INTRODUCTION

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ARTERIOLES ARE PRECAPILLARY microvessels that control local blood flow into capillary beds and exert a major influence on blood pressure (28). It is of interest, therefore, to understand the cellular and molecular mechanisms that determine the contractile state of arteriolar smooth muscle cells, which are the direct regulators of arteriolar diameter. One important cellular parameter is the smooth muscle cell membrane potential, hyperpolarization, for example, switching off Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (20, 18). This type of effect is thought to be dominant in resistance arteries and arterioles of the cerebral circulation where vessels have a marked sensitivity to dihydropyridine Ca²⁺ antagonists (6). Although there is compelling evidence that outward currents through large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and delayed rectifier (K_V) channels oppose depolarizing excitation currents (23, 13, 32), there is less clarity about mechanisms governing the resting potential when excitation is lacking. Outward current through inward rectifier K⁺ (K_IR) channels may perform this function in submucosal arterioles from the ileum because Ba²⁺, which blocks K_IR channels, causes depolarization (17). However, not all blood vessels express K_IR channels (33, 31), and in coronary arteries expressing K_IR channels, Ba²⁺ did not evoke depolarization when the extracellular K⁺ concentration was normal (24). A voltage-independent background K⁺ current has been identified in renal arcuate artery, and this may maintain the resting potential in this vessel (32). However, we have observed that cerebral precapillary arterioles have a particularly high membrane resistance near the resting potential and thus have hypothesized that Na⁺ pump electrogenicity may have an important role.

ATP-driven pumping of Na⁺ out and K⁺ into the cell creates the Na⁺ gradient necessary for depolarizing Na⁺ current and forward Na⁺-Ca²⁺ exchange, and the K⁺ gradient combines with K⁺ selectivity of the plasma membrane to generate a K⁺-dependent negative membrane potential. The Na⁺ pump is also intrinsically electrogenic, giving net efflux of positive charge. The influence of the resulting current on membrane potential depends on its amplitude relative to that of other currents, usually through ion channels. Na⁺ pump electrogenicity has a dominant role in cell types, including rod photoreceptors (37), peritoneal mast cells (11), and thalamic neurons (36), and is suspected to be important in visceral and conduit arterial smooth muscle tissues (10). Furthermore, Na⁺ pump functions are of specific interest in the vasculature: endogenous ouabain-like substances are implicated in the generation of hypertension (1), there is potential for the development of Na⁺ pump modulators as novel antihypertensive drugs (14), ouabain prevents loss of autoregulation caused by reoxygenation in pial arterioles (21), and the Na⁺ pump mediates part of the response to endothelium-derived hyperpolarizing factor (9). Also, a truncated isoform of vascular Na⁺ pump has been cloned and appears to be selectively expressed in some arteries (26). This suggests Na⁺ pumps may have specialized functions in some blood vessels.

Quinn and Beech (34) have established methodology for patch-clamp recording from short fragments of arteriole freshly isolated from rabbit pial membrane. The recordings are stable even during arteriolar constriction, and the membrane/patch pipette seal and membrane resistances are high. In this study we have combined the use of this electrophysiological method with diameter measurements from arterioles within intact pial membrane. The aim of the study was to determine whether Na⁺/K⁺ pump current could be detected and, because it could, to elucidate the consequences of this current for the control of membrane potential in arterioles under resting conditions.

	METHODS

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In accordance with the Code of Practice as set out by The United Kingdom Animals Scientific Procedures Act of 1986, male Dutch dwarf rabbits were killed before any other procedure by an intravenous overdose of heparinized pentobarbital sodium (70 mg/kg). The brains were removed and placed immediately in ice-cold Hanks' solution. Arterioles were prepared from pial membrane that had been removed from the surface of the cerebral cortex with the use of fine forceps. Pieces of the membrane were incubated with 0.032 mg/ml protease and 0.2 mg/ml collagenase type 1-A at 37°C for 10 min. The tissue was mechanically agitated and washed with an excess of Hanks' solution and centrifuged at 1,000 rpm for 1 min to concentrate the arteriolar fragments. Isolated arterioles were studied on the same day as the isolation procedure and stored at 4°C to minimize cell damage and de novo protein expression. Arterioles were reequilibrated at room temperature (25 °C) before experiments.

