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(Circulation Research. 2000;86:355.)
© 2000 American Heart Association, Inc.

MiniReview

Interactions Between Ca²⁺ and H⁺ and Functional Consequences in Vascular Smooth Muscle

Clare Austin, Susan Wray

From the Department of Medicine (C.A.), Manchester Royal Infirmary, Manchester, and The Physiological Laboratory (S.W.), The University of Liverpool, Liverpool, UK.

Correspondence to Dr Clare Austin, Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UK. E-mail caustin{at}fs1.cmht.nwest.nhs.uk

	Abstract

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
Abstract—Ca²⁺ and H⁺ ions can profoundly alter vascular tone. In many physiological and pathological processes, changes in the concentration of both ions occur. Thus, to understand the processes and mechanisms that modify force, it is necessary to understand what changes occur in these ions and, importantly, how they interact with each other. In this minireview, we highlight the quantitatively important mechanisms involved in the contractile responses of vascular tissues to pH change and discuss the cellular and molecular reasons underlying these responses.

Key Words: Ca²⁺ • H⁺ • pH • vascular smooth muscle

	Introduction

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
Calcium and H⁺ ions are important regulators of many cellular functions, including contractility. Changes in both Ca²⁺ and H⁺, such as those due to hypoxia and agonist stimulation, and their effects on vascular contractility may be of major physiological importance in distributing blood and maintaining normal blood pressure. Whereas it is clear that, individually, both ions are important modulators, Ca²⁺ and H⁺ are also connected in such ways that changes in one may alter the other. Thus, an understanding of the interactions that may occur between the 2 ions and the contractile apparatus is necessary. Because both ions can affect many cellular components, their influence is widespread, contributing to the often complex effects seen on vascular tone.¹ In intact vessels, it should also be noted that other cell types, eg, endothelial cells, may also influence the contractile responses to alterations in H⁺ or Ca²⁺. A discussion of these effects is beyond the scope of the present review.

	Vascular Tone

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
Smooth muscle contractility is primarily regulated by [Ca²⁺] around the myofilaments.² A rise in [Ca²⁺]_i causes the formation of the Ca²⁺-calmodulin complex and the subsequent activation of myosin light chain kinase, phosphorylation of myosin light chains, interaction with actin, and force production. Relaxation occurs when [Ca²⁺]_i falls and the myosin light chains are dephosphorylated. Receptor-mediated stimulation of vascular smooth muscle may also alter the sensitivity of the contractile fibers to Ca²⁺. These Ca²⁺-sensitizing pathways involve activation of kinases, eg, protein kinase C, mitogen-activated protein, and small G proteins, such as RhoA, a member of the Ras family.³ They may affect not only the balance between phosphorylated and nonphosphorylated myosin but also the thin-filament regulatory molecules, eg, calponin and caldesmon, and, subsequently, actomyosin ATPase activity. Much detail in these sensitizing pathways remains to be elucidated, and they will not be discussed further.

Effects of pH on Vascular Tone
The pH of blood and extracellular fluid (pH_o) is maintained within narrow limits at 7.4, although it may be altered with pathophysiological conditions, eg, respiratory and metabolic disorders and diabetes. The pH inside (pH_i) vascular smooth muscle cells is more acidic, 7.0 to 7.2.^{4 5} Changes in pH_i are limited by buffering and by pH regulatory mechanisms, as will be discussed later. Both pH_o and pH_i may alter tone; thus, when considering the effects of [H⁺] on vascular tone, they must be considered separately.

Changes in pH_o
The functional effects of pH_o on vascular smooth muscle are clear, and many studies have shown that a decrease in pH_o will relax systemic vascular tissues^{5 6 7 8 9} and that an increase in pH_o contracts tissues.^{5 10 11 12}

Differences exist as to the relative importance of pH_i and pH_o in the effects observed on contractility. In some tissues, the effects of pH_o can be accounted for by induced changes in pH_i. For example, in mesenteric vessels, increases in pH_o resulted in a large change in pH_i and contraction of the tissue. The contraction was abolished when the pH_i change was neutralized.⁵ In contrast, in cerebral arterioles, the relaxation associated with a decrease in pH_o was maintained if pH_i was kept constant.^{8 11} These different intrinsic properties of the vessels may be due to a number of factors, including permeability to protons and ion channels expressed in the membrane, and highlight the diversity of vascular smooth muscle.

