INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

IT IS WELL KNOWN that voltage-gated Ca²⁺ channel currents in a variety of tissues are modified by alterations in extracellular pH (pH_o). Reduction of pH_o below the control value of 7.2-7.4 (acidification) caused a decrease in the Ca²⁺ channel current amplitude in tunicate egg cells (20), guinea pig ventricular myocytes (13, 17, 29), and hybridoma cells (11). Alkalinization caused the opposite effect. The reduction/enhancement of the Ca²⁺ channel current during acidification/alkalinization was explained by a decrease/increase in the surface concentration of the charge-carrying ion near the Ca²⁺ channel mouth because of alterations in surface potential created by the interaction of protons with fixed negative charges on the outer surface of the cell membrane (11, 17, 20). Simultaneous displacements of the voltage dependencies of activation and inactivation were also ascribed to surface potential effects (17, 20). In addition, it has been shown that extracellular protons can have a direct action on Ca²⁺ channel conductance state (23).

Although it is known that acidosis is associated with vasodilatation, the relative roles of intracellular pH (pH_i) and pH_o in causing vascular relaxation are controversial (5, 8, 12, 27). Surprisingly, the effects of pH_o on voltage-gated Ca²⁺ channel currents in single vascular smooth muscle cells (SMCs) have received limited attention. Decreasing pH_o caused an inhibition of the Ba²⁺ current through voltage-gated Ca²⁺ channels in guinea pig cerebral arterial cells (30). Prominent changes in current kinetics were also described, but significant shifts in the current-voltage (I-V) relationship associated with changing pH_o were absent. On the other hand, alteration of pH_o caused changes in the Ca²⁺ channel current amplitude and in the voltage dependencies of channel activation and inactivation in single bovine pial and porcine coronary arterial myocytes (14). These divergent observations may have reflected differences in the regulation of activity of Ca²⁺ channels by pH_o in various vascular SMCs.

Therefore, to investigate the mechanism of the modulation of L-type Ca²⁺ channels by extracellular protons in SMCs isolated from human mesenteric arteries (HMAs), the effect of pH_o on the amplitude, kinetics, and voltage dependencies of activation and inactivation of the Ca²⁺ channel current was examined. The current was studied in the presence of physiological (1.5 mM) and high (10 mM) concentrations of Ca²⁺ and also in the same concentrations of Ba²⁺. The effect of pH_o on the noninactivating "window current" (25) measured in the presence of 10 mM Ba²⁺ was also studied. Our results suggest strongly that the alteration of the Ca²⁺ current by pH_o can be explained primarily by changes in the surface concentration of divalent cations in the vicinity of the mouth of the Ca²⁺ channel in these cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Human mesenteric (omental) arteries were obtained from the fat attached to sections of stomach or small or large bowel removed during routine surgery at St. Thomas's Hospital (London). This procedure was approved by the St. Thomas's Ethical Committee.

Methods of SMC isolation, current recording, and data analysis have been described in detail previously (25, 26). The external solution had the following composition (mM): 130 NaCl, 1 CsCl, 1.2 MgCl₂, 1.5 CaCl₂, 4 tetraethylammonium chloride, 10 HEPES, and 10 glucose. NaOH was used to adjust the pH of the solution to 6.2, 7.2, or 8.2. In some solutions, 1.5 mM Ba²⁺, 10 mM Ca²⁺, or 10 mM Ba²⁺ replaced 1.5 mM Ca²⁺. In 10 mM Ba²⁺- or Ca²⁺-containing solutions, NaCl was reduced to 120 mM. At pH 9.2, 5 mM HEPES was replaced with 5 mM 2-(N-cyclohexylamino)ethanesulfonic acid in the Ba²⁺-containing solution. The pH in this solution fell during experiments to 9.15-9.02, probably because of a slow precipitation of Ba²⁺ by hydroxide ions. This difference was considered negligible. The pipette solution contained (mM) 135 CsCl, 2.5 MgCl₂, 2 Na₂ATP, 10 HEPES, and 10 EGTA, and its pH was adjusted to 7.2 with NaOH. Basic chemicals were purchased from BDH (Poole, UK); HEPES, 2-(N-cyclohexylamino)ethanesulfonic acid, EGTA, and tetraethylammonium chloride were obtained from Sigma Chemical (Poole, UK).

