INTRODUCTION

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ALTHOUGH VASCULAR SMOOTH MUSCLE (VSM) cells effectively buffer changes in intracellular pH (pH_i) under physiological conditions (1), intracellular acidosis may be an important signal linking metabolic demand to blood flow during periods of metabolic challenge. Levels of pH_i measured in smooth muscle cells range between 7.0 and 7.2 under resting conditions (35). In pathophysiological states, tissue extracellular pH (pH_o) may fall by 0.5-0.7 unit (2, 18), producing large corresponding declines in VSM pH_i (26). Interestingly, decreases in arterial pH are associated with circulation-specific, and sometimes opposing, vasoactive effects in vivo. For example, arterial acidosis stimulates vasodilation of the coronary microcirculation (16), augmenting coronary blood flow to regions of ischemic myocardium. In contrast, acidosis constricts the pulmonary vasculature (35), thereby diverting blood toward better-ventilated alveoli to improve ventilation-to-perfusion matching.

Given the complexity of pH effects on the vasculature, it is not surprising that the cellular mechanisms by which acidosis exerts its contrasting influence on arterial muscle tone in the coronary and pulmonary circulations are incompletely understood. For example, changes in pH may regulate the contractile state of VSM by altering the release and reuptake of intracellular Ca²⁺, the activity of Ca²⁺-permeable ion channels, or the Ca²⁺ sensitivity of contractile proteins (2). A pH-sensitive release of vasoactive factors from the endothelium may further modulate vascular tone (2, 36). However, the primary vasoactive response to acidosis may rely on the pH-sensing properties of the VSM cells (16, 36). In fact, in vivo studies indicate that the effects of acidosis on arterial muscle tone may occur independently of muscarinic, -adrenergic, or sympathetic nervous system innervation and may be mediated by mechanisms inherent to the blood vessel wall (20).

In this respect, several lines of evidence suggest that VSM K⁺ channels may mediate pH-induced alterations of coronary and pulmonary vascular tone. First, changes in pH reportedly modulate the open-state probability of several K⁺ channel families, including high-conductance Ca²⁺-sensitive (BK_Ca), ATP-sensitive (K_ATP), and voltage-activated (K_V) K⁺ channel types (3, 7, 16). These same K⁺ channel families are reported to regulate the membrane potential (E_m) of coronary and pulmonary VSM cells (4, 23, 37). Second, VSM cells of rat cerebral arterioles hyperpolarize when bath pH is reduced from 7.3 to 6.8, indicating that factors that regulate resting membrane potential may be pH sensitive (9). Third, metabolic stimuli, other than pH, have been shown to differentially influence the activity of K⁺ channels expressed in different VSM cell membranes. For example, although hypoxia attenuates the open-state probability of K_V channels in cultured rat pulmonary VSM cells, K_V channels in mesenteric VSM cells are unaffected by hypoxia (37). These findings raise the possibility that a tissue-specific regulation of vascular K⁺ channel types also may be involved in mediating the opposing effect of acidosis on coronary and pulmonary arterial smooth muscle tone. However, little is known about the mechanisms by which acidosis may regulate K⁺ channels in coronary or pulmonary VSM cells or about the identity of the single channels that may represent the conducting pathways. Furthermore, because the ionic effects of changes in pH_o and pH_i generally are not examined independently (2), the membrane location of a pH-sensing site on the K⁺ channel protein remains hypothetical.

Hence, this study examined the effect of lowering pH_i on whole cell K⁺ currents in patch-clamped rat coronary and pulmonary VSM cells. We used a method based on the imposition of transmembrane NH₄Cl gradients to induce a selective decrease in the pH_i of the VSM cells, which permitted a focused, detailed analysis of the effect of this single intracellular metabolic stimulus on K⁺ channel current (12). As outlined below, our results provide initial evidence that a differential effect of pH_i on distinct K_V channel subtypes in coronary and pulmonary VSM cell membranes may provide one explanation for the opposing effect of acidosis on coronary and pulmonary vascular muscle tone.

