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Oxygen induces electromechanical coupling in arteriolar smooth muscle cells: a role for L-type Ca21 channels DONALD G. WELSH, WILLIAM F. JACKSON, AND STEVEN S. SEGAL The John B. Pierce Laboratory and Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06519 Welsh, Donald G., William F. Jackson, and Steven S. Segal. Oxygen induces electromechanical coupling in arteriolar smooth muscle cells: a role for L-type Ca21 channels. Am. J. Physiol. 274 (Heart Circ. Physiol. 43): H2018–H2024, 1998.—We tested whether O2-induced vasomotor responses of arterioles correspond to changes in membrane potential (Em ) of cells in the arteriolar wall. The cheek pouches of anesthetized male hamsters were prepared for intravital microscopy and intracellular recording. Microelectrodes containing Lucifer yellow dye were used to label smooth muscle cells (SMC) or endothelial cells (EC) during arteriolar responses to O2. During low- PO2 superfusion (,20 Torr; arteriolar diameter 55 6 2 µm), Em of SMC and EC averaged 237 and 236 mV, respectively. High-PO2 superfusion (,150 Torr) depolarized SMC (to 215 6 1 mV) with vasoconstriction (to 24 6 2 µm) and diameter cycled with Em of SMC during vasomotion. In contrast, the Em of EC did not change with PO2 nor during vasomotion, yet Em depolarized by 21 6 2 mV when the extracellular K1 concentration ([K1]o ) was raised to 55 mM. Superfusion with diltiazem (10 µM) or nifedipine (1 µM) abolished vasomotor and electrical responses to PO2 in SMC but did not eliminate depolarizations to elevated [K1]o. We conclude that, under physiological conditions, electrical and mechanical responses of arteriolar SMC to changes in PO2 are mediated through L-type Ca21 channels without corresponding electrical activity in EC. arteriole; blood flow control; membrane potential; microcirculation; oxygen reactivity; vascular smooth muscle

ARTERIOLES RESPOND to changes in PO2 during blood flow control. In peripheral tissues, a fall in PO2 elicits vasodilation, whereas the elevation of PO2 produces

vasoconstriction (5, 15). At present, the mechanism by which O2 induces vasomotor responses in resistance vessels remains unresolved. Vasomotor responses to O2 may involve the release of factors from parenchymal cells (5, 15), endothelial cells (EC) (18, 20), or red blood cells (5a) and their corresponding actions on smooth muscle cells (SMC). Alternatively, O2 may act directly on SMC to regulate their contractile activity (3, 6–8). In SMC of coronary (3, 23) and cerebral (8) arteries, a fall in PO2 has been found to activate ATP-sensitive K1 channels and Ca21-activated K1 channels (KCa ), respectively. The resulting hyperpolarization limits Ca21 influx through voltage-sensitive, L-type Ca21 channels to produce smooth muscle relaxation. In contrast, O2 may act directly on L-type Ca21 channels to regulate Ca21 entry into SMC independent of changes in K1 conductance (6, 7). In arterioles, it is unclear whether membrane potential (Em ) is influenced by the ambient PO2, particularly under physiological conditions. If so, then changes in PO2 could regulate arteriolar diameter via electromechanical coupling (22, 25). H2018

The goal of the present study was to determine whether O2-induced vasomotor responses of arterioles correspond to Em changes in cells of the arteriolar wall. In turn, we hypothesized that if Ca21 influx depends on changes in K1 conductance, then antagonism of L-type Ca21 channels should inhibit the vasomotor effects of O2 with little effect on depolarization. Alternatively, if L-type Ca21 channel activation underlies both electrical and mechanical responses, then antagonism of these channels should block both responses to O2. METHODS

