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CARDIOVASCULAR

Endothelium-Independent Relaxation by Adrenomedullin in Pregnant Rat Mesenteric Artery: Role of cAMP-Dependent Protein Kinase A and Calcium-Activated Potassium Channels

Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, Texas

Received January 23, 2006; accepted March 17, 2006.

	Abstract

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The mechanisms of relaxation of adrenomedullin were investigated in isolated mesenteric artery from pregnant rats. Adrenomedullin (1 nM–0.3 µM) produced concentration-dependent relaxation of endothelium-denuded mesenteric artery rings precontracted with norepinephrine at a concentration required to produce 70% of maximal response (ED₇₀). The concentration-response curve of adrenomedullin was shifted to the right by adrenomedullin receptor antagonist adrenomedullin_22–52 (10 µM) or calcitonin gene-related peptide_8–37 (1 µM). Inhibition of adenylate cyclase by 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) (10 µM) or protein kinase A [Rp-cyclic adenosine monophosphorothioate (Rp-cAMP); 10 µM] reduced the adrenomedullin-induced relaxation to the same magnitude. Adrenomedullin increased the intracellular cAMP level from 0.38 ± 0.07 to 2.00 ± 0.47 pmol/mg tissues, which was completely inhibited by adrenomedullin_22–52 (100 µM). Extracellular high potassium (80 mM), which inactivates the potassium channels, reduced the adrenomedullin-induced relaxation. Blockade of ATP-sensitive, voltage-gated, or inward rectifier potassium channels did not affect the adrenomedullin-induced relaxation. Blockade of calcium-activated K⁺ channels (K_Ca) by tetraethylammonium (1 mM) or iberiotoxin (100 nM) inhibited the adrenomedullin-induced relaxation, whereas there was no additional inhibition by SQ22536 or Rp-cAMP when K_Ca channels were already inhibited. In conclusion, this study provides evidence that cAMP-dependent protein kinase A and K_Ca channels seem to mediate as the cellular pathways in the adrenomedullin-induced endothelium-independent relaxation of mesenteric artery from pregnant rats.

Many studies have investigated the mechanism of the vasodilatory effect of adrenomedullin; however, the results differed depending on the animal species and vascular preparation. For example, in perfused rat mesenteric vascular beds, administration of adrenomedullin induced endothelium-independent vasodilation (Nuki et al., 1993). On the other hand, adrenomedullin-induced rat mesenteric artery relaxation is reported to be endothelium-dependent (Champion et al., 2001). Adrenomedullin binds to specific receptors in endothelial cells and elicits endothelium-dependent vasorelaxation mediated by nitric oxide. According to Shimekake et al. (1995), specific binding of adrenomedullin to bovine aortic endothelial cells was observed, and adrenomedullin induced intracellular cAMP accumulation in a dose-dependent manner. Adrenomedullin also dose-dependently induced an increase in intracellular free calcium in endothelial cells by phospholipase C activation and inositol 1,4,5-triphosphate formation, thereby activating nitric-oxide synthase (Shimekake et al., 1995). Nishimatsu et al. (2001) showed phosphatidylinositol 3-kinase/Akt-pathway involvement as another mechanism of endothelial nitric-oxide synthase activation. Some studies have shown that adrenomedullin caused endothelium-independent relaxation through the elevation of cAMP levels in vascular smooth muscle cells (Eguchi et al., 1994); whereas the downstream mechanism may vary depending on the vascular bed and species. The reported involvement of K⁺ channels in adrenomedullin-induced vasodilation is also puzzling, because adenosine triphosphate-sensitive potassium (K_ATP) channels seem to mediate the adrenomedullin actions in dog coronary artery (Sabates et al., 1997) but not in rat mesenteric artery (Champion et al., 2001). Although most of the reports available dealt with endothelium-dependent mechanisms, not much is known regarding the endothelium-independent mechanisms in adrenomedullin-induced vascular relaxation. Thus, in this study, we proposed to elucidate the cAMP-dependent downstream mechanisms involved with respect to potassium channels in adrenomedullin-induced vasodilation in rat mesenteric artery using endothelium-denuded vessel. Because plasma adrenomedullin levels are elevated in normal pregnancy (Jerat and Kaufman, 1998) and adrenomedullin-induced hypotension is due to reduced total peripheral resistance, we used a resistance vessel (mesenteric artery) from pregnant rats for this study.