	MATERIALS AND METHODS

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Experimental animals. Sprague-Dawley rats were obtained from Sasco/Charles River Laboratories (Wilmington, MA) at 8-12 wk of age. On the day of experiments, rats were killed with an overdose of pentobarbital sodium (60 mg/kg ip), and the heart and lungs were immediately removed and placed in a dissecting dish filled with ice-cold physiological saline solution (PSS) (11). The heart was examined at ×20 magnification to locate the proximal origin of the left anterior descending coronary artery. This artery was followed and dissected as it approached the cardiac apex, and arterial branches of 50- to 150-µm OD were dissected free and placed in a vial of cold PSS. The lung was similarly examined to locate the origin of the right and left main pulmonary arteries. These arteries were followed and dissected as they approached the lung apex, and fourth- and fifth-division arterial branches of 50- to 150-µm OD were dissected free and placed in cold PSS.

Cell isolation. Enzymatic isolation of VSM cells was performed as described recently in detail for rat microvessels (17). Briefly, small segments of rat coronary or pulmonary artery were placed for 10 min in a 1-ml aliquot of PSS containing 100 µM Ca²⁺ and 1 mg/ml BSA. Vascular segments were then transferred to a fresh 1-ml aliquot of the same solution containing 1.5 mg/ml papain and 1 mg/ml dithioerythritol (Sigma Chemical, St. Louis, MO), which was warmed to 37°C for 7-10 min. Segments were then incubated for 10-15 min in 1 ml of PSS containing (in mg/ml) 2 collagenase, 0.5 elastase, and 1 soybean trypsin inhibitor (Sigma Chemical). Single smooth muscle cells were released from the vessels by gentle trituration, and the resulting cell suspensions were stored at 4°C for up to 6 h. Only long, smooth, optically refractive cells were used for patch-clamp measurements.

Patch-clamp recording. Whole cell K⁺ currents were recorded in single coronary and pulmonary VSM cells using standard pulse protocols and a patch-clamp station previously described in detail (28, 29). The pipette solution contained (in mmol/l) 50 NH₄Cl, 1 Na₂ATP, 5 HEPES, 1 MgCl₂, 1 EGTA, 100 glutamate, and 104 K⁺ (pH 7.0). By including 50 mmol/l NH₄Cl in the pipette solution dialyzing the cells and maintaining the NH₄Cl concentration in the bath (extracellular) solution at 15, 7.9, or 4 mmol/l, we changed pH_i between calculated values of 7.0, 6.7, and 6.4, respectively, according to the following equation (12) where [NH⁺₄]_i and [NH⁺₄]_o represent intra- and extracellular concentration of NH⁺₄. As such, the bath solution composition for a calculated pH_i of 7.0 was (in mmol/l) 15 NH₄Cl, 100 HEPES, 1 MgCl₂, 2 CaCl₂, 1 EGTA, 45 NaCl, 10 glucose, and 2.4 K⁺ (pH 7.5). The calculated level of ionized Ca²⁺ in the bath solution was 1 mmol/l. By adjusting the NaCl concentration, a constant osmolarity of 290 mosmol/l was maintained when the NH₄Cl concentration in the bath solution was lowered from 15 mmol/l to 7.9 or 4 mmol/l to change pH_i. This method for intracellular acidification, illustrated in Fig. 1, is based on the imposition of transmembrane NH₄Cl gradients, taking advantage of the fact that NH₄Cl only traverses the cell membrane in its uncharged form (NH₃). This process leaves residual H⁺ in the cell interior. Hence, increasing the gradient for NH₃ efflux by reducing the NH₄Cl concentration in the bath solution results in predictable levels of intracellular acidification. Because bath pH (pH_o) is stabilized with 100 mmol/l HEPES to buffer changes in the extracellular H⁺ concentration, but the pipette solution contains a low concentration of HEPES (5 mmol/l), pH_i can be selectively and predictably modified in patch-clamped cells subjected to the whole cell or perforated-patch configurations. This method has been validated extensively by Grinstein et al. (12) using a pH-sensitive fluoroprobe in mouse peritoneal macrophages.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Method used to modify intracellular pH (pHi) of vascular smooth muscle (VSM) cells clamped in whole cell mode. By including a high concentration of buffer (100 mmol/l HEPES) in the bath solution and a low concentration of buffer (5 mmol/l HEPES) in the pipette solution dialyzing the cell, changes in transmembrane NH4Cl gradient can alter pHi without affecting extracellular pH (pHo) (12). Decreasing extracellular NH4Cl concentration ([NH4Cl]o) from 15 to 7.9 or 4 mmol/l at a constant intracellular NH4Cl concentration of 50 mmol/l enhances diffusion of NH3 out of cell. Residual intracellular H+ ions change estimated pHi from 7.0 to 6.7 or 6.4. [HEPES]o and [HEPES]i, extra- and intracellular HEPES concentration; [NH+4]o and [NH+4]i, extra- and intracellular NH+4 concentration.