Hamster cheek pouch preparation. All procedures were approved by the Animal Care and Use Committee of The John B. Pierce Laboratory and were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council. Washington, DC: Natl. Acad. Press, 1996). Male Golden hamsters (90–130 g, n 5 18; Charles River Breeding Laboratories) were anesthetized with pentobarbital sodium (60 mg/kg ip) and tracheotomized to ensure airway patency. A cannula secured in the left femoral vein enabled continuous replacement of fluids and maintenance of anesthesia throughout experiments (10 mg pentobarbital sodium/ml isotonic saline, infused at 0.41 ml/h). Esophageal temperature was maintained at 37–38° C with conductive heating. With the use of a stereomicroscope (model DR, Zeiss), the cheek pouch was exteriorized onto a Plexiglas board and superficial connective tissue was removed. The preparation was superfused continuously with a bicarbonate-buffered PSS (37°C; pH 7.4) of the following composition (in mM): 137.0 NaCl, 4.7 KCl, 1.2 MgSO4, 2.0 CaCl2, and 18.0 NaHCO3; salts were obtained from Sigma or J. T. Baker. Under control conditions the PSS was equilibrated in a 50-ml reservoir with 5% CO2-95% N2; this condition (referred to as ‘‘low’’ PO2) corresponded to a PO2 of ,20 Torr in the superfusate and ,20–25 Torr in the tissue (5, 15, 16). The preparation was placed on the stage of an intravital microscope (modified model 20T, Zeiss) and transilluminated with a 100-W halogen lamp (condenser NA 5 0.32, Zeiss) and observed through a long-working-distance objective (Leitz UM 32; NA 5 0.30). The image was coupled to a video camera (model NC-70X, Dage-MTI) with a total magnification of 3470 on the monitor screen (model PVM 122, Sony). Preparations were equilibrated for 60 min; all vessels studied showed brisk and reversible dilation (magnitude 20–30 µm) to sodium nitroprusside (10 µM; Sigma) in the superfusate. Typically, one or two cells were studied in a given vessel, with up to four cells per cheek pouch preparation; each cell was evaluated as a separate experiment. Membrane potential, intracellular dye injection, and diameter recordings. A reference electrode (Ag-AgCl pellet) was secured in the effluent superfusate. Recording microelectrodes were pulled (model P-87, Sutter Instruments) from borosilicate glass capillary tubes (GC120F-10, Warner Instruments) to produce long, flexible tips. The tip of a microelectrode (resistance ,300–400 MV) was backfilled with Lucifer

0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society

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OXYGEN RESPONSES OF ARTERIOLAR SMOOTH MUSCLE CELLS IN VIVO

yellow dye (lithium salt, 4% solution in deionized H2O; Sigma) and the remainder was filled with 150 mM LiCl2. The microelectrode was secured in a single-axis hydraulic micromanipulator (model MX510, SOMA) that was mounted on a three-way mechanical micromanipulator (model HS6, World Precision Instruments). Membrane potential was measured with an electrometer (Duo 773, World Precision Instruments). To record Em, a microelectrode was positioned above and parallel to the axis of a second- or third-order arteriole at a penetration angle of ,45°. While observed through the microscope, the tip of the microelectrode was lowered carefully onto the edge of the vessel and a cell was impaled by gradually advancing the hydraulic micromanipulator. The criteria for a successful penetration were 1) sharp, negative deflection of potential on entry, 2) stable recording of Em for at least 1 min, and 3) sharp, positive deflection on exit; tip potentials on exit averaged ,2 mV. Cell labeling often occurred with diffusion of Lucifer yellow dye from the microelectrode during the recording period (typically 5–20 min); in some experiments, dye was microinjected by passing negative current (5 nA for 1 min) through the recording microelectrode. The cell type recorded from was identified using epifluorescence (75-W Xenon lamp; Zeiss filter set 48 77 05), as viewed through a 340 immersion objective (NA 5 0.75, Zeiss) (24). Internal diameter (ID) of arterioles was measured from the monitor screen with the use of a video caliper (modified model 321; Colorado Video Instruments); spatial resolution was ,2 µm. Simultaneous outputs from the electrometer and video caliper were digitally recorded at 40 Hz (MacLab 8s, AD Instruments). For summary data (Table 1), respective values for Em and diameter were obtained by averaging data points for a 1,000-ms interval during a stable Em (,75% of experiments) or for 500-ms intervals coincident with the corresponding peaks of depolarization and vasoconstriction (remaining experiments). Microiontophoresis. Micropipettes (tip ID 1 µm) were fabricated by using the same capillary tubes and puller as for the recording microelectrodes; these were backfilled with phenylephrine hydrochloride (PE, 0.5 M; Sigma). The identity of SMC and EC was tested functionally by microiontophoresis of PE onto the abluminal surface of an arteriole. In the hamster cheek pouch, this selective a1-adrenoceptor agonist depolarizes arteriolar SMC but not EC (24). Experiment 1: Does O2 influence Em of cells in the arteriolar wall? Once a stable Em and vessel diameter were attained (,1 min), a PE micropipette was hydraulically positioned (model MO-102, Narishige) with its tip in close proximity to the