Ca²⁺ and Ba²⁺ currents through L-type Ca²⁺ channels were measured in whole cell voltage-clamp mode at room temperature and analyzed using pClamp 6.0 software (Axon Instruments). We previously described T- and L-type voltage-gated Ca²⁺ channel current in HMA myocytes. The L-type Ca²⁺ channel current, which dominated in these cells, was the main subject of the present investigation. T-type current was eliminated by using a holding potential of 60 mV (25). To compare activation for L-type Ca²⁺ channel currents, a standard voltage protocol was applied. Cells were held at 60 mV, and Ca²⁺ or Ba²⁺ current was recorded using 100-ms depolarizing voltage steps applied at 5-mV increments from 70 to +80 mV. The I-V relationship was initially recorded at pH_o 7.2 and then repeated at the test pH_o in the same cell. Peak current amplitude was measured at each membrane potential and converted to current density, which was calculated as the ratio of the current amplitude to the cell membrane capacitance with the assumption of a specific membrane capacitance of 1 µF/cm². The cell membrane capacitance was determined from the area under the capacitive transients elicited by a 10-mV hyperpolarizing pulse of 5-ms duration recorded with 50-kHz filtering.

Values are means ± SE. The significance of an experimental value was determined using Student's paired or unpaired t-test as appropriate, with P < 0.05 considered to be significant, unless otherwise stated.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effects of acidification and alkalinization on activation and inactivation of L-type Ca²⁺ currents. Figure 1A shows families of Ca²⁺ currents recorded in the same HMA cell at 6.2, 7.2, and 8.2 pH_o. A decrease in pH_o from 7.2 to 6.2 caused a diminution of the Ca²⁺ current from 57 to 33 pA at +10 mV and from 40 to 10 pA at 10 mV. Alkalinization to pH_o 8.2 produced the opposite effect on the Ca²⁺ current, increasing its amplitude by 1.4 and 1.9 times at +10 and 10 mV, respectively. The effects of altering pH_o were completely reversible in this and all other external solutions tested (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Effect of various extracellular pH (pHo) levels on Ca2+ channel current (ICa) at 1.5 mM external Ca2+. A: ICa recorded at pHo 6.2, 7.2, and 8.2 at 10-mV increments between 40 and 0 mV (top) and +10 and +40 mV (bottom). Holding potential was 60 mV; dashed lines, zero current. Currents at each pHo were recorded from the same cell with a membrane capacitance of 45 pF. B: averaged current-voltage (I-V) curves recorded at pHo 7.2 (n = 8), 6.2 (n = 5), and 8.2 (n = 6) and expressed as current densities. Vm, membrane potential. C: mean I-V curves normalized to peak ICa in each cell at pHo 6.2, 7.2, and 8.2.

(1)

(2)

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Modulation of steady-state inactivation for ICa (1.5 mM) by pHo. A: a family of ICa recorded at a 150-ms test potential (Vt) to +10 mV (pHo 7.2 and 8.2) and +20 mV (pHo 6.2) after a series of 30-s preconditioning steps to various voltages (Vc). Cell membrane capacitance was 48 pF. Dashed lines, zero current. Voltage protocol is shown at top. The interpulse interval was 30 ms. B: steady-state inactivation dependencies for ICa averaged for 5 (pHo 6.2), 13 (pHo 7.2), and 9 (pHo 8.2) cells. Solid lines, theoretical fit according to the Boltzmann equation with half-inactivation potentials (Vh) equal to 23.1, 32.4, and 36.3 mV and slope factors (kh) equal to 11.3, 9.3, and 9.2 mV at pHo 6.2, 7.2, and 8.2, respectively. Vertical dashed lines, Vh.