Drugs. All drugs were obtained from Sigma Chemical, except IBTX, which was obtained from Research Biochemicals International (Natick, MA). Drugs were reconstituted as concentrated stock solutions for direct dilution into the bath solution. Glibenclamide was dissolved as a 10 mM stock in 0.1 M NaOH. Nifedipine was reconstituted as a 10 mM stock solution in 70% ethanol. 4-AP was dissolved as 1 M aqueous stock solution in distilled H₂O, buffered to pH 7.4 with HCl. IBTX and -DTX were dissolved as 100 µM stock solutions in distilled H₂O. Addition of the drugs did not significantly affect the pH of the bath solution and resulted in 0.01% dilution of bath constituents.

Statistics. All averaged data are expressed as means ± SE. Statistical comparisons between groups were made with one-way repeated-measures ANOVA with subsequent Duncan's test. Significance was accepted at P < 0.05.

	RESULTS

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Intracellular acidosis differentially regulates outward current in coronary and pulmonary VSM cells. Representative traces in Fig. 2, A and B, show that incremental 10-mV depolarizing steps from 60 to 0 mV (at pH_i 7) elicited families of outward current in single coronary and pulmonary VSM cells, respectively. Between the two cell types, outward currents observed in coronary VSM cells at pH_i 7 generally exhibited a higher component of noisy current, although current appearance, kinetics, and density varied significantly even between cells from the same resistance artery. Under these conditions, peak current density at 0 mV averaged 2.20 ± 0.15 pA/pF in coronary VSM cells (range 0.32-7.44 pA/pF, n = 61) and 6.48 ± 0.8 pA/pF in pulmonary VSM cells (range 1.54-18.08 pA/pF, n = 65). Cell capacitance in the same cells averaged 17.4 ± 0.6 and 18.7 ± 2.3 pF, respectively. Although the enzymatic isolation of cells is a potential cause of variability in ion current levels, heterogeneity of channel currents between cell populations also has been documented in detail by other laboratories (4, 21).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   A and B: effect of changing pHi on outward current in coronary and pulmonary VSM cells, respectively. Progressive 10-mV depolarizing steps from 60 to 0 mV elicited outward currents in both cell types. Representative traces demonstrate that reducing pHi from a calculated level of 7.0 to 6.4 reversibly increased outward current in coronary VSM cells but reversibly decreased outward current in pulmonary VSM cells. Current-voltage (I-V) relationships show effect of lowering pHi from 7.0 (pre) to 6.4 and returning to pHi 7 (post) on K+ current density in coronary and pulmonary cells (n = 10 and 9, respectively). C and D: I-V relationships for coronary and pulmonary VSM cells, respectively, serially exposed to pHi 7, 6.7, and 7 (n = 7 and 8, respectively). * Current density at acidotic pHi was significantly different from that measured at initial pHi 7 at same membrane potential (Em).  Current density at acidotic pHi was significantly different from that measured on return to pHi 7 at same Em. NH4Cl gradient was 50 mM in pipette and 15 mM in bath to establish control pHi 7.0, 50 mM in pipette and 7.9 mM in bath to lower pHi to 6.7, and 50 mM in pipette and 4 mM in bath to provide pHi 6.4.

