Role of maxi-K+ channels in endothelin-induced vasoconstriction of mesenteric and submucosal arterioles

Ceredwyn E. Hill, Adam Kirton, Douglas D. Wu, and Stephen J. Vanner

Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital, Kingston, Ontario, Canada K7L 5G2

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The action of endothelin in small intestinal resistance vessels of the guinea pig was studied by examining submucosal arteriole vasoactivity in vitro and electrical properties of mesenteric arteriole smooth muscle cells. Endothelin-1 (ET-1) constricted submucosal arterioles with a half-maximal effective concentration of 170 pM. ET-3 caused detectable constriction with a minimum of 20 nM. The ET-1 response was prolonged, with a time to 90% relaxation of 41 ± 2.8 min after washout. The ETA antagonist BQ-123 (200 nM) decreased the sensitivity to ET-1 ~40-fold. Arterioles preconstricted with prostaglandin F2alpha did not relax when superfused with ET-1, ET-3, or an ETB agonist, IRL-1620, and pretreatment with the nitric oxide synthase inhibitor NG-monomethyl-L-arginine was ineffective in countering ET-1-induced constriction, indicating the absence of functional ETB receptors. Resting membrane potential in isolated cells was characterized by transient hyperpolarizing spikes (THs). ET-1 (20 nM) increased TH frequency and caused the emergence of a larger amplitude population. Under voltage clamp, spontaneous transient outward currents (STOCs) were seen that reversed at the K+ equilibrium potential. ET-1 increased STOC frequency and amplitude. Iberiotoxin (IBTX; 200 nM), a maxi-K+ channel antagonist, blocked the ET-1-induced THs and reduced STOC activity. IBTX or tetraethylammonium increased the rate and extent of ET-1-induced arteriole constriction. We suggest that ET-1-induced vasoactivity of ileal resistance arterioles involves ETA receptor-mediated early activation of maxi-K+ channels that serves to counter strong constriction.

smooth muscle; potassium channel; ileum; patch clamp

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ENDOTHELINS ARE A GROUP OF 21-amino acid residue peptides that have powerful vasoconstrictive effects on vascular smooth muscle (33). Endothelins are significant hemodynamic regulators in the microvasculature of the rat intestine (16), and they appear to mediate the decreased arteriolar blood flow and diameter that occurs during bacteremia (32). Endothelin is synthesized within the lamina propria cells of the gastrointestinal tract (19), and mRNA for its receptor has been demonstrated in the small and large intestine (3). However, the pharmacological properties of endothelin and the cellular effects it has on small resistance vessels within the gastrointestinal tract have not been characterized, although these arterioles serve as the primary sites for control of intestinal perfusion and ultimately the efficiency of mucosal absorption and secretion.

In contrast to the small resistance vessels of the intestine, the mechanisms involved in endothelin-dependent vasoactivity in other vascular tissue are more clearly established. In general, activation of endothelin-A (ETA) receptors on vascular smooth muscle cells leads to changes in membrane excitability and inositol phospholipid metabolism that result in increased cytosolic Ca2+ concentration and ultimately contraction (see Ref. 5 for review). Membrane potential of resting vascular smooth muscle is mainly contributed to by a variety of K+ channels (see Refs. 5, 23 for review). Current flow through large-conductance, voltage- and Ca2+-sensitive K+ channels or maxi-K+ channels is thought to serve as a negative feedback signal to repolarize the membrane and counter the actions of vasoconstrictors and to regulate myogenic tone in pressurized resistance vessels (6). Smooth muscle preparations from larger and conduit arteries depolarize in response to ET-1 (7, 11, 13, 15, 21, 26), although initial transient hyperpolarizations have been reported (20, 30). In comparison, there is a dearth of studies on the effects of ET-1 on membrane electrical properties of resistance arteriole smooth muscle. The role of maxi-K+ channels in endothelin-induced vasoconstriction is not well defined and is confounded by reports showing increased (12, 26), decreased (18), or transiently stimulated (11, 15) activity. Early activation of maxi-K+ channels may be important in regulating the activity of voltage-gated and other channels that are modulated by endothelin.