Effects of acidification and alkalinization on the Ca²⁺ channel current in high Ca²⁺- and Ba²⁺-containing solutions. Figure 3 presents the effect of varying pH_o on the I-V curves for the Ca²⁺ channel current recorded in 1.5 mM Ba²⁺, 10 mM Ca²⁺, and 10 mM Ba²⁺. As described in MATERIALS AND METHODS, the I-V relationship was initially recorded at pH_o 7.2 and then repeated at the test pH_o in the same cell with the use of a standard voltage protocol. For 1.5 mM Ba²⁺ and 10 mM Ca²⁺, the pH was altered from the control pH_o of 7.2 to 6.2 or 8.2; for 10 mM Ba²⁺, pH_o was altered from 7.2 to 6.2, 8.2, or 9.2. In Fig. 3, the current recorded at pH_o 7.2 is shown paired with the current obtained at one test pH_o. Figure 3 illustrates that the effect of changing pH_o on the I-V curve was very similar for each of the three external solutions examined and also closely resembled the effect recorded in the solution containing 1.5 mM Ca²⁺ (Fig. 1).

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Effect of various pHo on L-type ICa in the presence of low and high concentrations of divalent cations. A and C: Ba2+ currents (IBa) recorded in the presence of 1.5 and 10 mM Ba2+, respectively; B: ICa measured in 10 mM external Ca2+ with use of the same experimental protocol described in the legend of Fig. 1. Currents are expressed as current densities. Data represent means for 6 and 7 (A), 5 and 4 (B), and 10, 8, and 8 (C) paired human mesenteric arterial (HMA) cells studied at pHo 6.2 and 8.2 (A and B) and 6.2, 8.2, and 9.2 (C), respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Relationship between pHo and the peak L-type ICa measured at the nadir of the I-V curve. IP,pH/IP,7.2 represents the ratio of the peaks of ICa or IBa at test pHo to that at pHo 7.2, respectively, at each divalent cation concentration. The line was drawn according to the following equation: IP,pH/IP,7.2 = Amax/[1 + 10(pKa  pH0)n], where Amax, the maximal saturated ratio, was 1.6, the apparent pKa value was 6.8, and n, the apparent Hill coefficient, was 0.6.

Effects of changing pH_o on the Ca²⁺ channel window current. Figure 5 depicts the normalized steady-state inactivation dependencies together with the activation curves (generated using the mean values of k_m and V_m at each pH_o) for the Ca²⁺ channel current recorded in 10 mM Ba²⁺ physiological saline solution, the solution in which the greatest range of pH_o was studied. Figure 5 illustrates the extent to which activation and inactivation of the Ca²⁺ channel current were displaced to more-negative voltage ranges as pH_o was increased. The shift in the inactivation curve would be expected to have little effect on the current, since even at pH_o 9.2, Ba²⁺ current is almost completely available at a holding potential of 60 mV. On the other hand, the position of the activation curve is such that its displacement should have an important effect on current amplitude, especially at negative potentials.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effect of pHo on steady-state activation and inactivation of IBa in 10 mM external Ba2+. Activation and inactivation curves (lines) were generated using Boltzmann equations, with mean Vm and Vh values shown in Tables 1 and 2, respectively. Mean km values were 6.3, 6.4, 7, and 6.4 mV and mean kh values were 8.1, 8.2, 9.5, and 10.5 mV at pHo 6.2, 7.2, 8.2, and 9.2, respectively. Symbols show IBa normalized with respect to the current recorded after the conditioning potential to 100 mV. Dashed lines, half-activation and half-inactivation potentials.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Effect of pHo on the window IBa. A: IBa (10 mM) measured using 30-s steps to 10 mV from the holding potential of 60 mV at pHo 6.2, 7.2, and 8.2 and in the presence of 0.5 mM Cd2+. Cell capacitance = 32 pF. B: window IBa (measured as Cd2+-sensitive current at the end of 30-s voltage step and presented as current densities) recorded at test potentials of 10 and 0 mV at pHo 6.2, 7.2, and 8.2. Each symbol represents a different HMA cell. Solid lines connect mean values calculated for each pHo.