Recordings were made by application of the patch-clamp technique to a smooth muscle cell within intact electrically coupled arteriolar segments (34). Membrane potential (zero current mode) experiments were made by perforation of the cell-attached patch with 120 µg/ml amphotericin or by mechanical rupture of the patch (conventional whole-cell configuration). Voltage-clamp recordings were made by conventional whole cell methodology. The patch-clamp amplifier was either an Axopatch 1D or Axopatch 200A (Axon Instruments). In current clamp, data were filtered at 0.5 kHz (4-pole Bessel filter) and sampled digitally at 1 kHz for 1 s every 3 s by an analog-to-digital (A/D) converter (CED1401Plus, Cambridge Electronic Design) before being stored on a computer hard disk. Membrane potentials were plotted as joined data points sampled every 3 s. In voltage clamp, data were also filtered at 0.5 kHz and sampled at 1 kHz during the time period of the voltage-ramp command. Patch pipettes were pulled from borosilicate glass (Clark Electromedical Instruments) and fire polished before use. Command paradigms and data analysis were controlled by CED Patch and VClamp 6 software (CED). Curve fitting and data presentation were carried out with the use of Origin 4.1 software (MicroCal). Averaged data are given as means ± SE, where n is equal to the number of arterioles studied.

Voltage clamp was applied to arteriolar fragments that were always <300 µm and most often 100-150 µm in length. Thus the arterioles were finite cables with a length ~1/10 that of the arteriolar length constant (39). Endothelial cells are presumed to have had little influence on the observations, because immunological staining with endothelial nitric oxide synthase-specific antibody has revealed that these cells are most often extracted from short arteriolar fragments (data not shown).

Arteriolar constriction was measured by placement of pieces of pial membrane sheet in a modified culture dish on the stage of an inverted trinocular microscope (Nikon TMS) with an attached video camera (Sony). All recordings were made after equilibration at 37°C. External diameter was measured with the use of a video dimension analyzer (Living Systems Instrumentation) with off-line capture of signals via an A/D converter to a computer hard disk (Picolog software; Picolog Technology, Cambridge, UK).

Hanks' solution contained (in mM) NaCl 137, KCl 5.4, NaH₂PO₄ 0.34, K₂HPO₄ 0.44, D-glucose 8, HEPES 5, and CaCl₂ 0.01 (pH 7.4). Artificial cerebrospinal fluid (CSF) contained (in mM) NaCl 125, KCl 1.72, NaHCO₃ 24, MgSO₄ 1.74, KH₂PO₄ 1.17, D-glucose 5.35, CaCl₂ 2.47, and EDTA 0.023. The CSF was gassed continuously with a mixture of 5% CO₂ and 95% O₂. The standard 5 mM K⁺ bath solution contained (in mM) KCl 5, NaCl 130, glucose 8, HEPES 10, MgCl₂ 1.2, and CaCl₂ 1.5 (pH 7.4). When the bath K⁺ concentration was raised, the Na⁺ concentration was reduced by the same amount. For amphotericin-perforated patch-membrane potential recordings, patch pipettes had resistances of 5-20 M and contained (in mM) KCl 130, NaCl 5, glucose 8, HEPES 10, MgCl₂ 1.2, and CaCl₂ 1.5 (pH 7.4). Amphotericin B was prepared as a 60-mg/ml stock in 100% DMSO. For conventional "whole-cell" membrane potential, voltage-clamp recordings, and single-channel recordings, patch pipettes had resistances of 2-3 M and contained (in mM) KCl 130, HEPES 10, MgCl₂ 1.2, and EGTA 0.2 (pH 7.4). The recording chamber had a volume of ~150 µl. Solutions flowed into the chamber from one of six reservoirs that were above the recording chamber, and solution was removed from the chamber via a small tube attached to a vacuum pump. Complete solution exchange occurred in <1 min (see Fig. 4B, inset).