The aims of this study were to establish the functional role of endothelin in resistance arterioles of the guinea pig ileum and to define the contribution of maxi-K+ channels in ET-1-induced vasoactivity. An isolated submucosal preparation was used to measure vasomotor activity in the resistance vessels within the intestine, and membrane electrical properties were monitored in acutely dissociated smooth muscle cells from small mesenteric resistance arteries. The results show that ET-1 constricts ileal resistance vessels through ETA receptors and that early activation of maxi-K+ channels supplies a significant buffer countering the full constrictive potential of this peptide.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation and measurement of submucosal arteriole diameter in guinea pig ileum. Segments of ileum (8-10 cm) were removed from male albino guinea pigs (150-200 g; Charles River Canada, St. Constant, PQ, Canada) after cervical dislocation and carotid transection. This method was approved by the Animal Care committees of Queen's University and the Canadian Council. We isolated 1-cm2 sections of a submucosal preparation by removing the mucosa and, from the opposite surface, the circular, longitudinal, and myenteric plexus, as described previously (31). The submucosal preparations were pinned serosal side up in an organ bath and perfused at 15 ml/min with a physiological saline solution containing (in mM) 126 NaCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose, gassed with 95% O2-5% CO2 and maintained at 35-36°C. Peptides, antagonists, and modified saline solutions were applied by bath perfusion.

The outside diameter of submucosal arterioles was monitored using a video-imaging technique involving a video camera and monitor interfaced with a frame grabber (PC Vision Imaging Technology, Woburn, MA). The outer diameter of the imaged vessels was measured as the distance between two cursors using Diamtrack software (25) and displayed on a chart recorder. The amount of constriction in response to the peptides was expressed as the percentage of the resting vessel diameter. In some experiments, the recordings were normalized to the maximum amount of constriction induced by 2 nM ET-1 and the resting vessel diameter, so that the time course of constriction could be examined and statistically analyzed. At least three experiments were conducted for each condition. Representative traces are shown, and, where appropriate, mean and SE are reported.

Isolation of mesenteric arteriole smooth muscle cells and measurement of membrane potential and whole cell currents. Mesenteric arterioles (6 to 8 1-cm segments) consisting of the vasa recta were dissected from between the smallest arcades and the ileal border of the segments of ileum obtained previously and placed into N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered Hanks' saline containing 0.15% (wt/vol) bovine serum albumin (BSA) and (in mM) 136.9 NaCl, 4.2 HCO<SUP>−</SUP><SUB>3</SUB>, 0.8 MgSO4, 0.16 CaCl2, 5.9 KCl, and 19.8 HEPES, pH 7.4, and incubated for 10 min at 20°C. The vessels were then minced in fresh saline containing 1.3 mg/ml papain, 1 mM dithiothreitol, and 0.05% BSA and incubated at 37°C for 30 min at 75 revolutions/min (rpm). The tissue was collected and resuspended in fresh saline containing collagenase (type I; 2 mg/ml) and 0.05% BSA and incubated for 15 min at 37°C and 75 rpm. After gentle trituration and washing, the cells were resuspended in the initial Hanks' saline and plated onto poly-L-lysine-coated glass coverslips and allowed to adhere for at least 1 h at 4°C. Patch-clamp experiments were carried out at 20°C the same day on cells 10-20 µm wide and 20-40 µm long.

Whole cell voltage and current clamp measurements were recorded from single smooth muscle cells dialyzed with a pipette solution containing (in mM) 145 KCl, 5 HEPES, and 1 MgSO4, pH 7.4. Ca2+ was weakly buffered to 0.1 µM by the further addition of 7.5 µM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 5 µM CaCl2. The extracellular solution normally consisted of (in mM) 140 NaCl, 5 KCl, 5 HEPES, 1 MgSO4, 1 CaCl2, and 5 glucose, pH 7.4, although in some experiments KCl was increased to 115 mM with NaCl decreased to 30 mM. Heat-polished patch pipettes of thin-walled borosilicate glass (Kimax-51) had resistances of 2-3 MOmega in these solutions. Membrane potential and whole cell currents were measured using an Axopatch 200A amplifier under the current- and voltage-clamp configurations and a Digidata 1200 A/D board (Axon Instruments, Foster City, CA). Data were filtered (low pass, 3 dB cutoff) and sampled at 0.2 and 1 kHz respectively. Digitized records were analyzed using pClamp 6.0 (Axon Instruments) and Origin 4.1 (Microcal Software, Northampton, MA) software.

