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(Hypertension. 2001;37:301.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Angiotensin II Relaxes Microvessels Via the AT₂ Receptor and Ca²⁺-Activated K⁺ (BK_Ca) Channels

Christiana Dimitropoulou; Richard E. White; Leslie Fuchs; Hanfang Zhang; John D. Catravas; Gerald O. Carrier

From the Department of Pharmacology and Toxicology (C.D., R.E.W., L.F., J.D.C., G.O.C.) and the Vascular Biology Center (L.F., H.Z., J.D.C.), Medical College of Georgia, Augusta.

Correspondence to Dr Gerald O. Carrier, Department of Pharmacology and Toxicology, Medical College of Georgia, 1120 15th St, Augusta, GA 30912-2300. E-mail gcarrier{at}mail.mcg.edu

	Abstract

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Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II–induced vasoconstriction is mediated by the AT₁ receptor, whereas the relative expression and functional importance of the AT₂ receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT₁ receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT₂ receptors. In addition, reverse transcriptase–polymerase chain reaction studies revealed high amounts of AT₂ receptor mRNA in smooth muscle from these same microvessels. Ang II–induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BK_Ca) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BK_Ca channels. As in our tissue studies, the effect of Ang II on BK_Ca channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT₂ receptor with subsequent opening of BK_Ca channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.

Key Words: angiotensin II • receptors, angiotensin • potassium channels • patch-clamp techniques

	Introduction

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Many complications of hypertension and congestive heart failure are ameliorated by interdiction of the renin-angiotensin system, and agents that attenuate the effects of angiotensin II (Ang II) are now a first-line pharmacological antihypertensive intervention. Ang II induces constriction, hypertrophy, and proliferation of vascular smooth muscle via the AT₁ receptor. It is generally assumed that the therapeutic effects of AT₁ antagonists are due to direct receptor blockade; however, a secondary effect of these agents is increased plasma levels of Ang II,¹ which may then activate AT₂ receptors. This receptor bears only 34% sequence homology to the AT₁ receptor^{2 3} and mediates growth inhibition⁴ and apoptosis⁵ of vascular smooth muscle. In contrast, very little is known about the role of AT₂ receptors in regulating blood pressure. Both Ang II receptors are expressed in the vasculature, but AT₂ receptor expression is heterogeneous with respect to vascular bed, species, and developmental stage.⁶ Nonetheless, evidence suggests that AT₂ receptors modulate vascular contractility. AT₂ receptors mediate endothelium-dependent vasodilation of renal arterioles.⁷ Furthermore, AT₂-deficient mice exhibit an increased pressor response to Ang II,⁸ whereas overexpression of AT₂ receptors enhances Ang II–induced endothelium-dependent vasodilation.⁹ These findings suggest that AT₂ receptors mediate Ang II–induced vascular relaxation; however, the cellular/molecular basis of Ang II–induced vasodilation is not clearly defined, nor has an effector mechanism been identified.

The purpose of the present study was to investigate the expression and physiological role of AT₂ receptors in the microvasculature. We now report significant expression of AT₂ receptors in mesenteric microvessels from adult rats and also demonstrate that Ang II relaxes these vessels via AT₂ receptor–mediated stimulation of the large-conductance, calcium- and voltage-activated potassium (BK_Ca) channel. These findings provide evidence for an endothelium-independent vasodilatory effect of Ang II on vascular smooth muscle cells and may help to explain the salutary effects of AT₁ receptor antagonists on the cardiovascular system, (eg, reduced blood pressure and infarct size¹⁰ due to "indirect" stimulation of AT₂ receptors).

	Methods

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Isolated Microvessel Preparation
Fourth-order mesenteric arterial branches (200 to 300 µm in diameter) were isolated microscopically from male Sprague-Dawley rats (aged 14 to 16 weeks) in accordance with guidelines of the American Physiological Society for the humane treatment of experimental animals and prepared for measurement of intraluminal diameter by video dimension analysis as described previously.¹¹ Vessels were allowed to equilibrate for 30 minutes and were preconstricted to 45% of their resting diameter with endothelin-1 (ET-1). After the constriction reached a steady state level, a complete concentration-response relaxation to Ang II was performed. In additional vessels a concentration-response relationship for Ang II was measured in the presence of tetraethylammonium (TEA), iberiotoxin (IBTx), losartan, or PD123,319.