	Materials and Methods

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Animals. All procedures were approved by the Animal Care and Use Committee at the University of Texas Medical Branch in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Studies were performed in timed-pregnant Sprague-Dawley rats (250–280 g) obtained from Harlan (Houston, TX). All rats were maintained in the colony room with fixed photoperiod of 12-h light/12-h darkness, having access to water and rodent chow ad libitum. Rats were used on day 18 to day 20 of pregnancy.

Myograph Mounting of Arteries. The animals were killed by exsanguination under deep anesthesia with i.p. injection of ketamine (50 mg/kg) and xylazine (8 mg/kg). The mesentery artery was removed and placed in ice-cold physiological salt solution (PSS). The PSS contained the following composition: 114 mM NaCl, 4.7 mM KCl, 1.15 mM KH₂PO₄, 1.10 mM Na₂HPO₄, 1.18 mM MgSO₄·7 mM H₂O, 15 mM NaHCO₃, 1.15 mM CaCl₂, and 5.0 mM glucose. Segments (2 mm in length) of secondary branches of the superior mesenteric artery were isolated, cleaned of fat and connective tissue, mounted on a wire myograph (Kent Scientific, Litchfield, CT) using tungsten wires, and incubated for 15 to 30 min in PSS at 37°C, which was gassed with 95% air and 5% CO₂ to maintain pH 7.4. The segment was then stretched to a length that was equivalent to a diameter of 225 to 250 µm and incubated for another 15 min. The tissue was activated to contract by the addition of 5 µM norepinephrine until reproducible responses were obtained. The relaxation responses were measured at cumulative doses of adrenomedullin between 10^–9 and 3 x 10^–7 M on vessel segments precontracted with effective concentration of norepinephrine required to produce 70% of maximal response (ED₇₀) that was determined for each vessel.

Experiments were performed on endothelium-denuded arterial segments. The endothelium was removed by gentle rubbing of the intimal surface of the vessel with tungsten wire (size 1 µm) and confirmed by the failure of acetylcholine to relax the vascular segment precontracted by ED₇₀ of norepinephrine. The viability of endothelium-denuded arterial rings was assessed by relaxation induced by sodium nitroprusside (1 µM). Time controls were run to establish the repeatability of adrenomedullin and the stability of the contractile response to norepinephrine.

Experimental Protocol. Concentration-response curves to adrenomedullin (10^–9 to 3 x 10^–7 M) were established on endothelium-denuded artery segments precontracted by ED₇₀ of norepinephrine. The vessels were then washed thoroughly using PSS. After equilibration for 15 min, the arterial strips were incubated with various enzyme inhibitors, receptor antagonists, or channel blockers for 30 min before concentration-response curves to the adrenomedullin were constructed again. There are two types of receptors that mediate adrenomedullin actions. The association of calcitonin receptor-like receptor (CL) with receptor activity-modifying protein (RAMP) subtype 2 (RAMP₂) produces adrenomedullin receptor subtype 1 (AM₁) selective to adrenomedullin that can be antagonized by the weak adrenomedullin peptide antagonist adrenomedullin_22–52, and CL with RAMP₃ forms another adrenomedullin receptor subtype 2 (AM₂) that can respond to both calcitonin gene-related peptide (CGRP) and adrenomedullin, which can be more potently antagonized by CGRP_8–37 than by adrenomedullin_22–52. To assess the receptor mediation, adrenomedullin receptor antagonists adrenomedullin_22–52 (10 µM) and CGRP_8–37 (1 µM) were used. To rule out the endothelial nitric-oxide synthase involvement in adrenomedullin-induced vasodilation, N-nitro-L-arginine methyl ester (L-NAME) (100 µM) was used. To assess the role of adenylate cyclase and protein kinase A, we used 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) (10 µM) and 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMP) (10 µM), respectively. To determine the involvement of potassium channels, respective blockers were used, viz. 10 µM glibenclamide (K_ATP), 10 µM barium chloride (K_IR), 1 mM 4-aminopyridine (K_V), 1 mM tetraethylammonium chloride (K_Ca), and 1 µM paxilline (maxi-K). To confirm the involvement of potassium channels, adrenomedullin-induced relaxation was done in tissues precontracted by 80 mM potassium PSS, a condition in which all of the potassium channels are inactivated.