NaCl, KCl, D-glucose, and CaCl₂ were from BDH (British Drug House). NaH₂PO₄ was from Rectapur, and K₂HPO₄ was from May and Baker. HEPES, EGTA, EDTA, DMSO, tetraethylammonium (TEA⁺), penitrem A, glibenclamide, apamin, DIDS, niflumic acid, sodium aspartate, ouabain, and dihydroouabain (DHO) were from Sigma (Poole, UK). BaCl₂ (Ba²⁺) and tetramethylammonium (TMA⁺) were from Aldrich, and 3,4-diaminopyridine (3,4-DAP) was from Fluka. Levcromakalim was from SmithKline Beecham.

	RESULTS

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Isolated vessels with a characteristically thick single layer of smooth muscle cells were classed as arterioles (16, 34). They were consistently small, with external diameters ranging from 24 to 58 µm (n = 195). Isolated arterioles in Ca²⁺-containing solution had a mean ± SE diameter of 35.3 ± 0.8 µm (n = 49), which was similar to that of arterioles within intact pial membrane (36.8 ± 0.81 µm; n = 65). The resting membrane potential of isolated arterioles was 68.9 ± 1.8 mV (n = 119), close to that reported for other arteriolar preparations (8, 15, 19, 38).

Na⁺ pump activity was inhibited by 10 µM DHO, which has been reported to be just sufficient to inhibit Na⁺ pump current in ventricular myocytes (5). Experiments were performed in the presence of 0.1 mM Ba²⁺ to reduce contamination from inward rectifier K⁺ current. DHO reversibly induced a downward deflection in the current trace over a wide range of potentials (Fig. 1, A and B). The sensitive current was always outward but decreased in amplitude at negative membrane potentials, approaching an extrapolated zero current potential of about 160 mV (Fig. 1C). The slope resistance between 60 mV and 150 mV was 1.2 G in the presence of 10 µM DHO and 0.1 mM Ba²⁺ (Fig. 1D).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Na+/K+ pump current in arteriolar segments. The 5 mM K+ bath solution was used with 0.1 mM Ba2+ added to block inward rectifier K+ current. A: recording of current from an isolated arteriolar segment in voltage-clamp mode under control conditions and in presence of 10 µM dihydroouabain (DHO). Ramp protocol was a change in voltage from 50 to 195 mV applied over a 1-s period every 10 s, and holding potential was 50 mV. B: as in A, except a time (t)-series plot for current amplitude at 80 mV is shown. DHO (10 µM) was bath applied. C: mean (3 arterioles) DHO-sensitive current during a ramp from 50 to 150 mV. D: mean (3 arterioles) residual current remaining in presence of 10 µM DHO and 0.1 mM Ba2+. Fitted straight (dashed) line has a slope of resistance of 1.2 G.

Presence of significant Na⁺ pump electrogenicity was also investigated by measuring membrane potential and then removing extracellular K⁺ or applying 10 µM ouabain, procedures which inhibit the Na⁺ pump in rabbit vascular and cardiac muscles (31, 35). Both procedures routinely induced depolarization: K⁺-removal depolarizing arterioles by 37.0 ± 3.6 to 33.0 ± 2.6 mV (n = 20; Fig. 2A) and ouabain-depolarizing arterioles by 38.6 ± 7.7 to 39.7 ± 5.9 mV (n = 7; Fig. 2B). The effect of K⁺-free solution was rapid, occurring at about the same speed as changes in liquid-to-liquid junction at the ground agar bridge (see Fig. 4B, inset), and was readily reversible on returning 5 mM K⁺ to the bath solution (Fig. 2A).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Depolarization in response to inhibition of Na+/K+ pump. A: membrane potential recorded from an isolated arteriolar segment. K+ was omitted from standard 5 mM K+ bath solution as indicated. B: for same arteriole as in A, 10 µM ouabain was bath applied in standard 5 mM K+ bath solution.