recording microelectrode. While Em and diameter were monitored, a PE stimulus (500-nA, 500-ms pulse) was delivered. After 3–4 min of recovery, the superfusate was equilibrated with gas containing 21% O2 (balance 5% CO2-74% N2, referred to as ‘‘high’’ PO2); this produced a superfusate PO2 of ,150 Torr and a tissue PO2 of ,60–65 Torr (5, 15, 16). In cases where high PO2 recordings were maintained for at least 5 min, the superfusate was then reequilibrated with low PO2. If PE and elevated PO2 elicited vasomotor responses without a corresponding change in Em (see RESULTS ), KCl was added to the 50-ml reservoir to produce an extracellular K1 concentration ([K1]o ) of ,55 mM in the superfusate; depolarization confirmed successful impalement and intracellular recording (24). Experiment 2: Are O2-induced responses inhibited by antagonists of L-type Ca21 channels? Superfusate PO2 was increased from low to high to ascertain arteriolar constriction in response to elevated PO2. The cheek pouch was then reequilibrated (,20 min) under low PO2 with diltiazem (10 µM; Sigma) or nifedipine (1 µM; Sigma) in the superfusate. A cell in the arteriolar wall was impaled and a stable recording confirmed (as in experiment 1). Superfusate was raised to high PO2 for 5–10 min and then returned to low PO2 while Em and vessel diameter were recorded. In light of the effect of L-type Ca21-channel antagonists on the response to high PO2 (see RESULTS ), [K1]o was elevated (as in experiment 1) to ascertain the success of recording. Data presentation and analysis. Representative tracings were selected to illustrate the characteristic responses of arteriolar SMC and EC to experimental maneuvers. Typically, each trace represents observations from 4 to 11 experiments. Because Em was monitored from a single cell in each experiment, EC and SMC responses in each of Figs. 2–4 are from two separate experiments and are presented together for illustrative purposes. Summary data from all experiments are presented in Table 1. To evaluate the effect of O2 on measured variables, the response to elevation of PO2 was calculated as the difference between values recorded during low and high PO2. One-way analysis of variance was used to compare responses during control conditions with responses during treatment with diltiazem or nifedipine; Tukey’s comparison was used for post hoc analysis. A Kruskal-Wallis one-way analysis of variance on ranks was performed on all data that failed a test for normality; in these cases, Dunn’s method was used for post hoc analysis. Comparisons were considered statistically significant with P # 0.05.

Table 1. Membrane potential and diameter responses to changes in superfusate PO2 Endothelial Cells

Control Diltiazem Nifedipine

Smooth Muscle Cells

PO 2

Em , mV

Diameter, µm

Em , mV

Diameter, µm

Low High Response Low High Response Low High Response

235.7 6 1.0 (17) 236.4 6 0.9 0.7 6 0.4 235.8 6 1.5 (9) 235.7 6 1.8 20.1 6 0.6 235.6 6 2.0 (4) 235.9 6 1.9 0.3 6 0.2

52.3 6 2.5 21.9 6 1.9 30.3 6 5.2 52.3 6 3.4 49.6 6 3.2 2.6 6 1.0* 62.3 6 1.8 61.5 6 1.6 0.5 6 0.3*

237.1 6 2.8 (10) 215.2 6 0.6 221.9 6 2.6 235.3 6 1.3 (11) 231.4 6 1.0 23.9 6 1.0* 232.3 6 0.8 (7) 230.2 6 1.1 22.1 6 0.8*

58.1 6 4.6 26.3 6 5.9 31.8 6 3.0 57.9 6 3.5 55.1 6 3.7 2.9 6 0.5* 57.5 6 1.7 56.6 6 2.0 0.9 6 0.6*

Values are means 6 SE for number of observations (n) for each experiment, shown in parentheses. Membrane potential (Em ) and arteriolar diameter responses were monitored in separate experiments under control conditions (low PO2 ) in presence of diltiazem (10 µM) or nifedipine (1 µM) and while superfusate PO2 was increased from ,20 Torr (low) to ,150 Torr (high). ‘‘Response’’ was calculated as difference in values between low and high PO2 . See METHODS for details. * Significant difference from control response, P # 0.05.