Reverse Transcriptase–Polymerase Chain Reaction
Smooth muscle cells from 5 mesenteric arteries for each RNA isolation (n=15 rats, 3 RNA isolations) were harvested and pooled for total RNA measurement. As a negative control, RNA was also extracted from rat liver. Total RNA was isolated with the use of Trizol Reagent (GIBCO BRL). Phase separation was achieved by addition of chloroform, and RNA was precipitated from the aqueous phase with isopropyl alcohol. The amount of RNA was determined spectrophotometrically at A₂₆₀. Total RNA (1 to 1.5 µg) was treated with DNase I (GIBCO BRL) for 15 minutes at room temperature and reverse transcribed (RT) with oligo(dT) using SuperScript (GIBCO BRL). Aliquots of the RT products were amplified with Taq DNA polymerase (GIBCO BRL) with the use of gene-specific primers. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed as described previously with the use of primers derived from published cDNA sequences of the AT_1B (accession X64052) and AT₂ (accession D16840) receptors.¹² Primer sequences for AT_1B were 5'-GGC ATT ATC CGT GAC TGT GAA A-3' (forward) and 5'-CTG CTT AGC CCA AAT GGT CCT C-3' (reverse), and the final PCR product was 446 bp in length. Primer sequences for the AT₂ were 5'-GGA GCG AGC ACA GAA TTG AAA GC-3' (forward) and 5'-TGC CCA GAG AGG AAG GGT TGC C-3' (reverse), and the final PCR product was 446 bp in length. PCR negative controls were (1) amplification of H₂O and (2) non-RT samples that were obtained from the same RNA, processed in parallel with the RT reaction but without adding the RT enzyme. The PCR reaction mixtures were first denatured at 94°C for 4 minutes and then amplified for 35 or 38 cycles of 94°C for 40 seconds, 65°C for 1 minute, and 72°C for 1 minute. An aliquot of 10 µL of the RT-PCR amplified sample was fractionated by electrophoresis on 1.5% agarose gel and visualized with ethidium bromide stain.

Patch-Clamp Studies
Single myocytes were isolated by a modification of a procedure described previously,¹³ and potassium channel activity was recorded in either the whole-cell (amphotericin perforated patch) configuration or from cell-attached or inside-out patches as described previously.^{14 15} Briefly, for cell-attached patches several drops of cell suspension were placed in a recording chamber containing the following (mmol/L): 140 KCl, 10 MgCl₂, 0.1 CaCl₂, 10 HEPES, and 30 glucose (pH 7.4; 22°C to 25°C). Activity of single potassium channels was recorded in cell-attached patches by filling the patch pipette (2 to 5 M) with Ringer’s solution, as follows (mmol/L): 110 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, and 10 HEPES. In experiments on inside-out patches, the bathing solution exposed to the cytoplasmic surface of the membrane consisted of the following (mmol/L): 60 K₂SO₄, 30 KCl, 2 MgCl₂, 0.16 CaCl₂, 1 BAPTA (10^-7 M Ca⁺⁺), 10 HEPES, 5 ATP, and 10 glucose (pH 7.4; 22°C to 25°C). The pipette solution was the same Ringer’s solution described above. Current voltage relations were obtained from whole-cell or single-channel amplitudes at potentials between -50 and +50 mV, and single-channel conductance was calculated from the slope of the current-voltage relationship fitted by linear regression. Average channel activity (NPo) in patches with multiple BK_Ca channels was determined as described previously.¹⁴

Drugs
TEA was obtained from Aldrich. Ang II, dithiothreitol, amphotericin B, glibenclamide, 4-aminopyridine, and ET-1 were purchased from Sigma Chemical. PD123,319 was obtained from Research Biochemical International, papain from Worthington Biochemical Corp, and losartan from DuPont-NEN.