Measurement of cAMP Levels. The basal and adrenomedullin-stimulated intracellular cAMP levels in mesenteric arterial strips were measured by radioimmunoassay using cAMP [¹²⁵I] assay systems (Amersham Biosciences, Inc., Little Chalfont, Buckinghamshire, UK). In brief, the mesenteric arterial arcade was carefully removed, weighed, and equilibrated for 1 h in 5 ml of Krebs' buffer containing 100 µM isobutyl-1-methyl-xanthine, a phosphodiesterase inhibitor (Sigma-Aldrich, St. Louis, MO), at 37°C aerated with 95% O₂ and 5% CO₂. After equilibration, tissues were incubated with 100 nM adrenomedullin for 2 min and rapidly frozen in liquid nitrogen and homogenized in 1.2 ml of 10% trichloroacetic acid. Tissues used to determine the antagonism by adrenomedullin antagonist, adrenomedullin_22–52, were preincubated with 100 µM adrenomedullin_22–52 for 30 min before adding adrenomedullin. In the cAMP measurements, since we used only a single (higher) concentration of adrenomedullin (0.3 µM), we chose 100 µM adrenomedullin_22–52 because antagonists are generally used, at least approximately 2-fold more than the concentration of the agonists, to avoid the effect of the antagonist being surmounted by the higher concentration of agonist. The cAMP standards (2–128 fmol/tube) and samples (diluted 1:50) were acetylated by adding triethylamine/acetic anhydride [2:1 (v/v) 5 µl/tube]. Labeled cAMP bound to their respective antibodies were recovered by using magnetic beads with goat anti-rabbit IgG, and radioactivity was quantified in a gamma counter. cAMP levels are presented as picomole/milligram of tissue weight.

Drugs Used. Stock solutions of adrenomedullin (100 µM), norepinephrine (10 mM), adrenomedullin_22–52 (100 mM), CGRP_8–37 (1 mM), L-NAME (0.1 M), SQ22536 (10 mM), Rp-cAMP (10 mM), BaCl₂ (10 mM), 4-AP (1 M), tetraethylammonium (1 M), and iberiotoxin (100 µM) were dissolved in triple-distilled water aliquoted and stored at –80°C. Paxilline (1 mM) was dissolved in dimethyl sulfoxide. With the exception of adrenomedullin, adrenomedullin_22–52 and CGRP_8–37, which were purchased from American Peptide Co., Inc. (Sunnyvale, CA), all other chemicals were purchased from Sigma-Aldrich.

Statistical Analysis. Data are presented as mean ± S.E. Relaxation to adrenomedullin is expressed as 100 minus the percentage of the initial precontraction to norepinephrine. The data were analyzed by SigmaPlot 9.0 and Prism (GraphPad Software Inc., San Diego, CA) employing appropriate statistical tools. Means of different groups were analyzed by Student's unpaired t test or one-way ANOVA or two-way ANOVA with Bonferroni post hoc test. Student's paired t test or two-way repeated measures ANOVA with Bonferroni post hoc test was used when comparisons were made between control and drug treatments in the same preparation. p 0.05 was considered statistically significant. Individual concentration-response curves of adrenomedullin were subjected to linear regression analysis to determine EC₅₀, and data are expressed as pD₂ (negative logarithm of the molar concentration of the agonist required to produce half-maximal response).

	Results

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Effect of Adrenomedullin_22–52 and CGRP_8–37 on Adrenomedullin-Induced Vasodilation. The involvement of adrenomedullin receptors in adrenomedullin-induced vasodilation was assessed using the receptor antagonists adrenomedullin_22–52 and CGRP_8–37. Incubation of arterial rings in adrenomedullin_22–52 (10 µM) shifted the concentration-response curve of adrenomedullin to the right. The pD₂ and E_max values were 7.02 ± 0.10 and 72.84 ± 3.5% in the absence of adrenomedullin_22–52 and were 6.66 ± 0.12 and 58.08 ± 6.26% in the presence of adrenomedullin_22–52 (10 µM), respectively. Incubation of the artery rings with CGRP_8–37 (1 µM) also shifted the adrenomedullin concentration-response curve to the right (pD₂, 6.55 ± 0.15; E_max, 53.83 ± 6.78%) (Fig. 1A; Table 1). The maximal relaxation of mesenteric arterial strips by adrenomedullin was suppressed approximately 42 and 47% by adrenomedullin_22–52 (10 µM) and CGRP_8–37 (1 µM), respectively.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Effect of adrenomedullin receptor antagonists adrenomedullin22–52 (10 µM) and CGRP8–37 (1 µM) (A) and inhibition of nitric oxide synthesis by L-NAME (100 µM) (B) on concentration-dependent relaxation induced by adrenomedullin in endothelium-denuded day 18 pregnant rat mesenteric artery precontracted by ED70 concentration of norepinephrine. Values are mean ± S.E.M.; n = figure in parentheses. Data were analyzed by two-way repeated measures ANOVA followed by Bonferroni's post-test. a, p 0.05 versus adrenomedullin22–52; b, p 0.05 versus CGRP8–37.