Materials. ET-1, ET-3, NG-monomethyl-L-arginine (L-NMMA), and prostaglandin F2alpha (PGF2alpha ) were purchased from Calbiochem (San Diego, CA). IRL-1620 was a gift from CIBA Geigy Canada (Calgary, AB, Canada). Tetraethylammonium (TEA) chloride was from Research Biochemicals (Natick, MA). Papain and HEPES were purchased from Boehringer Mannheim, and collagenase was from GIBCO/BRL (Grand Island, NY). All other reagents were from British Drug Houses (Toronto, ON, Canada) or Sigma Chemical (St. Louis, MO) and were of the highest grade available.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Characterization of response of submucosal arterioles to endothelins. Superfusion of 2 nM ET-1 to submucosal preparations of the guinea pig ileum caused a decrease in arteriole diameter that reached a steady state in 20 min (Fig. 1A). The initial rapid phase of constriction lasted ~2 min and was followed by a second further decrease to a minimum at 20 min of exposure. At increasingly lower concentrations, the rate and extent of constriction were greatly reduced and delayed and became monophasic. For example, constriction in response to 60 pM ET-1 (Fig. 1A, top trace) occurred at ~5% of the rate and took almost three times longer to reach a steady state than that in reponse to 2 nM ET-1. To construct a concentration-response relationship, diameter was measured at the steady state (above 60 pM) or at 1 h in the presence of lower ET-1 concentrations, since vasoconstriction in response to 300 nM PGF2alpha or 50 mM KCl was not consistent after 90 min (data not shown).


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Fig. 1.   Concentration dependence of endothelin-1 (ET-1)-induced vasoconstriction of ileal submucosal arterioles. A: vessel diameter recordings of arterioles superfused with 60 pM (top) and 2 nM ET-1 (bottom). Resting outside diameters were 65 and 58 µm for top and bottom, respectively. B: concentration-response curves constructed from arterioles exposed to different ET-1 concentrations in absence or presence of 200 nM BQ-123. Preparations were superfused with BQ-123 for 5 min before and during ET-1 application. %Constriction was calculated as [(resting diameter - minimum diameter)/resting diameter] × 100.

Vascular constriction in response to endothelins is dependent on the receptor subtypes present and the relative affinities for different peptide isoforms. The concentration-response curves shown in Fig. 1B demonstrate that submucosal arterioles have a high affinity for ET-1 [half-maximal effective concentration (EC50) = 0.17 ± 0.1 nM; slope = 0.79 ± 0.4; n = 3 at each concentration]. Furthermore, the constrictive activity is likely due to activation of ETA receptors since superfusion of 200 nM of the ETA receptor antagonist BQ-123 5 min before and during ET-1 exposure displaced the dose-response curve for ET-1 to the right without significantly affecting the slope or maximum constriction (EC50 = 7.22 ± 1.4 nM; slope = 1.58 ± 0.4; n = 3 at each concentration). The relative affinity for the other constrictor isoform of endothelin (ET-3) compared with ET-1 was at least 10-fold less (Fig. 2A).


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Fig. 2.   Effects of ET-3 on constriction, and endothelin agonists on submucosal arterioles preconstricted with prostaglandin F2alpha (PGF2alpha ). Representative recordings of arteriole diameter during superfusion with 0.2, 2, or 20 nM ET-3 (A) or 20 nM ET-3, 2 nM ET-1, or 200 nM IRL-1620 (B) preconstricted with 300 nM PGF2alpha superfused from the arrow. Resting outside diameters of the vessels were, from left to right, 62, 58, and 65 µm in A and 62, 85, and 64 µm in B.