Statistical Analysis
All data were expressed as mean±SE except data from tension studies, which were expressed as percentage of maximum relaxation. Statistical significance between 2 groups was evaluated with Student’s t test, and a 1-way ANOVA was performed to evaluate significance between multiple groups. A P value <0.05 was considered to reflect a significant difference.

	Results

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Ang II Dilates Microvessels Via the AT₂ Receptor
Baseline intraluminal diameter of microvessels (intraluminal pressure=40 mm Hg) was 226±12 µm (n >30). Vessels were preconstricted to 45% of the original baseline diameter with ET-1 (diameter reduced from 229±14 to 126±8 µm). In the absence of Ang II, ET-1–induced contraction was maintained for >60 minutes. A complete concentration-response curve for Ang II was then obtained. A typical tracing of Ang II–induced microvascular relaxation is illustrated in Figure 1A. On average, Ang II (0.1 to 300 nmol/L) induced a concentration-dependent relaxation with a maximum of 72±5% (Figure 1B; n=19). Pretreating microvessels with N-nitro-L-arginine (0.1 mmol/L; 30 minutes) had no effect on Ang II–induced relaxation (78±6%; n=4). A similar response to Ang II (78±3%; n=5) was obtained in microvessels pretreated for 30 minutes with 10 µmol/L losartan, a selective AT₁ receptor antagonist (Figure 1B). In contrast, Ang II induced only 31±8% relaxation (n=10; Figure 1B) in microvessels pretreated for 30 minutes with 100 nmol/L PD123,319, a selective AT₂ receptor antagonist. Pretreatment with these blocking agents did not affect baseline intraluminal diameter (Table). These studies indicate involvement of the AT₂ receptor in Ang II–induced vasodilation, and subsequent molecular studies were designed to verify expression of this protein in rat mesenteric microvessels.

View larger version (36K): [in this window] [in a new window]   Figure 1. Ang II relaxes mesenteric microvessels via activation of AT2 receptors. A, Representative tracing of a concentration-response relationship to Ang II in a mesenteric small artery precontracted with ET-1. Ang II produced a concentration-dependent relaxation of 79%. Addition of acetylcholine (Ach) (30 µmol/L) further relaxed the vessel 100%. Intraluminal diameter (ID) in micrometers and a time scale in seconds are shown. B, Average concentration-response relationships for Ang II–induced vasodilation of microvessels under control conditions (n=13) or after a 30-minute pretreatment with either 10 µmol/L losartan (n=5) or 100 nmol/L PD123,319 (n=10). Each point represents the mean±SE relaxation response of n determinations. C, Analysis of Ang II type 1B or type 2 receptor mRNA by RT-PCR in rat mesenteric artery smooth muscle cells (SMC) and liver, as indicated by the lines above the gel. RT-PCR products (10 µL) were separated on 1.5% agarose gel by electrophoresis and stained with ethidium bromide. Arrows denote the DNA size marker. The sizes for the AT1B and AT2 receptors and ß-actin are 346, 446, and 276 bp, respectively. The gel is representative of 3 separate RNA samples from mesenteric arterial SMC.

View this table: [in this window] [in a new window]   Table 1. Intraluminal Diameters of Mesenteric Microvessels

Ang II Receptor mRNA
Representative gels of AT_1B and AT₂ receptor mRNA from isolated mesenteric microvascular smooth muscle cells analyzed by RT-PCR are provided in Figure 1C. Each isolation was examined microscopically to ensure against contamination with other cell types; nonetheless, it is possible that some minor degree of contamination may have been present. Analysis consistently detected AT₂ receptor mRNA. In contrast, amplification of liver RNA did not reveal any bands for Ang II receptor mRNA. Absence or very low copy of these Ang II receptors in liver is consistent with previous studies,^{16 17} whereas mRNA for ß-actin was well detected in liver by RT-PCR.