Effect of Inhibition of Nitric Oxide Synthesis on Adrenomedullin-Induced Vasodilation. To confirm the endothelial denudation and the lack of nitric oxide contribution to the adrenomedullin-induced vasorelaxation, concentration-dependent responses were developed both in the presence and absence of L-NAME (100 µM). L-NAME did not alter the adrenomedullin-induced concentration relaxation response in endothelium-denuded mesenteric artery rings. The pD₂ and E_max values were 6.99 ± 0.11, 6.9 ± 0.19, 72.41 ± 4.24, and 68.18 ± 8.32%, respectively, in the presence and absence of L-NAME (Fig. 1B; Table 1).

Effect of Inhibition of Adenylate Cyclase or Protein Kinase A on Adrenomedullin-Induced Vasodilation. The involvement of cAMP-protein kinase A pathway in the adrenomedullin-induced endothelium-independent vasodilation in the mesenteric artery was assessed by inhibiting adenylate cyclase or protein kinase A using SQ22536 or Rp-cAMP, respectively. The adrenomedullin-induced concentration-dependent response curve was shifted to the right to a similar extent by both SQ22536 (pD₂, 6.66 ± 0.07%; E_max, 52.24 ± 9.8%) and Rp-cAMP (pD₂, 6.75 ± 0.08%; E_max, 57.02 ± 8.86%) compared with the controls in the absence of inhibitors (pD₂ and E_max were 7.02 ± 0.10 and 72.84 ± 3.5%, respectively) (Fig. 2A; Table 1).

View larger version (13K): [in this window] [in a new window]   Fig. 2. A, effect of inhibition of adenylate cyclase by SQ22536 (10 µM) or protein kinase A by Rp-cAMP (10 µM) on the concentration-dependent relaxation of endothelium-denuded mesenteric artery from day 18 pregnant rats precontracted with ED70 concentration of norepinephrine. Data were analyzed by two-way repeated measures ANOVA with Bonferroni's post hoc test. a, p < 0.05 versus SQ22536; b, p < 0.05 versus Rp-cAMP. B, cAMP generation by day 18 pregnant rat mesenteric artery strips at 2-min incubation with 0.3 µM adrenomedullin with and without its antagonist adrenomedullin22–52 (100 µM). a, p 0.001 versus basal; b, p 0.001 versus adrenomedullin22–52 + adrenomedullin; one-way ANOVA with Bonferroni's multiple comparison test; n = number in parentheses.

Effect of Extracellular High Potassium (80 mM) on Adrenomedullin-Induced Vasodilation. Depolarization by high potassium (80 mM) physiological saline solution produced a phasic contraction followed by a sustained tonic contraction. Concentration-dependent relaxation response was elicited by cumulative addition of adrenomedullin. The relaxation response to adrenomedullin was reduced in potassium-depolarized tissue (E_max = 49.87 ± 3.64%, n = 4) compared with the responses in physiological saline solution (pD₂, 6.88 ± 0.04%; E_max, 70.66 ± 2.98%; n = 13) (Fig. 3A).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Effect of 80 mM K+ Cl (A) or inhibition of KATP (B), KV (C), or KIR (D) channels on adrenomedullin-induced concentration-dependent relaxation of day 18 pregnant rat endothelium-denuded mesenteric artery precontracted by ED70 concentration of norepinephrine. Values are mean ± S.E.M.; n = number in parentheses, two-way repeated measures ANOVA with Bonferroni's post-test.