An alternative and noninvasive method to measure the effect of the Na⁺ pump was to monitor the reversal potential of BK_Ca-channel unitary currents through cell-attached patches. These experiments also indicated whether there was significant K⁺ depletion during short-term pump inhibition. BK_Ca-channel currents were observed with a fixed concentration of 130 mM K⁺ in the patch pipette, and the patch-pipette voltage required to reverse the polarity of the unitary currents was measured. Iberiotoxin (100 nM), TEA⁺ (4 mM), or penitrem A (100 nM) abolished the currents, confirming they were carried by BK_Ca channels (data not shown). With 5 mM K⁺ in the bath solution, the reversal potential was 84.1 ± 1.7 mV (n = 11; Fig. 3A), and when K⁺ was removed from the bath solution, the reversal potential changed to 51.9 ± 1.4 mV (n = 9; Fig. 3B). The unitary conductance of the BK_Ca channels did not change (Fig. 3C). The effect of K⁺ removal on the reversal potential for BK_Ca channels occurred rapidly and was sustained (Fig. 3D). The K⁺-channel reversal potential (E_revK) remained at a hyperpolarized potential during short-term exposure to K⁺-free solution because the K⁺-channel-opener drug levcromakalim elicited hyperpolarization once depolarization had been induced by K⁺-free solution (Fig. 3E).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Electrogenicity of Na+/K+ pump. A and B: cell-attached patch recordings of single-channel currents through Ca2+-activated K+ (BKCa) channels with 130 mM K+ in patch pipette and 5 mM K+ (A) or 0 mM K+ (B) in bath solution. The x-axes show absolute command voltage applied to patch pipette (i.e., negative potential on outside of cell-attached patch). Linear leakage currents have been subtracted. Arrows mark pipette voltages at point of current reversal. C: data from A (thick line) and B (thin line) superimposed and aligned on x-axis such that 0 mV is reversal potential. Only current positive of reversal potential is shown. D: rapid and stable change in pipette voltage for reversal of BKCa channel current on removal of K+ from bath solution. E: membrane potential recorded from an isolated arteriolar segment. Standard 5 mM K+ bath solution was used initially, and then K+ was removed and 10 µM levcromakalim was added.

The marked effect of Na⁺ pump current on membrane potential suggested that ion-channel currents were small or nonexistent near the resting membrane potential. Therefore, we tested whether ion channels were indeed closed at these voltages. High concentrations of the Cl-channel blocker DIDS (1 mM) or 1 mM DIDS in combination with 1 mM niflumic acid (25) did not induce depolarization (n = 3 and n = 6, respectively; Fig. 4). It appeared that replacement of 130 mM Cl by the large, less-permeant anion aspartate evoked a small and reversible hyperpolarization (12.0 ± 1.0 mV, n = 6; Fig. 4B). However, this effect was explained mostly by a liquid-to-liquid junction potential arising at the ground agar bridge (8.6 ± 0.6 mV, n = 6; Fig. 4B, inset). K⁺ channels were investigated by bath application of a combination of six K⁺-channel inhibitors: TEA⁺ (4 mM) and penitrem A (100 nM) to inhibit BK_Ca channels; 1 µM glibenclamide to inhibit ATP-sensitive K⁺ channels (K_ATP); 1 mM 3,4-DAP to inhibit K_V channels; 100 nM apamin to inhibit small-conductance Ca²⁺-activated K⁺ channels; and 100 µM Ba²⁺ to inhibit K_IR channels such as K_IR2.1. In five out of six arterioles, the resting membrane potential was unaffected by the K⁺-channel blockers (Fig. 5A). The insensitivity to K⁺-channel blockade was not because the arterioles lacked K⁺ channels or because the K⁺-channel blockers were ineffective in our experimental conditions. Voltage-clamp recording enabled analysis of current over a broad range of potentials. Ba²⁺ inhibited current at 120 mV, and the addition of the other five K⁺-channel blockers strongly inhibited outward current at 0 mV, revealing inward cadmium-sensitive current (Fig. 5B). Outward current at 50 mV was unaffected by any of the blockers.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Lack of Cl channel current. A and B: resting membrane potential recorded from 2 separate isolated arteriolar segments. A: standard 5 mM K+ bath solution was used, and DIDS (1 mM) was applied in bath as indicated. B: standard 5 mM K+ bath solution was used, except as indicated by aspartate, when 130 mM NaCl was replaced by 130 mM sodium aspartate. DIDS (1 mM) and niflumic acid (1 mM) were applied to bath together after washout of aspartate. Inset: a liquid-to-liquid junction potential recording (cell-free) showing effect of changing from 130 mM NaCl to 130 mM sodium aspartate bath solution. Reference electrode contained 3 M KCl, and recording electrode contained standard bath solution.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Lack of K+-channel current near resting membrane potential. A: resting membrane potential recorded from an isolated arteriolar segment. Standard 5 mM K+ bath solution was used. Statement "6 K+ channels blockers" means addition of a mixture of 100 nM penitrem A, 4 mM tetraethylammonium (TEA+), 100 nM apamin, 1 µM glibenclamide, 1 mM 3,4-diaminopyridine (3,4-DAP), and 0.1 mM Ba2+. B: recording of current from an isolated arteriolar segment by use of conventional whole cell voltage clamp. A ramp change in voltage from 150 to 0 mV was applied over a 1-s period every 10 s, and holding potential was 50 mV. In time-series plot, absolute current amplitude is shown for 0 mV (), 50 mV (), and 120 mV (). To standard 5 mM K+ bath solution were added 0.1 mM Ba2+, 0.1 mM Ba2+ plus other 5 K+ channel blockers (see A), and then 1 mM Cd2+.