Changes in pH_i
Generally, resting force falls with intracellular acidification^{10 11 12 13 15 16} and increases with alkalinization.^{17 18 19 20 21} In unstimulated tissues, both increases and decreases in pH_i have been associated with no change in force^{5 9 10 11 12 13 15 16} ; relaxation may, of course, be difficult to observe in unstimulated tissues.

In many vessels activated by agonists or high K⁺, intracellular acidification decreases tone (as in unstimulated preparations) but produces an initial transient rise in tension.^{4 13 15} In vivo, where vessels are under continued tonic influence from agonists, similar responses have also been observed.^{23 24} Similar but opposite effects of alkalinization have been noted.¹⁵ Stimulation with high doses of K⁺ does not always produce the same effects on tone as agonist stimulation (eg, see Reference 25²⁵ ), presumably because of the latter influencing more intracellular pathways. The decrease in tone produced by pH_i will, of course, act synergistically with the effects of a decrease in pH_o.

From the above-mentioned findings, it can be appreciated that the responses of vascular smooth muscle to alterations in pH_i may depend on the method of stimulation or species. We next need to consider the mechanisms that can account for these differences; therefore, the effects of pH on [Ca²⁺]_i and then on myofilament Ca²⁺ sensitivity are discussed.

	Effects of pH on [Ca2+]i

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
From the available data concerning the effects of pH on [Ca²⁺]_i (Table), it is clear that alteration of pH may produce either parallel or reciprocal changes in [Ca²⁺]_i. Thus, the point is immediately made that the effect of pH_i on [Ca²⁺]_i in vascular smooth muscle cannot be predicted, as is the case also for the effect of pH_i on tone. However, what can be seen is that vascular tone, when measured, has mirrored the change in [Ca²⁺]_i; ie, force increased as [Ca²⁺]_i increased, in all the studies apart from one. To date, only one full study has made simultaneous measurements of pH_i, Ca²⁺, and force.⁴² It showed that alteration of pH_o initially changed pH_i, then [Ca²⁺]_i, and, subsequently, force. Such information is obviously valuable in providing mechanistic data.

View this table: [in this window] [in a new window]   Table 1. Effects of Decreased pH on [Ca+]i, Ion Channels, and Em in Vascular Smooth Muscle

Therefore, from the data available, it appears that tone follows the pH-induced changes in [Ca²⁺]_i in the majority of vessels. How then is pH producing a change in [Ca²⁺]_i? Figure 1 summarizes the known mechanisms by which acidification may alter [Ca²⁺]_i and thus increase or decrease force in vascular smooth muscle. Throughout the following discussion, reference will be made to this diagram, in which the various pathways/possible points of action are distinguished alphabetically in parentheses.

View larger version (34K): [in this window] [in a new window]   Figure 1. Effects of acidification on intracellular calcium. A, Effects of protons on Ca2+ homeostasis–promoting contraction. B, Effects of protons on Ca2+ homeostasis–promoting relaxation. The letters in parentheses are referred to throughout the text. A indicates agonist; R, receptor; PIP2, phosphatidylinositol 4,5,-bisphosphate.

	Mechanisms That Alter [Ca2+]i and Their Modulation by pH

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
[Ca²⁺]_i may change as a result of (1) the influx or efflux of Ca²⁺ across the plasmalemma or (2) the release or uptake of Ca²⁺ from intracellular stores. The general point should be noted that alteration of pH may affect the charge and, hence, the activity of many proteins, including the pumps, receptors, channels, and enzymes, in addition to the activity of the drugs used to study them.