Role of pH_i. Although the pipette solution contained 10 mM HEPES to buffer pH_i, it is possible that the changes in pH_o were causing corresponding alterations in pH_i that contributed to the observed effects of different pH_o on the Ca²⁺ channel current. To evaluate this possibility, the concentration of HEPES in the pipette solution was increased to 100 mM (the concentration of CsCl was reduced to 80 mM accordingly). Effects of pH_o 6.2, 7.2, and 8.2 on activation and inactivation of Ba²⁺ current in 10 mM Ba²⁺ were then compared using the same experimental approach described above. Acidification of the external solution to 6.2 decreased the peak Ba²⁺ current amplitude by 46 ± 7% (n = 4), and alkalinization increased the peak current by 38 ± 3% (n = 6; data not shown). These values were not significantly different from those measured with 10 mM HEPES in the pipette solution (54 ± 2 and 42 ± 7%, n = 10 and 8, respectively). In addition, similar shifts in activation and inactivation dependencies were found (Tables 1 and 2). These results strongly suggest that changes in the pH_i either did not occur in 10 mM HEPES or did not affect the properties of the current we recorded. This implies that the observed changes in the voltage-dependent characteristics during external acidification or alkalinization were entirely caused by the effects of external protons on L-type Ca²⁺ channels.

Apparent voltage dependence of the effect of pH_o is due to the shift in activation gating. As noted above, the effect of pH_o on Ca²⁺ channel currents was more prominent at negative than at positive potentials. A comparison of the pH_o dependency of Ca²⁺ (1.5 mM Ca²⁺) and Ba²⁺ (10 mM Ba²⁺) current ratios at 10 and +10 mV is presented in Fig. 7 (left). Ca²⁺ and Ba²⁺ currents were clearly more pH_o dependent at 10 than at +10 mV. This is unlikely to be due to differences in the concentration of divalent cations at the membrane surface (see below), since the surface potentials at 10 and +10 mV should be the same for any given pH_o. One possible explanation could be that the current amplitude at any one potential would be influenced by the pH_o-mediated shifts in current activation that we have described above (effects on current availability with a holding potential of 60 mV were small enough to be ignored). To examine this possibility, we divided Ca²⁺ or Ba²⁺ current at 10 mV by the fraction of Ca²⁺ channels activated at this potential (calculated using Eq. 2). This was done at each pH_o tested. The resulting currents thus adjusted for activation or inactivation were then expressed as a fraction of the adjusted current at pH_o 7.2. This process was then repeated for both currents at +10 mV. The resulting corrected pH_o dependencies for Ca²⁺ and Ba²⁺ currents are shown in Fig. 7 (right) and represent effects of pH_o on the current that were independent of shifts in current activation. This correction resulted in the pH_o dependencies of the current at 10 and +10 mV becoming almost superimposable (Fig. 7, right). Once corrected as described, the pH_o dependency of the current amplitude at various potentials followed closely that shown in Fig. 4 for the peak current. Similar results were also obtained from the analysis of Ca²⁺ and Ba²⁺ recorded in 10 mM Ca²⁺ and 1.5 mM Ba²⁺, respectively (not shown). These results therefore suggest that the apparent potential dependence of the effect of pH_o on Ca²⁺ and Ba²⁺ current amplitudes is mainly due to the shift in current activation.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Apparent potential dependency of the effect of pHo on L-type ICa. A and B: normalized ICa (1.5 mM) and IBa (10 mM), respectively, recorded at test potentials of 10 and +10 mV before (left) and after (right) correction for activation. The current amplitude at each test potential at a given pHo was normalized to that at pHo 7.2 and presented on left as ICa,pH/ICa,7.2 (A) or IBa,pH/IBa,7.2 (B). In each cell at each test potential, the current was divided by the fraction of channels activated at that potential (calculated using Eq. 2) to correct current amplitude for activation. The resulting values at each pHo were normalized to that at pHo 7.2 and are expressed as ICa,pH*/ICa,7.2* for ICa (A) and IBa,pH*/IBa,7.2* for IBa (B).