Ang II–Induced Vasodilation Involves K⁺ Channels
We have demonstrated previously that disrupting the gradient for potassium efflux with high extracellular [K⁺] inhibits potassium channel–mediated vasorelaxation.^{11 14 18} To investigate whether AT₂ receptor–mediated vasodilation is dependent on K⁺ efflux, microvessels were preconstricted with 80 mmol/L K⁺. Ang II did not affect KCl-contracted arteries (data not shown). In microvessels preconstricted with ET-1, Ang II–induced relaxation was measured after a 30-minute pretreatment with a potassium channel inhibitor: 1 mmol/L TEA (n=4; Figure 2A) or 100 nmol/L IBTx (n=4; Figure 2B). In the presence of these agents, even high concentrations of Ang II failed to produce significant relaxation. In contrast, subsequent treatment of microvessels with 10 µmol/L acetylcholine relaxed the microvessels 70±14% (TEA; n=4) or 73±10% (IBTx; n=4).

View larger version (18K): [in this window] [in a new window]   Figure 2. Ang II–induced relaxation of microvessels is mediated by potassium channels. A, Average concentration-response relationship for Ang II relaxation of mesenteric microvessels in the presence of 1 mmol/L TEA. After addition of 1 µmol/L Ang II had no effect, subsequent addition of 10 µmol/L acetylcholine induced significant relaxation of the vessels. Each point represents the mean±SE of 4 experiments. B, Average concentration-response relationship for Ang II relaxation of mesenteric microvessels in the presence of 100 nmol/L IBTx. After addition of 1 µmol/L Ang II had no effect, subsequent addition of 10 µmol/L acetylcholine induced significant relaxation of the vessels. Each point represents the mean±SE of 4 experiments. C, Superimposed whole-cell current tracings from single mesenteric myocytes recorded with the perforated patch configuration. Cells were voltage-clamped at a holding potential of -50 mV, and currents were elicited by 100 ms depolarizing pulses to -40, -10, 20, or 50 mV before or 10 to 15 minutes after exposure to 100 nmol/L Ang II. In the right panel, the effect of subsequent addition of 1 mmol/L TEA is illustrated. Dashed line indicates baseline current. D, Complete current-voltage relationship for steady state outward current (holding potential=-50 mV) before and 10 to 15 minutes after addition of 100 nmol/L Ang II. The effect of subsequent addition of 1 mmol/L TEA on the same cell is also plotted.

Ang II Increases Potassium Currents in Single Myocytes
Direct evidence that Ang II stimulated outward potassium currents in microvascular myocytes was obtained from whole-cell (perforated patch) patch-clamp recordings. Ang II (100 nmol/L) increased macroscopic outward current over the entire range of membrane voltages. For example, at +50 mV Ang II nearly tripled (287±35%; n=7; P<0.001) the steady state outward current. Representative tracings illustrating the effect of Ang II are provided in Figure 2C. The stimulatory effect of Ang II was reversed completely by subsequent treatment with 1 mmol/L TEA. A lower concentration of Ang II (10 nmol/L) nearly doubled (93±12% at +50 mV; n=3; P<0.05) steady state outward currents, and the effect was again abolished by 1 mmol/L TEA (decreased 27±3% below control values). The complete current-voltage relationship for these experiments is illustrated in Figure 2D. In contrast to TEA, neither 4-aminopyridine (1 mmol/L; n=3), a blocker of the delayed rectifier K⁺ channel, nor glibenclamide (10 µmol/L; n=3), a blocker of the ATP-sensitive K⁺ channel, affected Ang II–stimulated outward current (data not shown). These findings strongly suggested that the BK_Ca channel mediated Ang II–induced relaxation of microvessels, and subsequent single-channel studies were undertaken to prove this hypothesis.