Ca²⁺ Influx and Efflux Mechanisms
Several functional studies have found that contractile responses to changes in pH are abolished by Ca²⁺-free medium or by Ca²⁺ channel blockers,^{13 25 32 43} suggesting that Ca²⁺ influx is important for pH alteration of force. Ca²⁺ entry is largely regulated by mechanisms that alter the permeability of the membrane to Ca²⁺, whereas the removal of Ca²⁺ depends on energy-dependent processes.

Ion Channels
Voltage-Operated Ca²⁺ Channels
There are at least 2 types of Ca²⁺ channels sensitive to membrane potential (E_m) in vascular smooth muscle: T- and L-type voltage-operated Ca²⁺ channels (VOCs). Activation of VOCs results in an influx of Ca²⁺ across the cell membrane and thus an increase of [Ca²⁺]_i, predominately via L-type channels. This may result in the release of Ca²⁺ from intracellular stores by Ca²⁺-induced calcium release (CICR) (see below). Thus, any factor that influences the opening of VOCs will affect [Ca²⁺]_i.

H⁺ ions may interact with VOCs both internally and externally (letter a in Figure 1 and Table).^{17 31} In pial and porcine coronary arteries, an increase in pH_i potentiated, and a decrease in pH_i inhibited, the current through L-type channels.^{17 31} This appears to be due to altered gating properties of the channel rather than effects on channel conductance or lifetime of the open state.³¹ This has been attributed to an effect of protonation at a cytosolic binding site, possibly a histidine residue, that prevented the usual shift of the channels to their available states. The mechanism appears to reside in the ß subunit of the channel, inasmuch as channels not constituting this subunit were unaffected by pH_i.⁴⁴

Effects of pH_o on the L-type Ca²⁺ channel in vascular smooth muscle are less well documented (letter a in Figure 1). In addition, it is not always clear if the effects are due to pH_o directly or to an induced change in pH_i. For example, Klockner and Isenberg³¹ found that acidic pH_o decreased the Ca²⁺ current but attributed this to the rapid permeation of the cell membrane by H⁺, an effect previously reported in intact vessels.⁵ Inhibition of Ca²⁺ entry via VOCs by acidosis has also been reported in cerebral vessels³² and in several other smooth muscles; this effect is due to a change in the gating properties of the channel, with both activation and inactivation curves being shifted to more positive potentials.⁴⁵ In turn, these effects depend on the association of H⁺ with the charges on the surface groups around the channel, which may modify its conformational state and, thus, conductance. T-type channels appear to be less affected by pH than L-type Ca²⁺ channels.¹⁸

The inhibitory effect of reduced pH on the Ca²⁺ channel could clearly account for the decrease in [Ca²⁺] (the effect of decreased pH is described above) and also account for the decrease in tone with acidification, which has been reported in many studies (see Table and letter a in Figure 1). We suggest that this is one of the most important ways that a fall in pH_o or pH_i reduces [Ca²⁺]_i and tone.

Receptor-Operated Channels
Excitatory agonists can cause an influx of Ca²⁺ through channels that are not dependent on membrane depolarization; these channels are called receptor-operated channels (ROCs). The effect of pH_i on the opening of these channels has been little studied. pH_o has significant effects on receptor-mediated Ca²⁺ influx in A7r5 smooth muscle cells; acidosis decreases and alkalosis potentiates Ca²⁺ entry in the presence of a blocker of L-type Ca²⁺ channels⁴⁶ (letter b in Figure 1). Changes in pH_o may also alter channel activity via an effect on agonist binding to receptors¹⁹ (letter c in Figure 1). Thus, although the effects of pH_i and pH_o on ROCs remain to be elucidated, we suggest that acidification, by producing charge and conformational changes on the ROCs, will decrease [Ca²⁺]_i entry via this route and reduce force (letter b in Figure 1).