Role of the surface potential in the effects of pH_o on Ca²⁺ channel currents. It is generally accepted that the shift in I-V relationships for voltage-gated Ca²⁺ channels, caused by changing the extracellular concentration of divalent cations or protons, is due to the interaction of these cations with negative membrane surface charges that form a surface potential in the vicinity of the channel (6, 7, 20, 31). Increasing the concentration of protons, for example, results in a decline of the surface potential due to binding to and/or screening of negative surface charges. This leads to an increased transmembrane potential (as the potential between the bulk extra- and intracellular solutions remains the same) and, as a result, a shift of activation and inactivation dependencies toward more-positive voltages.

(3)

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Correlation between ICa and IBa peak amplitudes and the shifts in activation (Vm) and inactivation (Vh) dependencies in the presence of low (1.5 mM; A and C) and high (10 mM; B and D) concentrations of divalent cation. Shifts in activation for L-type ICa (mean ± SE) were determined using the differences between Vm at a given pHo and that at pHo 7.2 measured in individual cells. Shift in inactivation is presented as the difference between the mean Vh at a given pHo and that at pHo 7.2. The current peak ratio (ICa,pH/ICa,7.2 and IBa,pH/IBa,7.2) was normalized with respect to that at pHo 7.2. The correlation between the normalized peak IBa amplitude and the relative shifts in activation (open symbols) and inactivation (closed symbols) was plotted using a logarithmic ordinate. Lines in each plot show the predicted theoretical relationship described in Eq. 3 with a slope of 25 mV.

(4)

View larger version (19K): [in this window] [in a new window]   Fig. 9.   Comparison between pHo-dependent changes in the peak L-type ICa and predicted changes in the surface membrane concentration of divalent cations. Filled symbols and lines, ICa ( in A,  in B) and IBa ( in C,  in D; IP,pH) in the presence of 1.5 (A and C) and 10 (B and D) mM divalent cation. Open symbols, predicted changes in the surface membrane concentration for Ca2+ and Ba2+ (SC,pH). The ratio SC,pH/SC,7.2 was calculated according to Eq. 4 by using the mean shifts in activation, calculated using the differences between Vm at the test pHo and at 7.2 in individual cells (), and in inactivation, determined as the difference between the mean Vh at a given pHo and at pHo 7.2 (). The peak amplitude of ICa or IBa measured at a given pHo was normalized with respect to that at pHo 7.2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

These experiments provide the first detailed measurements of the effect of changing pH_o on the vascular smooth muscle L-type Ca²⁺ channel in the presence of a physiological concentration of Ca²⁺ (1.5 mM). The results suggest that in the presence of a normal or raised (10 mM) concentration of external Ca²⁺, as well as in the same concentrations of Ba²⁺, the degree of inhibition or enhancement of the Ca²⁺ channel current during acidification or alkalinization in HMA cells can be mainly explained in terms of the effect of protons on the surface membrane potential.

As described by the Gouy-Chapman equation (10, 11, 20), an increase in the proton concentration should screen negative membrane surface charges in the vicinity of the Ca²⁺ channel, resulting in a decrease in the surface potential and a consequent increase in the genuine transmembrane potential (since the potential between the bulk extra- and intracellular solutions is clamped). This would decrease the number of open Ca²⁺ channels in the physiological range of membrane potentials because of the shift of the activation to the right along the voltage axis (a "stabilizing" effect of protons on the cell membrane). Channel inactivation would also be shifted in a depolarizing direction. Opposite effects on gating would be expected for alkalinization. As summarized in Tables 1 and 2, alteration of pH_o shifted the activation and inactivation curves in HMA cells as expected in each of the four solutions examined. The magnitude of the shifts was comparable to those measured previously in a number of types of cells, including other vascular smooth muscles (14, 17, 28, 33).

