, as would be expected if the amount of EDHF released is reduced because of depletion of cytochrome P450 enzymes. The decrease in relaxation could be accounted for solely by a reduction in the hyperpolarization response. Attenuation of the relaxation response could be observed in the absence of L-NA. Enzyme depletion had no significant effect on the NO-mediated relaxation to ACh in K+-contracted arteries. Thus, changes in cytochrome P450 activity could affect the overall relaxation by modifying specifically the EDHF component without altering the EDRF/NO component. This finding reinforces the importance of taking into consideration the contribution of different factors in the study of endothelium-dependent relaxation. For example, decreases in endothelium-dependent hyperpolarization have been observed with aging and hypertension.24 In hypercholesterolemic rabbit carotid artery, normal relaxation to ACh is maintained by hyperpolarization-mediated relaxation despite a reduction in the contribution of the NO/cGMP component.25
The present study is the first to demonstrate modification of endothelium-dependent hyperpolarization by cytochrome P450 enzyme activity and how these changes in hyperpolarization could affect relaxation. Recently, EDHF has tentatively been identified as EETs, which are cytochrome P450 metabolites derived from arachidonic acid.8 Results from the present study support the hypothesis of EDHF being a cytochrome P450 metabolite. Thus, the amplitude of ACh-induced hyperpolarization varied in accordance with changes in cytochrome P450 enzyme activity. The effects were specific to the hyperpolarization induced by ACh, and the EDRF/NO component was not affected. It was also confirmed that in the absence of NO, relaxation was correlated to membrane hyperpolarization. Depletion of cytochrome P450 enzymes could reduce endothelium-dependent relaxation as a result of a decrease in the membrane hyperpolarization. In future studies, it would be ideal if hyperpolarization responses could be correlated to changes in endothelial cytochrome P450 enzyme activity and the production of an endothelium-derived cytochrome P450 metabolite such as EET.
Selected Abbreviations and Acronyms
|EDHF||=||endothelium-derived hyperpolarizing factor|
|EDRF||=||endothelium-derived relaxing factor|
This study was supported by a grant from the Heart and Stroke Foundation of Ontario.
- Received March 13, 1996.
- Accepted June 28, 1996.
Chen G, Cheung DW. Hyperpolarization and relaxation induced by acetylcholine and NO in the rat mesenteric arteries. In: Vanhoutte P, ed. Endothelium-Derived Hyperpolarizing Factor. In press.
Nagao T, Vanhoutte P. Hyperpolarization contributes to endothelium-dependent relaxations to acetylcholine in femoral veins of rats. Am J Physiol. 1991;261:H1034-H1037.
Hecker M, Bara A, Bauersachs J, Busse R. Characterization of endothelium-derived hyperpolarizing factor as a cytochrome P450-derived arachidonic acid metabolite in mammals. J Physiol (Lond). 1994;4812:407-414.
Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res. 1996;78:415-423.
Rosolowsky M, Falck JR, Willerson JT, Campbell WB. Synthesis of lipoxygenase and expoxygenase products of arachidonic acid by normal and stenosed canine coronary arteries. Circ Res. 1990;66:608-621.
Pinto A, Abraham N, Mullane K. Cytochrome P450-dependent monooxygenase activity and endothelial-dependent relaxations induced by arachidonic acid. J Pharmacol Exp Ther. 1986;236:445-451.
Pinto A, Abraham N, Mullane K. Arachidonic acid-induced endothelial-dependent relaxations of canine coronary arteries: contribution of a cytochrome P450-dependent pathway. J Pharmacol Exp Ther. 1987;240:856-863.
Oyekan AO, McGiff JC, Quilley J. Cytochrome P-450-dependent vasodilation of rat kidney by arachidonic acid. Am J Physiol. 1991;261:H714-H719.
Overby L, Nishio S, Weir A, Carver G, Plopper C, Philpot R. Distribution of cytochrome P450 1A1 and NADPH-cytochrome P450 reductase in lungs of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin: ultrastructural immunolocalization and in situ hybridization. Mol Pharmacol. 1992;41:1039-1046.
Kenakin T. Pharmacological Analysis of Drug-Receptor Interactions. New York, NY: Raven Press Publishers; 1987.
Weston A, Bray K, Duty S, McHarg A, Newgreen D, Southerton J. In vitro studies on the mode of action of pinacidil. Drugs. 1988;36(suppl 7):10-28.
Cheung DW. Electrophysiological properties of vascular smooth muscle in hypertension. In: Kwan CY, ed. Membrane Abnormalities in Hypertension. Boca Raton, Fla: CRC Press; 1989;1:1-11.
Kuriyama H, Ito Y, Suzuki H, Kitamura K, Itoh T. Factors modifying contraction-relaxation cycle in vascular smooth muscles. Am J Physiol. 1982;243:H641-H662.
Fujii K, Ohmori S, Tominaga M, Abe I, Takata Y, Ohya Y, Kobayashi K, Fujishima M. Age-related changes in endothelium-dependent hyperpolarization in the rat mesenteric artery. Am J Physiol. 1993;265:H509-H516.
Najibi S, Cowan C, Palacino J, Cohen R. Enhanced role of potassium channels in relaxations to acetylcholine in hypercholesterolemic rabbit carotid artery. Am J Physiol. 1994;266:H2061-H2067.