The possibility that endothelins could act through ETB receptors to dilate submucosal arterioles was tested by preconstricting vessels with 300 nM PGF2alpha followed by superfusion with ET-3, ET-1, or the ETB agonist IRL-1620 (Fig. 2B). There was no vasodilatory effect of any of these compounds over the time course examined. To confirm that nitric oxide (NO) was not being generated through ETB receptor activation, the preparation was exposed to the NO synthase inhibitor L-NMMA (300 µM) for 3 min before and during perfusion with 2 nM ET-1. The percent constriction in the presence of ET-1 alone (58 ± 9%; n = 3) was not significantly different from that recorded in the L-NMMA-pretreated tissue (54 ± 4%; n = 3) (data not shown).

A well-established characteristic of ET-1-induced vasoconstriction is its prolonged effect, even after extended washout of the peptide (33). The submucosal preparation also showed long-lasting constriction in response to ET-1 compared with several other vasoconstrictors. Figure 3A shows records of two different vessel measurements made in the presence of 10 µM norepinephrine or 0.2 nM ET-1. The time to 90% relaxation after removal of five different constrictors shows that ET-1, in contrast to each of the other compounds, took at least eight times longer (Fig. 3B).


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Fig. 3.   Time dependence of recovery from constriction of submucosal arterioles from ET-1 and other vasoactive compounds. A: representative recordings of arterioles superfused with 200 pM ET-1 (top) or 10 µM norepinephrine (NE; bottom). B: histogram shows means ± SE (n = 3 for each condition) of time to 90% relaxation calculated from onset of perfusion with compound-free perfusate (ET-1, 0.2 nM; NE, 10 µM; PGF, 300 nM PGF2alpha ; ET-3, 60 nM; VAS, 10 nM arginine-vasopressin).

Membrane potential and whole cell currents in mesenteric arteriole smooth muscle cells. Freshly isolated smooth muscle cells did not rest at a constant membrane potential. When dialyzed with a weakly buffered Ca2+ solution, membrane potential was characterized by aperiodic, transient hyperpolarizing spikes (THs) of variable amplitude. A representative recording shows that unstimulated cells rested at about -41 mV and had THs ranging from 5 to 40 mV in amplitude (Fig. 4A). Under voltage clamp, and at a holding potential of 0 mV, the same cell showed spontaneous transient outward currents (STOCs) of similar frequency and duration as the potential spikes (Fig. 4B). To demonstrate that these oscillations were mainly due to outward current through K+ channels, cells were bathed in medium containing 115 mM KCl rather than the usual 5 mM KCl. Under these conditions, the STOCs reversed near 0 mV so that STOCs were inwardly directed at -40 mV and outward at +40 mV (Fig. 4C).


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Fig. 4.   Whole cell recordings from single isolated mesenteric arteriole smooth muscle cells under current or voltage clamp. Cells were dialyzed with a weakly Ca2+-buffered, K+-rich solution and bathed in 5 mM (A, B) or 115 mM KCl (C). Membrane potential (A) and currents (B, C) were recorded, the latter at a holding potential of 0 mV (B) or ±40 mV (C). A and B are from the same cell and C is from a separate cell.