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RESULTS

Cell identification. Cell type was identified anatomically by examining the pattern of dye labeling after intracellular recording (Fig. 1). An SMC appeared as a narrow band wrapped circumferentially around the arteriole, with dye localized to the injected cell. In contrast, EC typically appeared as a narrow band parallel to the vessel axis, with dye spreading from the injected EC into many EC along and around the vessel lumen. This difference in dye-coupling properties between cell types has been reported in vivo (21, 24) and in vitro (17). In nine experiments, cell identity was further confirmed by the nature of intracellular recording during PE microiontophoresis (24). As shown in Figs. 2 and 3, PE consistently depolarized SMC (n 5 4) yet had no effect on the Em of EC (n 5 5). Control experiments verified that simply passing current through a microiontophoresis pipette filled with isotonic saline did not produce responses. Control Em and diameter. A summary of Em and diameter values recorded throughout these experiments is given in Table 1. During low-PO2 superfusion, resting Em averaged approximately 236 mV and was not different between EC and SMC. Experiment 1: Influence of O2 on cells in arteriolar wall. The elevation of superfusate PO2 elicited SMC depolarization and arteriolar constriction (Table 1). The mechanical responses to elevated PO2 were characterized as being essentially sustained (with slight oscillations, 20 of 27 arterioles; Fig. 2) or rhythmically cycling ‘‘vasomotion’’ (2–7 cycles/min, 7 arterioles; Fig. 3). Diameter changes in response to high PO2 were typically preceded (,2 s) by corresponding changes in the Em of SMC, with depolarization leading constriction and repolarization leading dilation. In marked contrast, high PO2 had no effect on the Em of EC. Nevertheless, EC promptly depolarized (with arteriolar constriction) by 21 6 2 mV (n 5 18) on elevation of [K1]o. Mechanical and electrical responses to high PO2 were consistently reversed on restoration of low PO2 (data not shown).

Experiment 2: Effects of L-type Ca21-channel antagonists on responses to O2. During low-PO2 superfusion, the addition of diltiazem or nifedipine to the superfusate produced a transient (2–3 min) dilation of arterioles, which then recovered. Thus antagonism of L-type Ca21 channels had no lasting effect on resting tone or Em (Table 1). This finding is consistent with the maintenance of tone in arterioles of the rat cremaster muscle during similar exposure to nifedipine (11). Taken together, these observations indicate pathways for Ca21 entry into arteriolar SMC other than through L-type Ca21 channels. Nevertheless, both diltiazem and nifedipine effectively prevented the depolarization and contraction of SMC in response to high PO2 (Fig. 4). In the presence of either diltiazem or nifedipine, raising [K1]o continued to produce sustained depolarization in SMC and EC. However, in contrast to the accompanying vasoconstriction seen under control conditions (Figs. 2 and 3), these depolarizations were associated with a slow and progressive vasodilation (Fig. 4) from a resting diameter of 57 6 2 to a peak of 79 6 2 µm (n 5 22; P , 0.05, paired t-test). In approximately one-half of the SMC recordings, elevated [K1]o initiated a slight, transient hyperpolarization before depolarization (Fig. 4) that occurred whether or not (data not shown) antagonists were present. Control experiments verified that arterioles regained sensitivity to O2 following the washout of diltiazem or nifedipine (data not shown). DISCUSSION

We present the first intracellular recordings from the wall of arterioles undergoing vasomotor responses to changes in ambient PO2. Our findings in the hamster cheek pouch demonstrate that SMC depolarized and arterioles constricted when superfusate PO2 was increased from ,20 Torr to ,150 Torr and tissue PO2 was increased by ,40 Torr (5, 16). This correspondence between depolarization and vasoconstriction with elevation of PO2 indicates that electromechanical coupling is the basis of the vasomotor response to O2 under physi-

Fig. 1. Identification of endothelial cells (EC) and smooth muscle cells (SMC) in hamster cheek pouch arterioles in vivo. Third-order arterioles were viewed using epifluorescent microscopy following Lucifer yellow dye microinjection (see METHODS and RESULTS for details). A: EC run parallel to vessel axis and show strong dye coupling. B: SMC wrap around arteriole; dye is constrained to each of two separately injected cells. Orientation and diameter (,40 µm) of arterioles is same in both A and B.