Effect of Blockade of Calcium-Activated Potassium Channel on Adrenomedullin-Induced Vasorelaxation. The involvement of K_Ca channels in adrenomedullin-induced vasorelaxation was assessed by blocking them with tetraethylammonium (1 mM). Incubation of arterial rings with tetraethylammonium for 30 min inhibited the E_max of adrenomedullin from 72.84 ± 3.5 to 43.92 ± 6.15% (Fig. 4A). We then confirmed the involvement of K_Ca channels using iberiotoxin (100 nM), a selective and reversible inhibitor of high-conductance calcium-activated potassium channels, which reduced the E_max of adrenomedullin to 58.92 ± 3.89% from 73.17 ± 7.96% (Fig. 4B). In addition, paxilline (1 µM), another selective blocker of high-conductance calcium-activated (Maxi-K) potassium channels, shifted the adrenomedullin-induced concentration-response curve to the right with a pD₂ of 6.75 ± 0.11% and E_max of 63.19 ± 6.93% (Fig. 4C). To assess the upstream dependence of K_Ca channels opening to cAMP action, adrenomedullin-induced concentration-response curve was developed in the presence of both SQ22536 (10 µM) or Rp-cAMP (10 µM) and tetraethylammonium (1 mM). The magnitude of shift of the concentration curve to the right was almost similar to the independent inhibition of either K_Ca channels or adenylate cyclase or protein kinase A. There were no additive effects by simultaneous inhibition of adenylate cyclase or protein kinase A and blockade of K_Ca channels on the inhibition of adrenomedullin-induced vasorelaxation (Fig. 4D; Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effect of inhibition of either KCa channels alone by tetraethylammonium (1 mM) (A), iberiotoxin (100 nM) (B), paxilline (1 µM) (C), or KCa (D) channels along with inhibition of adenylate cyclase or protein kinase A [SQ22536 (10 µM) + tetraethylammonium (1 mM) Rp-cAMP (10 µM) + tetraethylammonium (1 mM)] on adrenomedullin-induced concentration-dependent relaxation of endothelium-denuded day 18 pregnant rat mesenteric artery precontracted with ED70 concentration of norepinephrine. Values are mean ± S.E.M. n = values in parentheses. Two-way repeated measures ANOVA followed by Bonferroni's post-test. *, p 0.05; **, p 0.01 versus control.

	Discussion

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The distal portion of the secondary branches of mesenteric arteries (arterial diameter 250 µm) from pregnant rats was used in this study. The mesenteric feed arteries and microcirculatory vessels have been reported as resistance vessels in freely moving rats (Fenger-Gron et al., 1995). It is generally accepted that arteries of a diameter up to 300 µm are true resistance vessels (Christensen and Mulvany, 2001). Hence, we considered the vessel strip used in this study as a resistance vessel. Adrenomedullin mediates its effects through receptors, which are heterodimeric complexes of the CL together with RAMP₂ (AM₁) or RAMP₃ (AM₂) (McLatchie et al., 1998). The receptor AM₁ can be antagonized by the weak adrenomedullin peptide antagonist adrenomedullin_22–52, whereas the AM₂ receptors can respond to both CGRP and adrenomedullin, which can be antagonized more potently by CGRP_8–37 compared with adrenomedullin_22–52 (Brain and Grant, 2004). In this study, the relaxant response to adrenomedullin at lower concentrations was inhibited by adrenomedullin_22–52, only while the relaxant responses at higher concentrations were reduced by both CGRP_8–37 and adrenomedullin_22–52. It is possible that the threshold response to adrenomedullin is mediated mainly through AM₁ receptors, whereas the subsequent higher responses are mediated through both AM₁ and AM₂ receptors.

Vascular smooth muscle is known to relax in response to cAMP (Little et al., 1984), and it is reported that adrenomedullin has the ability to increase cAMP in human platelets (Kitamura et al., 1993). In this study, we found that inhibition of adenylate cyclase reduced the adrenomedullin-induced relaxation, indicating the involvement of cAMP. It is also supported by the increased cAMP levels in mesenteric arterial arcade after incubation with adrenomedullin. Previous studies have demonstrated that adrenomedullin caused increases in cAMP levels in cultured smooth muscle cells from the rat thoracic aorta (Eguchi et al., 1994; Ishizaka et al., 1994). This adrenomedullin-induced cAMP generation is receptor-mediated because adrenomedullin_22–52 (100 µM) was able to completely block the adrenomedullin-induced increases in cAMP levels. Inhibition of cAMP-dependent protein kinase A also reduced the adrenomedullin-induced vasorelaxation to a similar extent as that of inhibition of adenylate cyclase. This indicates a role of cAMP-protein kinase A pathway in the endothelium-independent mechanism of vascular relaxation caused by adrenomedullin.