Two observations were inconsistent with the notion that Na⁺ pump electrogenicity contributed to membrane potential. First, the resting membrane potential averaged 69 mV and thus was not driven close to the zero current potential for pump current (160 mV; Fig. 1C). Second, raised extracellular K⁺ levels depolarized the membrane potential as if the arterioles were almost pure K⁺-selective electrodes (Fig. 5A and Fig. 6A). These inconsistencies could be explained if there was a K⁺ current with a strong depolarizing, but weak hyperpolarizing, influence on the membrane potential. Because a strongly inwardly rectifying K⁺ current might serve such a function, we tested the effect of 10-100 µM Ba²⁺, which blocks inward rectifier K⁺ current (3) and attempted to detect the inward rectifier in voltage-clamp experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effect of blocking inward rectifier K+ channels. A: membrane potential sampled at 0.34 Hz from a single arteriole in standard 5 mM K+ bath solution, showing depolarizations in response to elevated levels of extracellular K+. Gap between traces is an interval of 11 min and 40 s. B: membrane potential of an isolated arteriole that hyperpolarized in response to 100 µM Ba2+ and was then exposed to increasing K+ concentrations from 5 to 10, 20, 40, and 60 mM while in continued presence of Ba2+. C: mean ± SE (n = 3-15) changes in membrane potential in response to elevated extracellular K+ concentrations ([K+]0) in absence () and presence () of 0.1 mM Ba2+. D: currents during 1-s ramp changes in voltage from a holding potential of 50 to 150 mV and in 5 mM K+ bath solution without (control) and with 0.1 mM Ba2+. Capacity and leakage currents have not been subtracted.

Ba²⁺ (100 µM) evoked hyperpolarization in 15 arterioles (15.5 ± 2.4 mV from an initial resting potential of 65.1 ± 2.1 mV; Fig. 6B), had no clear effect in three arterioles, and depolarized five arterioles (10.2 ± 2.0 mV from an initial resting potential of 67.0 ± 3.9 mV). The effect of 10 µM Ba²⁺ was similar, with hyperpolarization occurring in six arterioles (10.5 ± 3.8 mV from an initial resting potential of 67.7 ± 1.4 mV) and depolarization in two arterioles (8 and 20 mV). In some arterioles, the membrane potential reached values considerably negative of 80 mV in the presence of Ba²⁺ (Fig. 6B). Ba²⁺ (100 µM) also inhibited the depolarizing effect of raised extracellular K⁺ levels, and 10 or 20 mM K⁺ elicited hyperpolarization (Fig. 6C). Although 60 mM K⁺ still induced depolarization in the presence of Ba²⁺, the time before depolarization occurred was greater at 91.7 ± 19.2 s (n = 10) compared with 52.5 ± 8.4 s (n = 13; Fig. 6, compare A and B). Membrane resistance measured from electrotonic potentials elicited by 10-pA current steps increased concomitantly with Ba²⁺-induced hyperpolarization (0.6 ± 0.1 to 1.1 ± 0.2 G; n = 11), indicating that Ba²⁺ was inhibiting ionic current (data not shown).