Ang II Stimulates BK_Ca Channel Activity
Ang II stimulated the activity of single potassium channels in mesenteric myocytes. As illustrated in Figure 3A, there was minimal channel activity in cell-attached patches under control conditions (NPo <0.001 at +50 mV). However, channel activity was increased dramatically by 100 nmol/L Ang II (NPo 0.92; 15 minutes; Figure 3A), and this effect was concentration dependent (Figure 3B). Identification of this protein as the BK_Ca channel was demonstrated from experiments on cell-free inside-out patches in symmetrical concentrations of potassium (140 mmol/L). A complete current-voltage relationship for channel activity (Figure 3C) yielded a high single-channel conductance of 158±5 picoSiemens (n=3 to 5 inside-out patches). In addition, raising the concentration of calcium at the cytoplasmic surface of the membrane from 100 nmol/L to 100 µmol/L produced a substantial increase in channel gating behavior (Figure 4, top panel). These experiments identify this protein as the high-conductance, Ca²⁺-activated (BK_Ca) channel. In contrast to its effects on BK_Ca channels in cell-attached patches, addition of 100 nmol/L Ang II to the cytoplasmic surface of an inside-out patch had no effect on channel activity (n=3).

View larger version (33K): [in this window] [in a new window]   Figure 3. Ang II stimulates the activity of single BKCa channels in myocytes from mesenteric microvessels. A, Current tracings from the same cell-attached patch (+50 mV) before and 10 minutes after addition of 100 nmol/L Ang II. Channel openings are upward deflections from baseline (closed) state, as indicated by dashed line. B, Average activity (NPo) of BKCa channels recorded from cell-attached patches before and 10 to 15 minutes after addition of either 6 (n =4), 60 (n=4), or 100 nmol/L (n=6) Ang II. *Significant increase in channel NPo compared with control level. C, Average current-voltage relationship for single-channel activity recorded from inside-out patches with symmetrical (140 mmol/L) K+. Each point represents the mean±SE current of 3 to 5 patches, with the line fit by linear regression.

View larger version (18K): [in this window] [in a new window]   Figure 4. AT2 receptors mediate the stimulatory effect of Ang II on BKCa channels. Channel openings (+50 mV) are upward deflections from baseline (closed) state, as indicated by the dashed line. Top tracing, Recordings from the same inside-out patch with either 100 nmol/L or 100 µmol/L [Ca2+] in the solution at the cytoplasmic surface of the membrane, as indicated by the arrows. Ang II (100 nmol/L; 15-minute pretreatment) was also present in the bath solution. Second tracing, Recordings from the same cell-attached patch before and 10 minutes after addition of 100 nmol/L Ang II to the batch solution, as indicated by arrow. Third tracing, Recordings from the same cell-attached patch. Cell was pretreated with losartan (10 µmol/L; 30 minutes), and recordings were then taken before and 15 minutes after subsequent addition of 100 nmol/L Ang II, as indicated by arrow. Bottom tracing, Recordings from the same cell-attached patch. Cell was pretreated with PD123,319 (100 nmol/L; 30 minutes). Recordings were then taken before and 15 minutes after subsequent addition of 100 nmol/L Ang II, as indicated by arrow.

AT₂ Receptors Mediate Ang II Stimulation of BK_Ca Channels
Ang II receptor antagonists were used to determine which receptor mediated the stimulatory effect of Ang II on BK_Ca channels. Channel activity stimulated by 100 nmol/L Ang II is illustrated in Figure 4 under control conditions (panel 2) or after a 30-minute pretreatment with 10 µmol/L losartan (panel 3). Blockade of AT₁ receptors with losartan did not affect Ang II–induced channel activity. On average, control NPo was 0.001±0.02, and after exposure to 100 nmol/L Ang II NPo increased to 0.86±0.02 (n=4). Blockade of AT₂ receptors, on the other hand, completely prevented the stimulatory effect of Ang II. In the presence of PD123,319 (100 nmol/L, 30 minutes), 100 nmol/L Ang II had no effect on BK_Ca channel activity (NPo PD123,319, <0.001; PD123,310+Ang II, <0.001; n=4; bottom panel).