This type of shift in gating parameters should not greatly affect the amplitude of the current at the peak of the I-V curve, since this should be little affected by shifts in activation gating and since steady-state inactivation at a holding potential of 60 mV is almost pH_o insensitive (Fig. 5). However, in addition to modifying the voltage dependency of channel gating, acidification-mediated diminution of the surface potential should also reduce the concentration of divalent cations near the extracellular channel mouth, depressing the current amplitude (3, 9). This phenomenon has been visualized as a reduction in the amplitude of the unitary current on the single Ca²⁺ channel level, in, for example, guinea pig ventricular (29) and bovine and porcine vascular myocytes (14). Similarly, acidification of the trans-chamber, which corresponds to the external side, reduced the unitary conductance of the cardiac and skeletal sarcoplasmic reticulum Ca²⁺ release channels incorporated into planar lipid bilayers (24).

The extent of this reduction in the divalent cation concentration at the external surface of the membrane can be predicted using the shift in gating parameters, as described by Eq. 4, and assuming also that the channel mouth and the gating mechanism "see" similar surface potentials. If it is assumed that the current amplitude is linearly related to the surface concentration of divalent cation, it is possible to compare the observed effects of pH_o on peak current amplitude with the effects that are expected on the basis of surface potential-mediated changes in the surface divalent cation concentration. As illustrated in Fig. 9, a generally good correlation between changes in peak current amplitude and the predicted changes in the surface concentration near the channel mouth was found for both concentrations of Ca²⁺ and Ba²⁺ tested. This result is analogous to the similarity between the observed change in the unitary channel conductance and the surface divalent cation concentration calculated from the shift in activation dependencies that was previously found in bovine pial and porcine coronary arterial cells (14).

An alternative explanation for the effect of pH_o on Ca²⁺ channel currents is that a competition between protons and divalent cations for binding to negatively charged sites near or within the channel pore would directly or indirectly block the movement of Ca²⁺ through the channel. However, most of the reports that have considered this type of interaction have concluded that significant alterations in Ca²⁺ permeation would not occur unless pH_o is decreased to <6 or unless highly artificial conditions are employed (e.g., Ca²⁺-free conditions in which a monovalent cation is used as the permeant species) (14, 17, 21-23, 33). On the other hand, Klöckner et al. (16) more recently observed a progressive decrease in the conductance of the human cardiac L-type channel at pH_o > 6 when 10 mM Ba²⁺ was used as the charge carrier, which was abolished by replacement with alanine of one of the array of four glutamate residues thought to constitute the intrapore Ca²⁺ selectivity filter (32). This study (16) therefore suggested that a direct effect of protons on the permeation mechanism might occur with physiological levels of pH_o and extracellular Ca²⁺ concentration.

It is unlikely, however, that this type of interaction can explain the effects we have demonstrated in HMA cells, at least in the pH_o range of 6.2-9.2. The most striking finding of this study is that the relationship between pH_o and the amplitude of the Ca²⁺ channel current at the peak of the I-V curve was very similar in Ca²⁺ and Ba²⁺ and at 1.5 and 10 mM. Under conditions in which a competition between protons and divalent cations is influencing the permeation mechanism, the effect of pH_o on channel conductance should be sensitive to the identity and concentration of the divalent cation. The affinity of Ba²⁺ for the binding sites within the L-type channel that govern permeation is lower than that of Ca²⁺ (3, 9), and protons should be less able to competitively displace Ca²⁺ and Ba²⁺ from common binding sites when these divalent cations are present at higher concentrations. Thus, if protons compete for binding sites related to the permeation mechanism, it would be predicted that the effect of pH_o on the peak current amplitude should be larger in Ba²⁺ than in Ca²⁺ and should also be larger at the lower concentration of both ions. Instead, the effect of pH_o was similar, even when the extreme cases of 1.5 mM Ba²⁺ and 10 mM Ca²⁺ were compared. This finding is therefore consistent with the previous suggestions that a competition between protons and Ca²⁺ for the permeation pathway does not make an important contribution to the effect of pH_o on the Ca²⁺ current under normal conditions. By the same token, this invariance of the effects of pH_o on the current through L-type Ca²⁺ channels under all conditions tested further suggests that pH_o-dependent changes in the concentrations of divalent cation at the outer mouth of the channel are likely to be due to screening of the surface potential rather than a competition for binding sites.