Regardless of their initial level, outward currents in coronary and pulmonary VSM cells responded predictably to NH⁺₄-induced decreases in pH_i. The original traces in Fig. 2A show that reducing the NH₄Cl concentration in the bath solution from 15 to 4 mmol/l to lower the calculated pH_i from 7 to 6.4 increased the amplitude of voltage-activated outward current in coronary VSM cells. This effect was reversed on return to pH_i 7. In contrast, Fig. 2B shows that exposing pulmonary VSM cells to a similar change in NH₄Cl gradient to induce intracellular acidosis reversibly attenuated outward current amplitudes. The corresponding current-voltage (I-V) curves for coronary and pulmonary VSM cells in Fig. 2, A and B, respectively, further demonstrate these relationships between outward current amplitude, E_m, and pH_i. Outward current amplitudes reversibly increased in coronary VSM cells during intracellular acidification, and the current density elicited at 0 mV was 23 ± 3% higher at pH_i 6.4 than at pH_i 7 (n = 10). In pulmonary VSM cells (Fig. 2B) the reduction in outward current accompanying intracellular acidosis reversibly depressed the I-V curve at more positive potentials, and outward current density at 0 mV declined by 21 ± 3% when the calculated pH_i was lowered from 7 to 6.4 (n = 9). A smaller decrease in predicted pH_i from 7 to 6.7 also reversibly modulated the level of voltage-elicited outward current in coronary and pulmonary VSM cells, as shown by the I-V relationships in Fig. 2, C and D, respectively. Lowering pH_i from 7 to 6.7 reversibly increased current density at 0 mV by 15 ± 5% in coronary VSM cells (n = 7). In pulmonary VSM cells a similar decline in pH_i to 6.7 reversibly decreased outward current elicited at more positive voltages and reduced current density at 0 mV by 18 ± 3% (n = 8). In other experiments the K⁺ selectivity of outward currents obtained at calculated pH_i of 7.0 and 6.4 was verified by performing tail current analysis at external K⁺ concentrations ([K⁺]_o) of 5, 10, and 30 mmol/l. With the use of a published voltage protocol (28), the average reversal potential obtained for each log [K⁺]_o/[K⁺]_i (where [K⁺]_i is intracellular K⁺ concentration) was compared with the reversal potential predicted for a K⁺-selective channel by the Nernst equation (15). Under these conditions, reversal potentials in coronary and pulmonary VSM cells (n = 5-10 cells) were not different from those predicted by the Nernst equation for a purely K⁺-selective ion channel, implicating K⁺ as the primary charge carrier for outward current observed at both levels of pH_i. Further experiments demonstrated that the L-type Ca²⁺ channel antagonist nifedipine (1 µmol/l) did not prevent NH₄Cl-induced changes in outward current levels in either VSM cell type.

High intracellular HEPES prevents NH₄Cl-induced changes in K⁺ current. Subsequent experiments verified that the NH₄Cl-induced effects on outward current in coronary and pulmonary VSM cells were associated with pH_i change and did not occur as a consequence of the NH₄Cl method distinct from pH manipulation. In these experiments the same pulse protocol of 10-mV steps was used to elicit K⁺ currents between 60 and 0 mV. However, the pipette solution dialyzing the cells contained 50 mmol/l (rather than 5 mmol/l) HEPES, to buffer the intracellular H⁺ generated when the transmembrane NH₄Cl gradient was increased. The intracellular osmolarity was maintained at 290 mosmol/l by substitution of HEPES for glutamate in the pipette solution. Under these conditions, Fig. 3A shows that when the NH₄Cl concentration in the bath solution was changed from 15 to 4 mmol/l to generate intracellular H⁺, the level of K⁺ current was not altered in coronary VSM cells dialyzed with 50 mmol/l HEPES, although it was enhanced in similar cells dialyzed with only 5 mmol/l HEPES (Fig. 2A). Similarly, Fig. 3B shows that outward current elicited in pulmonary VSM cells dialyzed with 50 mmol/l HEPES was not affected by reducing the NH₄Cl concentration in the bath solution, whereas a similar intervention significantly attenuated outward current in pulmonary VSM cells dialyzed with 5 mmol/l HEPES (Fig. 2B). The average I-V relationships obtained from coronary and pulmonary VSM cells dialyzed with 50 mmol/l HEPES (Fig. 3) confirm that outward current did not change in either VSM cell type in response to lowering [NH⁺₄]_o from 15 to 4 mmol/l. Hence, NH₄Cl-induced changes in the level of pH_i, rather than nonspecific effects of the NH₄Cl technique, apparently triggered the contrasting changes in coronary and pulmonary VSM currents shown in Fig. 2.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effect of altering transmembrane NH4Cl gradient, in presence of elevated [HEPES]i, on whole cell K+ currents in coronary (A) and pulmonary (B) VSM cells. Currents were elicited by 10-mV depolarizing steps from 60 to 0 mV. In cells dialyzed with pipette solution containing 50 mmol/l HEPES to minimize changes in pHi, alternating [NH4Cl]o between 15 and 4 mmol/l had no effect on K+ current amplitudes. I-V relationships indicate that lowering [NH4Cl]o from 15 to 4 mmol/l did not affect K+ current densities measured in coronary (n = 6) or pulmonary (n = 6) VSM cells dialyzed with 50 mmol/l HEPES.