	Discussion

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We have demonstrated that Ang II relaxes mesenteric microvessels via a mechanism involving the AT₂ receptor and BK_Ca channel activity, and this mechanism of Ang II action on intact tissues was confirmed by molecular studies on isolated microvascular myocytes demonstrating (1) expression of AT₂ receptor mRNA and (2) AT₂ receptor–mediated stimulation of BK_Ca channel activity in cell-attached patches. These findings provide evidence for a novel endothelium-independent effect of Ang II on the microvasculature. Previous studies suggest that Ang II relaxes cerebral microvessels^{19 20} or renal afferent arterioles⁷ by an endothelium-dependent mechanism, probably involving release of bradykinin and endothelium-derived dilators (eg, NO).^{9 10} In the present study, however, pretreating mesenteric microvessels with N-nitro-L-arginine did not affect Ang II–induced relaxation, suggesting that endothelium-derived NO does not play a role in the response of this vascular bed to Ang II. More directly, in the absence of endothelium Ang II clearly stimulated activity of BK_Ca channels in isolated myocytes.

Previous studies indicated that Ang II stimulates both whole-cell²¹ and single-channel²² potassium currents in cultured neurons via the AT₂ receptor; however, this is the first study on the effects of Ang II on K⁺ channels in smooth muscle cells. Our studies demonstrate that Ang II increases both whole-cell and single-channel BK_Ca currents in mesenteric myocytes. Instead of traditional whole-cell recordings that require dialysis of the cytoplasm with exogenous calcium buffers, we used the perforated patch configuration to obtain whole-cell currents from "metabolically intact" myocytes. These findings indicated that Ang II increases a TEA-sensitive outward current in these cells, and subsequent single-channel studies revealed that this channel had a high conductance (>150 picoSiemens) and was stimulated by increasing "intracellular" calcium levels in inside-out patches. Therefore, we have identified this protein as the BK_Ca channel that is highly expressed in vascular smooth muscle and other cell types. Like other vascular smooth muscle cells, myocytes from mesenteric microvessels possess substantial outward K⁺ currents composed primarily of K⁺ efflux through BK_Ca channels.¹¹ Because of their large conductance and high density of expression, these channels help to set and maintain the resting membrane potential of vascular smooth muscle.²³ When intracellular [Ca²⁺] levels increase during contraction, BK_Ca channels provide an important repolarizing negative feedback mechanism that helps to reverse active contraction. Given the importance of BK_Ca channels in regulating vascular tone, these proteins constitute a powerful effector mechanism that mediates microvascular relaxation induced by a variety of vasodilatory agents, eg, nitrovasodilators.¹¹ Our studies indicated that microvascular relaxation required physiological gradients of [K⁺] suitable for potassium efflux, suggesting involvement of potassium channels. Although several species of K⁺ channels are also expressed in vascular smooth muscle, we did not observe significant effects of Ang II on other channel species at the single-channel level. Moreover, neither glibenclamide (K_ATP channel antagonist) nor 4-aminopyridine (delayed rectifier channel antagonist) affected Ang II–stimulated whole-cell currents. In contrast, selective blockade of BK_Ca channels (with either 1 mmol/L TEA or 100 nmol/L IBTx; Figure 2) abolished Ang II–induced relaxation. Although TEA can block several species of potassium channels, at this low concentration it exhibits selectivity for the BK_Ca channel. IBTx, on the other hand, is a highly selective BK_Ca channel antagonist. Moreover, our single-channel patch-clamp data clearly identified the BK_Ca channel as a primary target of Ang II action in microvascular myocytes, and our findings that PD123,319 (but not losartan) inhibited this stimulatory effect of Ang II further implicate the BK_Ca channel as an effector molecule for AT₂ receptor–mediated vasodilation. Although our findings clearly demonstrate an endothelium-independent effect of Ang II on microvascular myocytes, the vasodilatory response to Ang II in vivo would probably involve both endothelium-dependent and -independent mechanisms. In either case, our data obtained from intact tissues or single myocytes indicate that BK_Ca channels expressed in microvascular smooth muscle cells are the critical effector molecules involved in AT₂ receptor–mediated relaxation of microvessels.