Adenosine triphosphate-sensitive potassium channels are activated by protein kinase A in several arterial smooth muscle cells (Way et al., 1983). Adrenomedullin-induced relaxation was unaffected by glibenclamide at a concentration that blocks vascular K_ATP channels (Quayle et al., 1994), indicating that K_ATP channels are not involved. The lack of a role for K_ATP channel is also reported in rat mesenteric artery (Champion et al., 2001), similar to the current study. In contrast, K_ATP channels have been shown to mediate adrenomedullin-induced relaxation in dog coronary artery (Sabates et al., 1997). Inward rectifier potassium channels and K_V channels are involved in the arterial relaxation caused by several vasodilators (Zhao et al., 1997; Huang et al., 2002). Concentrations of BaCl₂ (Robertson et al., 1996) or 4-AP (Yuan, 1995) that selectively block vascular K_IR or K_V channels, respectively, did not modulate the adrenomedullin-induced relaxation in mesenteric artery, indicating their lack of involvement. On the other hand, blockade of K_Ca channels reduced the adrenomedullin-induced relaxation. We confirmed the involvement of K_Ca channels by using iberiotoxin, a selective and reversible inhibitor of high-conductance K_Ca channels and tetraethylammonium, at concentrations that inhibited single arterial BK channels (Langton et al., 1991). Paxilline, another selective blocker of high-conductance K_Ca channels, also reduced the relaxant responses to adrenomedullin. These novel observations support the notion that adrenomedullin may activate K_Ca channels and that the resultant membrane hyperpolarization would inhibit calcium influx via voltage-gated calcium channels. The shift of the concentration-response curve of adrenomedullin is similar in both independent blockade of K_Ca channels or inhibition of adenylate cyclase or protein kinase A and combined K_Ca channel blockade and adenylate cyclase or protein kinase A inhibition. Although there is no direct electrophysiological study using adrenomedullin, studies with CGRP, a peptide related to adrenomedullin, are reported with similar findings. In cultured smooth muscle cells from a porcine coronary artery, extracellular application of CGRP activated both K_ATP and K_Ca channels, only if the protein kinase A was not inhibited in cell-attached patch configuration (Miyoshi and Nakaya, 1995). In excised inside-out patches, application of cAMP or protein kinase A to the cytoplasmic side of the membrane activated the K_Ca channel (Minami et al., 1993). Hence, it is more likely that cAMP-dependent protein kinase A plays a role as intracellular messenger pathway to adrenomedullin receptors and activates K_Ca channels, which in turn reduces the intracellular calcium and causes the relaxation of the artery. Previously, our laboratory reported that infusion of pregnant rats with CGRP_8–37 increased blood pressure and fetal mortality and retarded fetal growth (Gangula et al., 2002). Because CGRP_8–37 can also block AM₂ receptors (Brain and Grant, 2004), it is possible that endogenous adrenomedullin plays a significant role in the gestational vascular adaptation.

In conclusion, the present study demonstrates a significant role for K_Ca channels in adrenomedullin-induced endothelium-independent relaxation in the isolated mesenteric artery from pregnant rats. Adrenomedullin may activate K_Ca channels but not other potassium channels, mostly through a cAMP-dependent protein kinase A-dependent cellular mechanism. The relaxation effects of adrenomedullin on mesenteric artery, a resistance vessel from pregnant rats, may contribute to the marked vascular adaptations during pregnancy.

	Acknowledgements

 
We thank Cheryl Welch for administrative support during the preparation of this article.

	Footnotes

 
This work was supported in part by the National Institutes of Health Grants HL-58144 and HL-72650.

doi:10.1124/jpet.106.101790.

ABBREVIATIONS: K_ATP, adenosine triphosphate-sensitive potassium channel; PSS, physiological salt solution; CL, calcitonin; RAMP, receptor activity-modifying protein; CGRP, calcitonin gene-related peptide; L-NAME, N-nitro-L-arginine methyl ester; AM₁, adrenomedullin receptor subtype 1; AM₂, adrenomedullin receptor subtype 2; K_V, voltage-gated potassium channel; K_IR, inward rectifier potassium channel; K_Ca, calcium-activated potassium channels; Rp-cAMP, Rp-cyclic adenosine monophosphorothioate(s); SQ22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine; 4-AP, 4-aminopyridine; ANOVA, analysis of variance.

Address correspondence to: Dr. Chandra Yallampalli, Department of Obstetrics and Gynecology, University of Texas Medical Branch, 301 University Blvd., MRB, 11.138, Rt. 1062, Galveston, TX 77555-1062. E-mail: chyallam{at}utmb.edu

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