Identification of the K⁺ channel type regulated by intracellular acidosis. The identity of the conducting pathways for K⁺ current activated at the control pH_i of 7.0 was explored using specific pharmacological K⁺ channel blockers. A similar approach was used to identify the K⁺ channel types underlying the pH_i-induced changes in outward current levels. On the basis of reports suggesting that several K⁺ channel types are coexpressed in coronary and pulmonary VSM membranes (3, 4, 23), we selected 1 µmol/l glibenclamide, 100 nmol/l IBTX, and 3 mmol/l 4-AP to antagonize K_ATP, BK_Ca, and K_V channels, respectively (23). The I-V relationships in Fig. 4, A and B, show that, under our control conditions of pH_i 7, addition of 1 µmol/l glibenclamide to the bath solution to block K_ATP channels did not alter K⁺ current levels measured in coronary or pulmonary VSM cells, respectively. The sample traces in Fig. 4, C and D, and the corresponding I-V curves plotted beneath, further demonstrate that glibenclamide did not prevent the opposing regulation of K⁺ current amplitude in coronary and pulmonary VSM cells triggered by lowering pH_i from calculated levels of 7 to 6.4 (n = 10 and 8, respectively).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   A and B: glibenclamide (1 µmol/l) did not affect I-V relationships for macroscopic K+ current observed at a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n = 6 and 5). C: actual traces and I-V curve for averaged data show that glibenclamide did not block the reversible increase in K+ current density associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n = 10). D: current traces and I-V relationship for averaged data show that glibenclamide did not block the reversible decrease in K+ current density associated with lowering pHi from 7 to 6.4 in pulmonary VSM cells (n = 8). * Current level at pHi 6.4 was significantly different from that measured at initial pHi 7 at same Em.  Current level at pHi 6.4 was significantly different from that measured on return to pHi 7 at same Em.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   A and B: I-V relationships showing effect of 100 nmol/l iberiotoxin (IBTX) on averaged K+ current density measured at a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n = 6 and 7). * IBTX significantly reduced K+ current density at indicated Em. C: actual traces and I-V curve for averaged data show that IBTX did not block the reversible increase in K+ current density associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n = 7). D: current traces and I-V relationship for averaged data show that IBTX did not block the reversible decrease in K+ current density associated with lowering pHi from 7 to 6.4 in pulmonary VSM cells (n = 6). * Current level at pHi 6.4 was significantly different from that measured at initial pHi 7 at same Em.  Current level at pHi 6.4 was significantly different from that measured on return to pHi 7 at same Em.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   A and B: I-V relationships showing effect of 3 mmol/l 4-aminopyridine (4-AP) on averaged K+ current density measured at a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n = 7 and 9). * 4-AP significantly lowered current level at indicated Em. C: actual traces and I-V relationship for averaged data show that 4-AP prevented the increase in K+ current density associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n = 6). D: current traces and I-V relationship for averaged data show that 4-AP blocked the decrease in K+ current density associated with lowering pHi from 7 to 6.4 in pulmonary VSM cells (n = 7).