In summary, our overall results suggest that the inhibition or enhancement of Ca²⁺ channel currents during extracellular acidification or alkalinization in HMA myocytes occurs mainly via alteration of the membrane surface potential, resulting in a shift in potential-dependent channel gating and in changes in the concentration of divalent cation near the external Ca²⁺ channel mouth.

The proposed explanation of the effects of pH_o on the Ca²⁺ channel currents in HMA myocytes can be directly related to the regulation of vascular tone in general, since the mechanism of action of pH_o described above reflects a common feature of Ca²⁺ channels in a number of different tissues. It has been shown that Ca²⁺ channels are slightly activated at the resting membrane potential in vascular SMCs and that this activation is likely to play a role in the regulation of vascular tone by vasoconstrictors and vasodilators (19). We previously described in HMA cells a window current that could contribute to a steady-state Ca²⁺ influx over the physiological range of the membrane potentials (25). The results of the direct measurement of the effect of pH_o on the noninactivating window Ba²⁺ current through L-type Ca²⁺ channels showed that this occurs in HMA myocytes (Fig. 6). An analogous effect on the window Ca²⁺ current would be also predicted, since a similar pH_o dependence of the peak amplitude of Ca²⁺ current and significant shifts in its activation dependencies were observed. In this case, even a quite small effect of pH_o on the voltage dependency of current activation that we have measured will lead to marked alterations in Ca²⁺ influx.

Despite the well-known fact that acidosis causes vasodilatation and alkalosis causes vasoconstriction in isolated arteries and also in vivo (1), controversy exists as to whether these effects are due to changes in pH_o or in pH_i. Under our experimental conditions, the effects of pH_o were independent of the changes of pH_i, as our results with 10 and 100 mM HEPES in the pipette solution demonstrated. In intact vessels, however, effects on pH_i associated with changes in pH_o (4) could also have an important impact on the overall effects of acidification and alkalinization. It has been recently shown that intracellular acidification also suppresses the Ca²⁺ current in vascular SMCs, largely by reducing channel availability (15). It is interesting, however, that Iino et al. (12) showed a marked suppression of the voltage-gated Ca²⁺ current in rabbit portal vein cells during intracellular acidification with propionate and, at the same time, reported that high K⁺ depolarization-mediated increases in intracellular Ca²⁺ concentration and tension development in the intact tissue are paradoxically enhanced under these conditions (12). Tian and co-workers (27) showed that reduction of pH_o under conditions where pH_i was kept constant caused vasodilatation. Furthermore, although application of the sodium salts of the weak acids lactate and butyrate caused vasodilatation of rat mesenteric resistance arteries, this response persisted even if the resulting intracellular acidification was prevented by simultaneous addition of ammonium chloride to the solution (2, 18). These observations point to a predominant role of pH_o, rather than pH_i, in mediating the vasodilating effects of acidosis. In agreement with this possibility, the present results and, in particular, the steep dependency on pH_o of the Ca²⁺ channel window current (Fig. 6) indicate that physiologically relevant levels of acidification are likely to result in significant inhibition of Ca²⁺ influx and a resulting vasodilatation. The relative contribution of pH_i vs. pH_o to the control of vascular tone during acidification and alkalinization remains, therefore, a subject worthy of additional investigation.