K⁺ Channels and E_m
Because the opening of Ca²⁺ channels is voltage dependent, any effect of pH on E_m will influence them and, thus, Ca²⁺ entry. E_m in vascular smooth muscle cells is -65 to -45 mV, and K⁺ channel activity (of ATP-, Ca²⁺-, and voltage-sensitive channels) is considered to make the most significant contribution to this value. Clearly, any effect of H⁺ on K⁺ channels may affect E_m and, hence, the opening of VOCs.

Apart from one study²⁰ (see Table), a decrease in pH_i increases the activity of all 3 types of K⁺ channels in cells from systemic vessels (letter d in Figure 1). This activation would be expected to lead to hyperpolarization and, hence, vasorelaxation. Whether the effects of acidification on the channels occur via protonation of residues in the pore-forming subunit or via the modification of the ß subunits remains to be seen. The pH sensitivity of cloned inwardly rectifying cardiac K⁺ is being elucidated,²¹ and presumably, data will soon be available for other channels.

In both coronary and cerebral vessels, acidosis activates K⁺ channels (particularly ATP-sensitive K⁺ channels) and, hence, decreases tone (letter d in Figure 1).^{9 37} In patch-clamp studies of cerebral arterial cells, acidosis increased the peak outward K⁺ current.³⁵ Again, this effect may be due to effects on the membrane surface charge, which changes the potential sensed and, hence, shifts the voltage dependence of the activation and inactivation curves. A positive shift in the inactivating curve will increase the availability of channels; hence, the K⁺ current will increase. In cerebral vessels, acidosis increases the slope of the relation between E_m and [K⁺]_o, consistent with an increase in K⁺ conductance.⁴⁷ The transient outward current (I_to) in human ventricular myocytes has been shown to be less sensitive to acidosis than that the I_to in rat ventricular myocytes. These authors point to the finding that only the Kv4.3 isoform contributes to I_to in human myocytes, whereas both Kv4.3 and Kv4.2 are essential components of I_to in rat myocytes; they note that this may explain the different sensitivity to pH_o. Similarly, Steidl and Yool⁴⁸ have examined Kv1.2 and Kv1.5 and have found only Kv1.5 to be susceptible to pH_o alteration as a result of a histidine residue in the third extracellular loop. Thus, differences in channel isoform expression and their pH sensitivity, although not yet studied, may also be expected in different vascular tissues.

Hypercapnic acidification is associated with a hyperpolarization (Table), which can be attributed to the increase in K⁺ channel activity and K⁺ permeability.^{32 41} This will reduce Ca²⁺ entry and decrease tone. Under normocapnic conditions, however, a decrease in pH_i appears to produce either little change in E_m³⁸ or a depolarization.²⁷ Despite this, a decrease in [Ca²⁺]_i was observed.²⁷ Because both normocapnic and hypercapnic acidosis reduce vascular tone, it appears that pH_i may alter tone by both membrane potential–dependent and –independent mechanisms. Irrespective of the mechanisms, extracellular acidosis in all these studies reduced [Ca²⁺]_i, and tone followed.

From the available data, we suggest that the activation by pH of K⁺ channels plays a critical role in bringing about the physiologically important changes in vasotonus that are required with increased activity or altered CO₂. The inhibitory effects of pH on Ca²⁺ channels described earlier will be additive to those on K⁺ channels in these vessels.

In pulmonary vessels, the response of K⁺ channels to pH change may be different and contribute to their different functional response to hypoxia, ie, vasoconstriction. Unlike systemic vessels, extracellular acidification reduced the voltage-activated K⁺ channel (K_V) current and produced a positive voltage shift in steady-state activation (letter e in Figure 1).³⁶ This is consistent with an increase in tone as pH_o falls with hypoxia in pulmonary vessels. Intracellular acidification increased K_V currents, as found in systemic vessels,³⁶ although other studies have reported a reduction in K_V in pulmonary cells³³ (letter e in Figure 1). These differences may be related to differences in channel subtype expression^{49 50} in different-sized vessels. Berger et al³³ also compared the responses of coronary cells to the pulmonary cells and found that K_V increased by intracellular acidification in the former, in agreement with earlier data as shown in the Table. 4-Aminopyridine abolished the effects of acidification in both types of cells. However, -dendrotoxin, which blocks only some subtypes of K_V, was effective only in blocking the effects of pH in the pulmonary cells.³³ This again suggests that subtypes (isoforms) of channels are expressed in different vessels and that this can lead to functionally opposing effects of pH. The direct comparison of 2 vessel types, made possible by the study of Berger et al, makes this one of the most important pieces of evidence available illustrating this effect at the subcellular level.