-DTX selectively prevents NH₄Cl-induced decreases in pulmonary arterial K⁺ current. To further identify which K_V channel subtypes account for the 4-AP- and pH_i-sensitive K⁺ current component in coronary and pulmonary VSM cells, we studied the effect of -DTX on the opposing regulation of K⁺ current by intracellular acidosis. -DTX is reported to preferentially block a subset of K_V channels, including K_V1.1, K_V1.2, and K_V1.6, with high affinity (6). The I-V relationships in Fig. 7, A and B, show the sensitivities of coronary and pulmonary VSM K⁺ currents, respectively, to the blocking action of this peptide toxin. At the baseline pH_i of 7, the addition of 100 nmol/l -DTX reduced the level of K⁺ current density activated at 0 mV by 6 ± 9% in coronary VSM cells (n = 7) and by 23 ± 3% in pulmonary VSM cells (n = 6). This inhibition was more pronounced and only significant in the pulmonary VSM cells. Importantly, the sample traces and corresponding I-V curve in Fig. 7C illustrate that 100 nmol/l -DTX failed to prevent the enhancement of coronary VSM K⁺ current associated with lowering pH_i from 7 to 6.4. In contrast, -DTX blocked the acidosis-induced decrease of K⁺ current in pulmonary VSM cells exposed to the same conditions (Fig. 7D).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   A and B: I-V relationships showing effect of 100 nmol/l -dendrotoxin (-DTX) on averaged K+ current density measured at a calculated pHi of 7 in coronary and pulmonary VSM cells, respectively (n = 7 and 6). * -DTX significantly reduced K+ current density at indicated Em. C: current traces and I-V relationship for averaged data demonstrate that -DTX did not block the increase in K+ current density associated with lowering pHi from 7 to 6.4 in coronary VSM cells (n = 7). D: actual traces and I-V curve for averaged data demonstrate that -DTX prevented the decrease in K+ current density associated with lowering pHi from 7 to 6.4 in pulmonary VSM cells (n = 8). * Current level at pHi 6.4 was significantly different from that measured at initial pHi 7 at same Em.  Current level at pHi 6.4 was significantly different from that measured on return to pHi 7 at same Em.

	DISCUSSION

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The new findings of this study demonstrate that 1) lowering pH_i from a calculated value of 7.0 to 6.7 or 6.4 produces a graded activation of whole cell K⁺ current in coronary VSM cells but a graded depression of macroscopic K⁺ current in pulmonary VSM cells, 2) the pH_i-induced changes in K⁺ current amplitude in coronary and pulmonary VSM cells are blocked by 4-AP, an inhibitor of K_V channels, and 3) -DTX, a preferential blocker of distinct K_V channel subtypes, prevented acidosis-induced changes in K⁺ current only in pulmonary VSM cells. To our knowledge, these data provide the first detailed evidence to suggest that the differential regulation of distinct K_V channel subtypes by intracellular acidosis may be a mechanism for the opposing effect of reduced pH_i on coronary and pulmonary arterial muscle cell excitability.

Evidence for pH regulation of K⁺ channels. A number of recent reports suggest that several types of membrane K⁺ channels may be sensitive to pH_i in nonvascular cell types (7, 19, 22, 25). Kume et al. (19) observed that lowering pH_i from 7.4 to 7.0 in rabbit tracheal smooth muscle cells decreased the open-state probability of BK_Ca channels. Similarly, Copello et al. (7) reported a decline in BK_Ca channel activity in Necturus gall bladder epithelial cells with intracellular acidosis. K_ATP channels also may be modulated by pH_i. In inside-out patches excised from rat pancreatic -cells, Misler et al. (22) showed that K_ATP channel activity decreased in the presence of 50-100 µmol/l ATP as pH_i was lowered from 7.3 to 6.25. However, applying a similar protocol for intracellular acidification to -cells from the mouse pancreas, Proks et al. (25) observed an enhanced open-state probability of K_ATP channels. Taken together, these findings suggest the plausibility of a direct effect of intracellular acidosis on K⁺ channel gating in membranes from several types of nonvascular cells and also imply that several K⁺ channel types may show pH_i sensitivity as a property.