The signaling process linking AT₂ receptors to BK_Ca channels in vascular smooth muscle is poorly understood. For example, metabolites of arachidonic acid might play a role in the response of BK_Ca channels to Ang II in mesangial cells; however, it is unclear whether the response of BK_Ca channels in these cells is mediated via AT₁ and/or AT₂ receptors.²⁴ Therefore, it is difficult to draw strict comparisons between these 2 studies on different cell types and species. Nonetheless, because arachidonic acid metabolites open BK_Ca channels in other arteries,²⁵ a potential role of arachidonic acid in mediating the response of mesenteric microvessels to Ang II remains a possibility. Alternatively, BK_Ca channels could be opened indirectly by Ang II–stimulated intracellular calcium. Although we have not tested this hypothesis conclusively, we do not believe that direct activation by calcium plays a major role because of the following: (1) Ang II induces vasodilation; therefore, the putative increase in calcium would have to be minimal and probably localized to the more peripheral regions of the cytoplasm near the BK_Ca channels. Such a "calcium-spark" model is possible, but we are unaware of any studies suggesting that Ang II stimulates such a mechanism in microvessels. (2) Ang II produced no obvious shift in the voltage sensitivity of outward currents, whereas increased [Ca²⁺]_i shifts the sensitivity of BK_Ca channels to more negative potentials. In contrast, the effect of Ang II appears to be due mainly to increased current amplitude rather than increased voltage sensitivity. In contrast to the aforementioned mechanisms, it is clear that cyclic nucleotide–dependent vasodilators open BK_Ca channels in vascular smooth muscle.¹⁵ In the present study, however, inhibitors of either the cAMP- or the cGMP-dependent protein kinase had no effect on Ang II–stimulated BK_Ca channel activity (data not shown). These data suggest that a more novel mechanism of action underlies the effect of Ang II in microvessels; however, further experiments are necessary to elucidate the transduction mechanism coupling AT₂ receptors to BK_Ca channels.

It is clear that AT₂-induced vasodilation affects blood pressure. Animals lacking AT₂ receptors exhibit an enhanced pressor response to Ang II,²⁶ whereas overexpression of AT₂ receptors antagonizes AT₁ receptor–mediated pressor effects.⁹ AT₂ receptor–mediated vasodilation, particularly of microvessels, may serve as a negative feedback mechanism to counterbalance the potent vasoconstrictor effect of AT₁ receptor activation. For example, Ang II levels are increased during exercise,²⁷ and AT₂ receptor–mediated dilation of precapillary vessels might help to offset potentially dangerous effects of diminished capillary perfusion in face of excessive AT₁ receptor stimulation. Therapeutically, it seems clear that AT₂ receptors mediate salutary responses. For example, AT₂ receptor stimulation may play a part in the antihypertensive effects of AT₁ receptor antagonists, which increase plasma Ang II levels. In addition, our proposed "endothelium-independent" vasodilatory effect of Ang II could constitute a protective vasodilatory mechanism to preserve tissue perfusion when the endothelium is damaged as a result of atherosclerosis and/or hemodynamic stress. Interestingly, activation of AT₂ receptors reduces infarct size.¹⁰ The present findings are the first to provide direct experimental evidence for a novel molecular effector (the BK_Ca channel) that can mediate endothelium-independent relaxation of vascular smooth muscle via Ang II stimulation of the AT₂ receptor. Future studies will identify and characterize the postreceptor signal transduction cascade stimulated by Ang II in these microvessels.

	Acknowledgments

 
This work was supported by grants from the American Heart Association (R.E.W., G.O.C., L.F.) and the National Institutes of Health (HL-54844, R.E.W.; HL-64779, R.E.W., G.O.C.; HL-52958, J.D.C.).

Received May 15, 2000; first decision June 6, 2000; accepted August 29, 2000.

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