ET-1 stimulates iberiotoxin-sensitive transient hyperpolarizations and STOCs with little effect on averaged membrane potential. The effects of ET-1 and the specific maxi-K+ channel blocker iberiotoxin (IBTX) on the resting properties of acutely isolated arteriole smooth muscle cells were assessed by continuously recording membrane potential or current before and during exposure to the peptides. Six of nine cells recorded from responded to the compounds. Figure 5A shows that 20 nM ET-1 increased TH amplitude and decreased baseline membrane potential by ~2.5 mV. Relatively high concentrations of IBTX were required to inhibit the THs, although it is apparent that 40 nM IBTX decreased TH frequency. The THs were almost fully blocked by 200 nM IBTX. To quantitate the effects of ET-1 and IBTX (200 nM) on smooth muscle cell membrane potential, the TH data from each responding cell were binned from identical record lengths in the absence or presence of the peptides and expressed as fractions of the total number of events. The data were pooled from all cells for each condition, and the means ± SE (n = 6) were plotted as a function of TH amplitude (Fig. 5B). The control data fell roughly within a single Gaussian distribution, with the peak number of events falling between 5 and 7.5 mV amplitude. ET-1 broadened and shifted the peak to a midpoint between 15 and 17.5 mV, with some new THs >30 mV. Subsequently, 200 nM IBTX reduced TH amplitude and frequency to <10 mV and 7% of the total number of events. The fraction of events during control, ET-1, and 200 nM IBTX intervals was 0.50 ± 0.2, 0.45 ± 0.2, and 0.07 ± 0.1, respectively. Averaged membrane potential of the pooled data (baseline plus THs) was depolarized by 0.51 ± 3.7 and 1.06 ± 3.7 mV (n = 6) in the presence of ET-1 and IBTX, respectively, relative to controls. The averaged potential of the recording shown in Fig. 5A was -43.3, -41.1, and -39.2 mV during the control, ET-1, and 200 nM IBTX intervals, respectively. One-way analysis of variance demonstrated that each of these values was significantly different (P < 0.01) from the others.


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Fig. 5.   Effects of ET-1 and iberiotoxin (IBTX) on frequency and amplitude of transient hyperpolarizations in single smooth muscle cells. A: cells were dialyzed with a weakly Ca2+-buffered, K+-rich solution and bathed in 5 mM KCl, and at the large bipolar spikes ET-1 and IBTX were applied to cell surface through a pressure-ejection pipette. Membrane potential was recorded under current clamp configuration of patch-clamp technique; arrow indicates -50 mV membrane potential. B: histogram shows that the fractional number of events from each of 6 responding cells were pooled over control, ET-1, and 200 nM IBTX intervals and plotted as a function of the transient hyperpolarization amplitude; bin width = 2.5 mV.

The effects of ET-1 and IBTX on the transient outward currents underlying the STOCs were observed by recording membrane current across a range of potentials between -40 and +40 mV. Representative traces from one cell are shown in Fig. 6. The results show that ET-1 increased the frequency and amplitude of STOCs, whereas subsequent addition of IBTX reduced these parameters to less than control values. These results indicate that STOCs were sensitive to IBTX and were enhanced by ET-1.


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Fig. 6.   ET-1 increases and IBTX decreases frequency and amplitude of spontaneous transient outward currents. Cells were dialyzed with a weakly Ca2+-buffered, K+-rich solution and bathed in 5 mM KCl in absence (control; left) or presence of 20 nM ET-1 (middle), or both ET-1 and 200 nM IBTX (right). Representative records from a single cell show membrane current under voltage clamp between -40 and +40 mV.

Contribution of a TEA- and IBTX-sensitive component to ET-1-induced vasoconstriction. Although the role of maxi-K+ channels in classic vasoconstrictor responses is fairly well defined, it has not been widely studied in the ET-1 stimulus-response pathway using parallel vessel and single cell models. TEA (10 mM) increased the extent and almost doubled the mean rate of contraction of seven submucosal arteriole preparations in response to 2 nM ET-1 (Fig. 7A, top). A similar trend was observed with IBTX (88 nM), although the smaller sample size (n = 3) resulted in an increased error about the mean (Fig. 7A, bottom). The IBTX- and TEA-sensitive components, obtained by subtracting the mean of the control records from those obtained in the presence of the blockers, had rapid onsets and attained steady states within 1-2 min of the initiation of constriction (Fig. 7B). Superfusion of 10 mM TEA for 60 min, or 88 nM IBTX for 5 min, in the absence of ET-1 had no effect on vessel diameter (not shown).