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Fig. 2. Membrane potential (Em ) and diameter responses in arterioles exposed to high PO2. SMC and EC were impaled during low PO2; responses to phenylephrine (PE), high PO2, or 55 mM extracellular K1 concentration ([K1]o ) were then monitored (see METHODS and RESULTS for details). In these recordings, high PO2 elicited sustained (with oscillation) vasoconstriction and depolarization in SMC; in contrast, EC had no electrical response to PE or to high PO2, yet prompt depolarization occurred on exposure to 55 mM [K1]o.

ological conditions. Moreover, both depolarization and contraction of SMC were prevented by antagonism of L-type Ca21 channels, indicating that the O2-induced influx of Ca21 is central to both electrical and mechanical events. Furthermore, EC remained electrically quiescent during changes in PO2, providing additional evidence (24) against electrical coupling between EC and SMC layers during blood flow control.

Specificity of cellular responses. Raising superfusate PO2 constricted arterioles and depolarized SMC, with recordings confirmed anatomically by dye labeling (Fig. 1) and functionally by responses to PE (Figs. 2 and 3). The elevation of superfusate PO2 elicited vasoconstriction that was either sustained with slight oscillation (74% of arterioles; Fig. 2) or cycled dramatically (26% of arterioles; Fig. 3) with smooth muscle Em. Whereas

Fig. 3. Rhythmic cycling of Em and arteriolar diameter in response to high PO2 (see METHODS and RESULTS for details). In SMC, note correspondence between cycling of Em and diameter; mechanical responses lag electrical responses by ,2 s. In contrast, Em of EC was unaffected by high PO2 in presence of vasomotion. EC promptly depolarized on exposure to 55 mM [K1]o.

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Fig. 4. Effect of nifedipine (1 µM) or diltiazem (10 µM) on Em in SMC and arteriolar diameter during exposure to high PO2 (see METHODS and RESULTS for details). Preparations were equilibrated with these L-type Ca21-channel antagonists during low PO2. At times indicated by arrows, high-PO2 superfusion was begun, and then (after 5 min) low PO2 was restored. Either diltiazem or nifedipine inhibited O2-induced vasoconstriction and depolarization. Note dilation with depolarization in presence of 55 mM [K1]o (compare with Figs. 2 and 3).

analogous vasomotor responses to elevated PO2 have been documented (5, 13, 15, 18, 20), this is the first study performed in vivo that demonstrates the correspondence between O2-induced changes in arteriolar diameter and the electrical activity of SMC in the arteriolar wall. The Em of EC [confirmed by dye labeling (Fig. 1) and by lack of response to PE] was unaffected by changes in superfusate PO2 (Figs. 2 and 3). This lack of response cannot be explained by the inability of EC to depolarize, because elevating [K1]o to 55 mM reduced the Em of EC by an average of 21 mV. Our finding that electrical responses to O2 were confined to SMC argues further that SMC are not electrically coupled to EC under physiological conditions (24). This conclusion is supported by recent in vitro studies of rat iridial arterioles (12) yet contrasts with the proposal of heterologous coupling between EC and SMC in isolated cheek pouch arterioles (17, 26). The changes in Em with constriction and dilation could reflect a piezoelectric effect associated with bending of the microelectrode tip during intracellular recording. If this were true, however, then O2-induced vasomotion should elicit similar changes in Em irrespective of the cell type from which they were recorded. As noted earlier, recordings from EC were consistently stable during pronounced changes in diameter (Figs. 2 and 3). Dissociation of mechanical and electrical events was also observed when [K1]o was raised; cells consistently depolarized, yet arterioles constricted under control conditions and dilated in the presence of diltiazem or nifedipine. Furthermore, changes in Em consistently preceded those in diameter by ,2 s (Ref. 24 and present