Summary
It appears that subtypes of ion channels, which may have vessel-specific expression and distribution, differ in their sensitivity to pH. In this way, the effects of pH (as well as other parameters, eg, Ca²⁺ and voltage) on the channels will depend on the vessel under investigation. Therefore, if we consider contraction to be the end result of several steps, then it becomes clear that the effects of pH on channel subtypes build into an effect on a particular channel, which, in turn, influences the conductance of the ion, leading to an effect on the level of [Ca²⁺]_i and, hence, force. There is ample opportunity to vary the effects of H⁺ between vessels. In time, as more detail of the level of channel expression in different vessels becomes known, our detailed understanding of the effect of pH will increase.

Capacitative Ca²⁺ Entry
Many agonists affect vascular tone by causing a release of Ca²⁺ from the internal Ca²⁺ store, the sarcoplasmic reticulum (SR). Emptying of this store causes Ca²⁺ influx across the surface membrane by a mechanism still being elucidated. This has been termed capacitative Ca²⁺ entry, and the current associated with it is called I_CRAC. External acidosis decreased this capacitative Ca²⁺ entry in cultured vascular A7r5 cells,⁴⁶ an effect not mimicked by intracellular acidification (letter f in Figure 1). Similar effects were observed on nonselective cation currents (I_CAT). This effect of pH_o on I_CRAC will presumably decrease Ca²⁺ and will be additive to the reduced Ca²⁺ entry via VOCs (letter a in Figure 1) and, hence, could contribute to the vasorelaxation seen with extracellular acidification.

Passive Entry
At rest, the vascular smooth muscle cell membrane is not entirely impermeable to ions, and small amounts of Ca²⁺ (and H⁺) move down their electrochemical gradients into the cell (letter g in Figure 1) as well as through L-type Ca²⁺ channels. This movement may contribute to the maintenance of resting levels of [Ca²⁺]_i. The routes by which this passive entry takes place are unknown, although it has been shown that the rate of Ca²⁺ entry can be reduced (by up to 65%) by increasing [H⁺]_i.⁵¹ Thus, interactions between H⁺ and Ca²⁺ ions may modify tone to a small extent by this route (letter g in Figure 1). It is unknown whether changes in [Ca²⁺] alter the permeability of the cell membrane to protons, but it is clearly possible that they enter via the same route and may simply compete for entry.

Ca²⁺ Efflux Mechanisms
Na⁺-Ca²⁺ Exchanger
Ca²⁺ may be moved in and out of cells via plasma membrane ion-exchange mechanisms. The electrogenic Na⁺-Ca²⁺ exchanger has been identified in several vascular smooth muscles.⁵² It is now recognized that submembrane [Ca²⁺]_i may be higher than bulk cytosolic [Ca²⁺]_i; therefore, during cell stimulation the exchanger may be activated despite its low affinity for Ca²⁺..^{52 53} In particular, the SR may vectorially release Ca²⁺ to the Na⁺-Ca²⁺ exchanger.⁵¹ The effect of pH on the exchanger in vascular smooth muscle is unclear, but presumably, as with cardiac muscle, both external and internal acidification will have an inhibitory effect.⁵⁴ Inhibition of Ca²⁺ extrusion during acidic conditions could be one mechanism whereby Ca²⁺ and, hence, tone are promoted (letter h in Figure 1).