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Fig. 7.   Tetraethylammonium (TEA)- or IBTX-induced increase in rate and extent of constriction of submucosal arterioles. Arterioles were superfused with 2 nM ET-1 from 0 min in absence or presence of 10 mM TEA added 5 min earlier and maintained over course of experiment, or 88 nM IBTX added 5 min before and the first 8 min during ET-1 exposure. Resting vessel diameter ranged from 38 to 87 µm. Vessel diameter was normalized by plotting resting diameter × maximum constriction + 2 nM ET-1. TEA and IBTX data were normalized using the resting diameter to determine appropriate scaling factor. A: pooled recordings (means ± SE) made in presence of ET-1 with or without TEA (top, n = 7) or IBTX (bottom, n = 3). B: TEA- and IBTX-sensitive components were calculated as ET-1 + TEA or IBTX - control from A. Dashed line indicates a normalized vessel diameter of 1.0.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

This study shows that ET-1-induced constriction of small intestinal resistance arterioles involves ETA receptors linked to early activation of TEA- and IBTX-sensitive K+ channels. We conclude that opening of maxi-K+ type channels serves to attenuate the force of contraction induced by ET-1. Because neither the averaged and pooled membrane potential in the isolated cells nor basal tone in the intact vessels was greatly affected by the maxi-K+ blockers, it is suggested that these channels do not contribute significantly to resting membrane potential but are important in modulating the strong vasoconstrictive effects of ET-1 and that depolarization is not a prerequisite for the latter.

ETA receptors in resistance arterioles of the guinea pig ileum. The functional studies reported here demonstrate that submucosal arterioles of the guinea pig ileum contract in response to ET-1 through ETA receptors. This conclusion is supported by the observations that the specific ETA receptor antagonist BQ-123 shifted the concentration-dependence curve to higher concentrations of ET-1, the ETB agonist IRL-1620 did not cause vasoconstriction, and the relative affinity of the constriction for ET-1 was roughly 100-fold greater than that for ET-3. These properties are similar to ETA receptor-mediated responses in a variety of other cells and tissues. ETA receptors in different vascular tissues vary between 10- and 100-fold higher affinity for ET-1 over ET-3 (1, 3, 8, 10, 14). The EC50 for high-affinity ET-1 receptor responses falls within 0.18 and 0.62 nM (3, 10, 14, 33), further demonstrating the similar nature of the submucosal receptors characterized here (0.17 nM). Our results with the NO synthase inhibitor and the ETB agonist in the submucosal preparation do not support the existence of functional ETB receptors linked to vasodilation, through activation of endothelial cells, although there is evidence for such a mechanism in arterial tissue (17, 21).

Submucosal and mesenteric arteriole endothelin receptors linked to maxi-K+ channel activation and membrane potential stabilization. Our results show that ET-1-induced constriction of submucosal arterioles (<100 µm diameter) was enhanced in the presence of 10 mM TEA or 88 nM IBTX, suggesting that maxi-K+ channels moderate the constrictive effect of ET-1. Furthermore, the kinetics of the blocker-sensitive components were identical to that observed for the ET-1-stimulated increase in cytosolic Ca2+ concentration in endothelium-denuded, fura-2-loaded, mesenteric arteriole strips (34). In both studies, maximum contraction was attained much later than the plateau of the Ca2+ concentration or TEA/IBTX-sensitive constriction. Attenuation of constriction may be due to the hyperpolarizing effect of increased K+ channel open probability and hence buffering of the depolarization resulting from cation entry or Cl- release through nonselective or Ca2+-activated channels, respectively, and/or decreased open probability of voltage-gated Ca2+ channels.

We also show that isolated smooth muscle cells from mesenteric arterioles do not undergo significant depolarization in response to ET-1. This contrasts with substantial evidence that saturating concentrations of ET-1 depolarize, by 20 to 30 mV, vascular smooth muscle cells from large, conduit arteries or derived cell lines (7, 11, 13, 15, 24, 26, 30). These studies used a variety of techniques to measure membrane potential, including whole cell and intracellular electrode recording and potential-sensitive dyes, so it is unlikely that the experimental approach used here (whole cell dialysis with weakly buffered Ca2+ solutions) could account for the differences. Rather, this may be due to the fact that the cells were derived from resistance arterioles rather than larger vessels. There is some evidence that responses to ET-1 and other vasoactive agents may be phenotype dependent since two populations of cells isolated from cultured thoracic aorta smooth muscle either depolarize or hyperpolarize when exposed to ET-1 (24). From the data available, it is not possible to determine the reasons for the quite different electrical responses to vasoconstrictors in different vascular smooth muscle, although their potential importance in the physiology and pharmacology of the microvasculature is evident.