data), which is inconsistent with an electrical event arising from mechanical deformation. Lastly, control experiments in which the tip of a microelectrode was positioned in the tissue and moved to approximate displacement during vasomotion had no effect on the potential recorded. We conclude that our electrophysiological measurements are free from mechanical artifact. Role for L-type Ca21 channels in arteriolar smooth muscle responses to O2. In coronary (3, 23) and cerebral (8) arteries, SMC depolarize in response to elevated PO2. This depolarization, which activates L-type Ca21 channels and elicits vasoconstriction, has been attributed to reductions in K1-channel conductance (gK ) (3, 8, 23). In the present study, if alterations in gK were the basis of O2-induced depolarization of arteriolar SMC, then depolarization should still have occurred, even if contraction were prevented by the inhibition of Ca21 influx. Figure 4 shows that nifedipine abolished both vasomotor and electrical responses to elevated O2; similar results were obtained with diltiazem (Table 1). Such consistent findings with both antagonists support the conclusion that electrical and mechanical responses of arteriolar SMC to O2 reflect the primary involvement of L-type Ca21 channels, independent from changes in gK. The mechanism by which molecular O2 acts on L-type Ca21 channels to govern the contractile activity of arteriolar SMC remains unclear. It is possible that these channels are involved in the release of paracrine factors from parenchymal cells (5, 15), EC (18, 20), or red blood cells (5a) in response to changing PO2, with such factors eliciting electrical responses in SMC. Alterna-


tively, O2 may directly interact with L-type Ca21 channels in the SMC membrane and influence their gating characteristics, as recently reported for SMC isolated from arteries (6, 7). Nevertheless, the correspondence between vasomotor and electrophysiological events in arteriolar SMC (e.g., Fig. 3) indicates that the actions of PO2 were ultimately expressed via electromechanical coupling (22, 25). In turn, the phase lag between electrical and mechanical responses reflects the intracellular events that occur between membrane excitation and contraction coupling (22). The activation of L-type Ca21 channels during high PO2 could depolarize SMC by different mechanisms. First, the inward Ca21 current could produce depolarization. If current through L-type Ca21 channels was the basis of SMC depolarization, then the Ca21 equilibrium potential must be relatively high under the conditions of our experiments. However, given the small magnitude of this current under physiological conditions [1–2 pA; (10)], depolarization would only occur if the input resistance of SMC was sufficiently high or if global changes in PO2 activated a sufficient proportion of the available channels. Alternatively, increases in intracellular Ca21 could indirectly depolarize SMC by influencing the conductance of other ion channels. For example, physiological increases in intracellular Ca21 (e.g., from 100 to 500 nM) may limit the activity of voltageactivated K1 channels (9) as well as augmenting the conductance of Ca21-activated Cl2 channels (19). Such events would lead to SMC depolarization, particularly if the rise in intracellular Ca21 did not activate KCa. This latter condition appears likely because the Ca21 threshold for activating KCa isolated from arteriolar SMC (, 3 µM) is above physiological levels (14). It is clear that resolving the specific actions of O2 on the membrane properties of arteriolar SMC requires additional study. The elevation of [K1]o consistently depolarized SMC and EC. However, in contrast to the vasoconstriction observed under control conditions (Figs. 2 and 3), depolarization in the presence of L-type Ca21-channel antagonists was accompanied by slow and progressive vasodilation. Although the nature of this response is beyond the focus of this study, we offer the following explanation. The discontinuity between electrical and mechanical responses during high [K1]o may have been induced by the release of vasodilator peptides from depolarized sensory nerves (which are insensitive to diltiazem or nifedipine) that course through the cheek pouch (J. L. Morris, D. J. Grasby, and S. S. Segal, unpublished observations). Alternatively, the depolarization of EC by elevated [K1]o may have increased intracellular Ca21 (1, 2), thereby stimulating nitric oxide production and SMC relaxation (4). In summary, we have recorded Em from defined cells in the wall of arterioles controlling blood flow to the hamster cheek pouch in response to physiological changes in ambient PO2. Elevating superfusate PO2 produced SMC depolarization and vasoconstriction; when vasomotion was induced, arteriolar diameter cycled with the Em of SMC. Remarkably, the Em of EC