The only ionic current we found to be sensitive to 100 µM Ba²⁺ was a strongly inwardly rectifying K⁺ current. This current occurred in 27 out of 29 recordings. The reversal potential of the Ba²⁺-sensitive current could not be determined in 22 of the experiments, because Ba²⁺-sensitive outward current was undetectable (i.e., less than a few picoamperes; see the current recordings at 50 mV in Fig. 5B). However, the voltage at which inward current was no longer detectable was estimated to vary between 70 and 85 mV. An experiment in which there was clear reversal of the Ba²⁺-sensitive current is shown in Fig. 6D. The current reversed at 88 mV, and the Ba²⁺-sensitive current at 60 mV was 9 pA, or 5% of the current amplitude at 116 mV. (Note that in these recordings, unsubtracted capacity current during ramp changes in voltage displaced the zero current potential from the resting membrane potential.)

To investigate whether the effects described above might be physiologically significant, we measured arteriolar diameter in pial membrane sheets equilibrated in artificial CSF at 37 °C. K⁺ removal (9 out of 13 arterioles) or application of 10 µM ouabain or 10 µM DHO (7 out of 14 arterioles) evoked arteriolar constriction (Fig. 7, A and B, respectively). Spontaneous and evoked oscillatory constrictions are evident in these recordings. In contrast, arterioles were unresponsive to the broad-spectrum K⁺-channel blockers (8 mM TEA⁺ and 8 mM TMA⁺; Fig. 7C). Furthermore, in five out of seven arterioles, there was no effect of a cocktail mixture of specific K⁺ blockers, which included 100 µM Ba²⁺, even after long periods of equilibration at 37°C (Fig. 7C). The two responsive arterioles constricted (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Arteriolar diameter measurements in intact pial membrane sheets. Each experiment is from a separate piece of pial membrane, at 37°C, bathed in artificial cerebrospinal fluid. A: constriction evoked by removal of K+ from bath solution. B: constriction evoked by adding 10 µM DHO to bath solution. C: lack of effect of TEA+ (8 mM), tetramethylammonium (TMA+, 8 mM), or a mixture of 100 nM penitrem A, 4 mM TEA+, 100 nM apamin, 1 µM glibenclamide, 1 mM 3,4-DAP, and 0.1 mM Ba2+. Constriction was evoked by 40 mM K+. D: lack of a clear constrictor effect of bath-applied 0.1 mM Ba2+ and lack of effect of 20 mM K+ in presence of Ba2+. After washout of Ba2+, 20 mM K+ evoked a constriction of ~5 µm.

Constriction evoked by K⁺ removal or ouabain could be explained by inhibition of Na⁺ extrusion rather than by depolarization resulting from loss of Na⁺ pump electrogenicity. Therefore, we further investigated the physiological role of Na⁺ pump electrogenicity by testing whether Ba²⁺ inhibited K⁺-induced constriction, as it had inhibited K⁺-induced depolarization (Fig. 6). In this series of experiments, 100 µM Ba²⁺ had no constrictor effect in 13 out of 14 arterioles, which did, nevertheless, constrict in response to 20 or 40 mM K⁺ after washout of Ba²⁺. Although Ba²⁺ had no effect on its own, it abolished constriction induced by 20 mM K⁺ in five out of five arterioles (Fig. 7D) or 40 mM K⁺ in five out of nine arterioles (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Na⁺/K⁺ pump current has been detected in short fragments of cerebral precapillary arteriole, and membrane potential was shown to be vulnerable to the hyperpolarizing effect of pump current. This vulnerability is suggested to arise because of high membrane resistance near the resting membrane potential. The data further suggest that excess hyperpolarization resulting from pump electrogenicity is prevented by inward (depolarizing) current through K_IR channels. Diameter measurements from arterioles in intact pial membrane sheets indicate that these electrical effects may occur under physiological conditions.