Our results, that STOCs in mesenteric arteriole smooth muscle cells are blocked by IBTX, increase in frequency and amplitude with membrane depolarization, and reverse at the K+ equilibrium potential, confirm earlier reports that STOCs are mainly carried by maxi-K+ channels (2, 22, 28). Furthermore, STOCs have been shown to be inhibited by TEA in a concentration-dependent manner, with 10 mM causing more than 90% block (4, 9, 28). In contrast, low-conductance K+ channels of smooth muscle are generally much less sensitive to TEA (29; see Ref. 23 for review). We therefore suggest that the majority of the TEA-induced constriction of the submucosal arterioles, as confirmed by the similar pattern of constriction by IBTX, was due to block of maxi-K+ channels activated by endothelin.

A few studies have looked at the effects of ET-1 on vascular smooth muscle cell STOC activity. Resting coronary, mesenteric, and renal arterial smooth muscle cells dialyzed with 20 or 30 µM EGTA and no added Ca2+ exhibited STOCs that, when exposed to saturating concentrations of ET-1, transiently increased in both amplitude and frequency and then disappeared (11, 15). In contrast, we found that ET-1 caused a sustained increase in amplitude and frequency of STOCs and transient hyperpolarizations in mesenteric arteriole cells. We used a mixture of 5 µM CaCl2 and 7.5 µM EGTA to weakly buffer internal Ca2+ to 0.1 µM, to be more representative of the state in vivo. Taken together, these results suggest that the relative complement of active maxi-K+ and depolarizing channels determines the presence of STOCs and the membrane potential response to ET-1, and that the increase in cytosolic Ca2+ caused by ET-1 may have been curtailed in the studies in which cells were dialyzed with EGTA.

Role of maxi-K+ in the stimulus-contraction coupling pathway for ET-1 in resistance arterioles. ET-1 is thought to stimulate contraction of smooth muscle cells through a sequence of events involving ETA receptor activation and inositol 1,4,5-trisphosphate synthesis, leading to increased cytosolic Ca2+ concentration as a result of sarcoplasmic reticular release and plasma membrane influx (see Ref. 17 for review). Although some of the ET-1-regulated channels have been identified (e.g., see Ref. 11 for review), there is conflicting evidence regarding the modulation, and hence physiological role, of maxi-K+ channels by ET-1 in vascular smooth muscle (11, 18, 26). At saturating concentrations of ET-1, we observed that submucosal arteriole constriction was biphasic, and this pattern was accentuated in the presence of maxi-K+ channel blockers. All of the endothelins have biphasic pressor effects in anesthetized rats (14). Similar effects of hypoxia on intrapulmonary artery tension in vitro are paralleled by biphasic increases in cytosolic Ca2+ (27). There is some evidence that the second phase involves Ca2+ sensitization of the contractile apparatus (27). The prolonged response to endothelins is also correlated with increased Ca2+ sensitivity of the myosin light chain (34). Our maxi-K+ blockade experiments in both the intact arterioles and single cells suggest that the first phase of ET-1-induced constriction is partially due to activation of voltage-gated Ca2+ channels, with the major complement of cytosolic Ca2+ derived from voltage-insensitive Ca2+ inflow and release from intracellular stores. We speculate that the hyperpolarizing influence of early activation of maxi-K+ channels would provide an increased driving force for current flow through nonselective channels, a neutralizing counterion for Ca2+-activated Cl- release, and/or a feedback mechanism for countering depolarization caused by the increased inward current induced by endothelin.

    ACKNOWLEDGEMENTS

We thank Margaret Bolton and Emma Hollingsworth for expert technical assistance.

    FOOTNOTES

This research was supported by the Medical Research (S. J. Vanner) and Natural Sciences and Engineering Research (C. E. Hill) Councils of Canada.

Address for reprint requests: C. E. Hill, GI Research, Hotel Dieu Hospital, 166 Brock St., Kingston, Ontario, Canada K7L 5G2.

Received 12 December 1996; accepted in final form 4 August 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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