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remained stable during O2-induced constriction and vasomotion, indicating that respective cell layers are electrically isolated from each other in vivo. Antagonism of L-type Ca21 channels with diltiazem or nifedipine prevented SMC depolarization, contraction, and vasomotion in response to elevated PO2. Thus O2 (or a PO2-linked substance) may regulate the flux of Ca21 into SMC through L-type Ca21 channels. The correspondence between O2-induced changes in smooth muscle Em and the diameter of arterioles in vivo indicates that electromechanical coupling is integral to the physiological control of tissue blood flow. This study was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-41026 and RO1-HL-56786 and was completed during the tenure of an Established Investigatorship Award (to S. S. Segal) from the American Heart Association and Genentech, Inc. W. F. Jackson was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-32469. Present address of W. F. Jackson: Dept. of Biological Sciences, Western Michigan Univ., Kalamazoo, MI 49008 (E-mail: [email protected]). Address for reprint requests: S. S. Segal, John B. Pierce Laboratory, Yale Univ. School of Medicine, 290 Congress Ave., New Haven, CT 06519 (E-mail: [email protected]). Received 22 December 1997; accepted in final form 13 February 1998. REFERENCES 1. Bkaily, G., P. D’Orleans-Juste, R. Naik, J. Perodin, J. Stankova, E.Abdulnour, and M. Rola-Pleszczynski. PAF activation of a voltage-gated R-type Ca21 channel in human and canine aortic endothelial cells. Br. J. Pharmacol. 110: 519–520, 1993. 2. Bossu, J. L., A. Elhamdani, and A. Feltz. Voltage-dependent calcium entry in confluent bovine capillary endothelial cells. FEBS Lett. 299: 239–242, 1992. 3. Daut, J., W. Maier-Rudolph, N. von Beckerath, G. Mehrke, K. Gunther, and L. Goedel-Meinen. Hypoxic dilation of coronary arteries is mediated by ATP-sensitive potassium channels. Science 247: 1341–1344, 1990. 4. Dora, K. A., M. P. Doyle, and B. R. Duling. Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles. Proc. Natl. Acad. Sci. USA 94: 6529–6534, 1997. 5. Duling, B. R. Oxygen sensitivity of vascular smooth muscle. II. In vivo studies. Am. J. Physiol. 227: 42–49, 1974. 5a.Ellsworth, M. L., T. Forrester, C. G. Ellis, and H. H. Dietrich. The erythrocytes as a regulator of vascular tone. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H2155–H2161, 1995. 6. Franco-Obregon, A., and J. Lopez-Barneo. Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries. J. Physiol. (Lond.) 491: 511–518, 1996. 7. Franco-Obregon, A., J. Urena, and J. Lopez-Barneo. Oxygensensitive calcium channels in vascular smooth muscle and their possible role in hypoxic relaxation. Proc. Natl. Acad. Sci. USA 92: 4715–4719, 1995. 8. Gebremedhin, D., P. Bonnet, A. S. Greene, S. K. England, N. J. Rusch, J. H. Lombard, and D. R. Harder. Hypoxia increases the activity of Ca21-sensitive K1 channels in cat cerebral arterial muscle cell membranes. Pflu¨gers Arch. 428: 621–630, 1994. 9. Gelband, C. H., T. Ishikawa, J. M. Post, K. D. Keef, and J. R. Hume. Intracellular divalent cations block smooth muscle K1 channels. Circ. Res. 73: 24–34, 1993. 10. Gollasch, M., J. Hescheler, J. M. Quayle, J. B. Patlak, and M. T. Nelson. Single calcium channel currents of arterial smooth muscle at physiological calcium concentrations. Am. J. Physiol. 263 (Cell Physiol. 32): C948–C952, 1992. 11. Hill, M. A., and G. A. Meininger. Calcium entry and myogenic phenomena in skeletal muscle arterioles. Am. J. Physiol. 267 (Heart Circ. Physiol. 36): H1085–H1092, 1994.

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