Ca²⁺- and H⁺-ATPase
Ca²⁺ is actively transported out of the cell by plasmalemmal Ca²⁺-ATPase, which has been reported to be most abundant in larger arteries.⁵⁵ Ca²⁺-ATPase is important in both the maintenance of relatively low [Ca²⁺]_i at rest and the extrusion of Ca²⁺ after elevation. Ca²⁺-ATPase is electroneutral, extruding 1 Ca²⁺ in exchange for 2 protons. Its stimulation has been shown to reduce pH_i.^{43 56} This dual effect of the Ca²⁺-ATPase, ie, reducing [Ca²⁺] and pH_i, will clearly be important for relaxation.

It is clear that as H⁺ is transported, any changes in pH_o or pH_i will affect the activity of the ATPase (letter i in Figure 1). Alkaline pH activates the pump via increasing its affinity for Ca²⁺ rather than altering V_max.⁵⁷ Hence, intracellular alkalinization will favor pump activity and, thus, the efflux of Ca²⁺ and relaxation. Extracellular alkalinization, however, will reduce pump activity and favor Ca²⁺ retention and, thus, contraction. Therefore, this could contribute to vasoconstriction when pH_o rises and vasodilation when pH_o falls, such as has been observed in coronary arteries.⁶ We suggest that this pump will be an important target for agonist modulation of tone, not just via alteration of [Ca²⁺] but also via alteration of [H⁺].

Intracellular Release and Sequestration
Activation of SR Ca²⁺ Channels
IP₃-Induced Release
In agonist-induced contractions of vascular smooth muscle, activation of G-protein–coupled receptors results in hydrolysis of phosphatidylinositol 4,5,-bisphosphate by phospholipase C (PLC) and release of inositol 1,4,5-trisphosphate (IP₃), which results in Ca²⁺ release from the SR, and diacylglycerol. It has been suggested that there are 2 forms of PLC in smooth muscle and that pH and Ca²⁺ differentially regulate these forms.⁵⁸ Diacylglycerol, which also arises from phosphatidylcholine hydrolysis by PLC, stimulates PKC production, which activates the Na⁺-H⁺ exchanger (discussed below).

The available data concerning the effects of pH on IP₃ in vascular tissue are limited and conflicting: increased pH_o, but not pH_i, increased [IP₃] and [Ca²⁺]_i in cerebral vessels,⁵⁹ whereas reduced pH_o, but not pH_i, increased [IP₃] and [Ca²⁺] in umbilical, but not aortic, smooth muscle cells⁶⁰ (letter j in Figure 1). Such differences may again contribute to the varying contractile responses of different vascular smooth muscle cells to alterations in pH. Presumably pH_o, but not pH_i, may alter the protonation of a functional group in a PLC-coupled membrane protein and thus alter IP₃ production. The increase in [IP₃] with extracellular alkalosis found by Albuquerque et al⁵⁹ could constitute an important mechanism whereby alkalosis produces vasoconstriction.

pH_i can alter IP₃-induced Ca²⁺ release; alkalinization increases the sensitivity of Ca²⁺ release in some vessels but decreases it in others.^{61 62} Although different experimental approaches in these studies cannot be ruled out as the explanation for these opposite findings, they may result from different subtypes of the IP₃ receptor, as has been demonstrated in different vessels,⁶³ even though within the same vessel they are considered homogeneous. An increasing affinity of IP₃ for its receptor with pH alteration may be due to the effect of pH on the ionization of the phosphate groups of IP₃. However, Tsukioka et al,⁶¹ who found that alkalinization enhanced IP₃ binding, also considered that pH was working by producing a conformational change in the SR receptor and, thereby, altering its gating kinetics. Because the contribution of IP₃-induced Ca²⁺ release to Ca²⁺ mobilization and, hence, contraction varies between smooth muscles,⁶⁴ this may well contribute to the tissue-specific effects of pH between vessels.