Mechanisms for differential regulation of K_V channels by intracellular acidosis. Our new findings provide initial evidence that pH_i may act at an intracellular site to differentially regulate K_V channels in coronary and pulmonary VSM cells. In the present study, pharmacological block of BK_Ca channels by IBTX and of K_ATP channels by glibenclamide failed to prevent the increase in coronary or the decrease in pulmonary VSM K⁺ current accompanying intracellular acidosis. After selectively blocking K_V channels in these VSM cell membranes with 3 mmol/l 4-AP (3, 23, 31), however, we no longer observed changes in outward current amplitude related to lowering levels of pH_i in either cell type. In contrast, blocking K_V1.1, K_V1.2, and K_V1.6 channels with -DTX (6) prevented only the pulmonary VSM K⁺ current response to intracellular acidosis. These observations, indicating that an -DTX-sensitive channel provides the pH_i-sensitive K⁺ current in pulmonary, but not in coronary, VSM cells, suggest that the opposing effects of pH_i on distinct 4-AP-sensitive K⁺ channels may constitute a novel mechanism to differentially regulate VSM excitability in the coronary and pulmonary circulations under conditions of acidotic stress.

Methodological considerations. Several different techniques have been used to induce intracellular acidosis in VSM cell or tissue preparations in earlier studies (1, 2, 35). Traditionally, levels of pH_i have been lowered by equilibrating the extracellular solution with gases containing elevated levels of CO₂ (1, 2, 8, 10, 33, 35). However, this method does not permit predicted levels of pH_i to be achieved, and interpretation of findings is complicated by the fact that elevated CO₂ levels per se may directly regulate VSM excitability (1, 20). For these reasons, changing the external concentration of the permeable weak conjugate base NH₃ in the presence of a large reservoir of the intracellular H⁺ equivalent NH⁺₄ has been used to produce calculated reductions in the level of pH_i in VSM preparations. However, although this technique has been carefully documented (12), the quantitative relationship between NH₄Cl transmembrane gradients and pH_i values in this study was not measured directly and should be regarded as representing calculated, rather than absolute, pH_i levels.

Physiological relevance. Although investigators have observed the opposing effects of acidosis on coronary and pulmonary vascular tone for many years (8, 10, 16, 30, 33, 35), controversy remains as to whether acidosis modulates coronary and pulmonary vascular tone at intracellular or extracellular sites. Recent reports indicating that pH_i levels in VSM cells may be particularly sensitive to environmental acidosis, with 70-80% of pH_o change transmitted to the cytoplasm (26), suggest that pH_i represents a potential stimulus for modulating vascular reactivity. Using the fluorescent indicator carboxy-seminaphthorhodafluor to measure pH_i in rat coronary VSM cells, Ramsey et al. (26) measured a decline in pH_i from 6.97 to 6.56 as pH_o was lowered from 7.4 to 6.9. Although further studies in vascular systems are required to clarify the physiological relevance of pH_i-induced changes in K_V current to the contrasting regulation of coronary and pulmonary arterial tone, our patch-clamp findings are consistent with a possible role for K_V channels. Notably, an amplification of outward K_V current in coronary VSM cells in response to intracellular acidification would favor VSM cell hyperpolarization and coronary vasodilation. Conversely, a decline of K_V current in pulmonary VSM membranes by intracellular acidosis would mediate depolarization and constriction of small pulmonary arteries. Furthermore, expression of distinct K_V channels in coronary and pulmonary resistance arteries may provide differential molecular targets for vasodilator therapies specific for these two circulations. A detailed characterization of these K_V channels and the subsequent development of K⁺ channel-opening drugs with site-specific actions could lead to significant therapeutic advances for pathologies in which blood flow to the coronary and pulmonary vasculatures is diminished.