The pharmacology and voltage dependence of the pump current of arterioles resembled that previously described in cardiac myocytes (12, 35) and in a recent study of smooth muscle cells from guinea pig mesenteric artery (30). We suggest that activity of the pump affected the membrane potential because of its electrogenicity and not because of its other function to maintain Na⁺ and K⁺ gradients. First, the background membrane resistance is theoretically high enough for the small pump current to generate a significant potential difference (~30 mV). Second, there was a large depolarization in response to K⁺-free solution that occurred rapidly and reversibly, effects that are unlikely to be explained by intracellular K⁺-depletion. Third, membrane potential was commonly resistant to K⁺-channel blockade, and it retained a negative value even when K⁺ dependence of the membrane potential had been inhibited by 100 µM Ba²⁺, sometimes exceeding reasonable predictions for the K⁺ equilibrium potential (E_K). Even if inhibition of the pump led to a positive shift of E_K, it would be of no consequence because the membrane potential was not strongly influenced by K⁺-channel activity. Furthermore, depletion of intracellular K⁺ to 31 mM would need to occur if the E_revK was to depolarize even to 80 mV in the absence of extracellular K⁺ (assuming Na⁺ permeability/K⁺ permeability = 0.01). K⁺ depletion is also unlikely because there was no change in the conductance of BK_Ca channels in cell-attached patches when the pump was inhibited (Fig. 3C).

If the effect of pump electrogenicity is unimpeded, it will result in extreme hyperpolarization, perhaps in excess of 100 mV. This is presumably unwanted, and our data suggest that arterioles have a built-in mechanism to prevent it. This mechanism involves strongly rectifying K_IR channels. Figure 6D shows a recording of the K⁺ current through K_IR channels in which E_revK was clearly at 88 mV. E_K is negative with regard to this value, and thus intracellular K⁺ must have been high in the experiment. In this experiment, the outward current through the channels was smaller than the pump current, and in the majority of experiments, the outward current was undetectable. The inward current at voltages negative to E_revK was much larger. Thus we suggest that hyperpolarization negative of E_revK is inhibited by inward (depolarizing) K⁺ current through K_IR channels. Blockade of the K⁺ channels with Ba²⁺ will remove this natural safety net and release the membrane, allowing it to go to extreme potentials (Fig. 6B). The safety net is not always needed (e.g., if the membrane potential is positive of E_revK), and this explains why Ba²⁺ had no effect or evoked a small depolarization in other experiments (e.g., Fig. 5A). The idea of a safety net against pump electrogenicity is not new. Torre (37) described this concept for rod photoreceptors, except in photoreceptors it was the hyperpolarizing current that "saved" the membrane potential rather than the inward rectifier K⁺ current. The explanation for Ba²⁺ inhibition of K⁺-induced depolarization in arterioles is similar, except there is no requirement for the membrane potential to be driven negative of E_revK under control conditions. The raising of extracellular K⁺ to 20 mM will shift E_K to about 47 mV. We suggest that inward current through K_IR channels depolarizes the membrane potential from the resting potential (e.g., 69 mV) toward 47 mV. Blocking of the channels with Ba²⁺ prevents the depolarization and removes the K⁺ dependence of the membrane potential. We suspect that 60 mM K⁺ did evoke depolarization in the presence of Ba²⁺ (albeit, after a delay; Fig. 6B) because raised extracellular K⁺ levels inhibit Ba²⁺ block of K_IR channels.

Once K_IR channels are blocked, it is theoretically possible for Na⁺ pump electrogenicity to drive the membrane potential as negative as 160 mV if it is the only conductance in the membrane. The fact that this did not happen indicates there was a small background depolarizing current present in the arterioles. This current might arise through basally active Cl channels, because we observed a slight hyperpolarizing effect of Cl-channel blockade (Fig. 4B), receptor-operated cation channels (16), or Na⁺-Ca²⁺ exchange (Guibert and Beech, unpublished observations).

The contribution of Na⁺/K⁺ pump current to the resting potential of arteriolar fragments averaged about 35 mV. Although such a large contribution may seem surprising, previous publications on the contribution of pump current in smooth muscle suggest a range of effects from nothing to as much as 30-40 mV (2, 4, 10, 29). This variation may arise because of differences between types of smooth muscle or different conditions. One variation between types of muscle is the background membrane resistance, a factor that is influenced by the level of basal ion channel activity. It is probably significant that the resistance of cerebral arteriolar fragments was high at rest (Fig. 1) (16). A similar high-resistance domain has been described for coronary arteriolar fragments (22), whereas the resting membrane resistance of mesenteric arterioles is substantially lower (40). This difference is intriguing because, in mesenteric arterioles, there is clear evidence of outward (hyperpolarizing) current through K_IR channels (7), and a depolarizing and constricting effect of Ba²⁺ has been shown (17). Thus outward current through K_IR channels is dominant in mesenteric arterioles and sets the resting potential. Cerebral, and possibly coronary, arterioles appear to be different and more susceptible to the effects of Na⁺ pump electrogenicity.

Experimental protocols or different physiological conditions may modify the contribution of Na⁺ pump electrogenicity. For example, isolated arteriolar fragments were stored at 4°C to minimize damage immediately after isolation. Incomplete recovery of intracellular Na⁺ levels during the rewarming procedure may have enhanced pump activity. However, pump activity is also lower at 25°C in our electrophysiological measurements than at 37°C (30), and various physiological effects such as Na⁺ influx via a colocalized Na⁺-Ca²⁺ exchanger (12, 27) and agonist-activated nonselective cation channels (2, 16), and elevated extracellular K⁺ levels (Ref. 31 and the references therein), will stimulate the pump. Furthermore, high resistance at the resting potential of arterioles will automatically confer vulnerability to small currents such as those from the Na⁺ pump, an enzyme that is active in all but the most extreme conditions. And the data of Edwards et al. (8) and our data obtained under quasi-physiological conditions are further evidence for a physiological role of pump electrogenicity. Because Edwards et al. (8) had previously reported sharp microelectrode data from cerebral arterioles (see below), we chose to investigate the function of pump electrogenicity with the use of noninvasive diameter measurements from arterioles still within intact pial membrane sheets and equilibrated at 37°C. We reasoned that Ba²⁺ should prevent the constrictor effect of 20 mM K⁺ if Na⁺ pump electrogenicity could maintain the resting potential negative of the threshold for activation of L-type Ca²⁺ channels, independently of K⁺-channel current. Ba²⁺ did inhibit K⁺-evoked constriction (Fig. 7D). Furthermore, Ba²⁺ failed to elicit constriction despite long periods of equilibration at 37°C (Fig. 7, C and D), in contrast to the effect of Ba²⁺ on mesenteric arterioles (17).

Edwards et al. (8) did not propose a significant contribution from Na⁺ pump electrogenicity or an interrelationship with inward rectifier K⁺ current, but there are many similarities between our observations from rabbit cerebral precapillary arterioles and those of Edwards et al. (8) on rat proximal cerebral arterioles. We suggest that the previous interpretation of these data is inconsistent with the small amplitude of outward current detected through K_IR channels and the large amplitude of outward current inhibited on lowering the extracellular K⁺ concentration (8). Furthermore, it was observed (8) that lowering the extracellular K⁺ concentration to 3 mM (the concentration in CSF) shifted the activation curve for K_IR channels to the left, so that activation was not apparent until voltages were negative of 100 mV. Thus there can be no outward current through these K_IR channels under this condition.

Membrane potential is set by a balance between the relative amplitudes of various ionic currents. We do not conclude that K⁺ channels cannot hyperpolarize the membrane in cerebral precapillary arterioles. Indeed, K_ATP channels are present in these arterioles, the current can be larger than the Na⁺ pump current, and activation of the channels can induce arteriolar relaxation (34). Under other circumstances, elevation of intracellular Ca²⁺ levels or depolarization evoked by a stimulant (e.g., endothelin-1) may activate Ca²⁺- and voltage-dependent K⁺ channels and hyperpolarize the membrane. Na⁺ pump electrogenicity may have the most effect in arterioles receiving minimal stimulation from stretch or vasoconstrictor agonists or those exposed to elevated extracellular K⁺ levels or in which forward Na⁺-Ca²⁺ exchange is stimulated. We demonstrate for the first time that cerebral precapillary arterioles are vulnerable to Na⁺ pump electrogenicity and that inward (depolarizing) K⁺ current has a function to prevent excess Na⁺ pump-induced hyperpolarization.