Ca²⁺-Induced Ca²⁺ Release
As with the IP₃ receptor, multiple types of ryanodine channels are expressed in vascular smooth muscle.⁶⁵ In saponin-skinned portal vein,³⁰ a decrease in pH (from pH 7.3 to pH 6.7) increased CICR. This additional Ca²⁺ release is therefore a route whereby decreased pH can lead to vasoconstriction (letter k in Figure 1); however, because decreased pH_i inhibits L-type Ca²⁺ entry, this will reduce or even abolish the stimulus for CICR (letter a in Figure 1). Therefore, the increased release at low pH, seen in permeabilized preparations, may be a way of maintaining Ca²⁺ release by CICR when Ca²⁺ entry is reduced.

The IP₃-mediated increases in [Ca²⁺]_i may induce further Ca²⁺ release from the SR by CICR. Thus, any interactions between H⁺ and Ca²⁺ ions at the IP₃ receptor or Ca²⁺ receptor may also influence release from the other receptor. Whether the IP₃- and ryanodine-sensitive Ca²⁺ stores are separate compartments and how much functional and spatial overlap there is between the two^{30 66} are areas of much present research.

Ca²⁺ Uptake by SR
Sequestration of Ca²⁺ by the SR is regulated by an ATP-dependent Ca²⁺ pump.⁵¹ Ca²⁺ movement is coupled to H⁺; Ca²⁺ uptake occurs with H⁺ efflux^{67 68} ; and the stoichiometry of this process is either 1H⁺:1Ca²⁺ or 3H⁺:2Ca²⁺ (ie, it is not electroneutral). Pollock et al⁶⁹ have recently shown that Cl^- plays an important role in charge compensation during SR Ca²⁺ uptake.

Given the importance of H⁺ efflux for Ca²⁺ uptake, it follows that Ca²⁺ and H⁺ ions may interact at the level of this pump; an increase in [Ca²⁺]_i or activity of SR Ca²⁺- ATPase will decrease pH_i. It has been shown that the SR Ca²⁺ pump activity in some vascular smooth muscles may be stimulated at acidic pH levels.^{7 70} Clearly, this stimulation as pH falls could contribute to the vasorelaxation seen in many vessels (letter l in Figure 1).

Ca²⁺ Buffering
Not all the Ca²⁺ that enters the cell remains in free form; some binds to certain intracellular sites from which it may subsequently be released after cell stimulation.⁵¹ If the intracellular binding/buffering of H⁺ and Ca²⁺ ions occurs at similar sites, there will be competition between the two. This may be a means whereby a decrease of pH_i can produce an increase in [Ca²⁺]_i and contraction (letter m in Figure 1).¹

Mitochondria
Recent data have suggested that mitochondria may play an important role in removing Ca²⁺ from the cytoplasm after cell stimulation. In vascular smooth muscle cells, mitochondrial Ca²⁺ was shown to increase when SR Ca²⁺ release was induced.⁷¹ Mitochondria have also been shown to regulate the IP₃-sensitive SR in colonic myocytes,⁷² an effect thought to be due to mitochondrial Ca²⁺ uptake regulating the [Ca²⁺] near the IP₃ receptor.⁷¹ Because mitochondrial functioning depends on a proton gradient, there may be an influence of H⁺ on mitochondrial Ca²⁺ homeostasis. There have been reports of mitochondrial inhibition affecting membrane Ca²⁺ currents in vascular smooth muscle. For example, McHugh and Beech⁷³ reported a decrease in Ca²⁺ current that was possibly due to elevated [Mg²⁺]_i when oxidative phosphorylation was blocked. Others have reported that glycolysis supports the Ca²⁺ current and Ca²⁺ entry in vascular smooth muscle.⁷⁴

Summary
From the above discussion, it is clear that the vascular smooth muscle cell has several mechanisms for increasing and decreasing [Ca²⁺]_i and that each of these may be affected by pH, as shown in Figure 1. Different vascular smooth muscles may have different functional responses to alterations in pH. Differences in the expression of ion channels and exchangers, position in the vascular tree, and age will undoubtedly contribute to some of the observed differences; clearly, this is an area in which progress will be made by further systematic quantitative cellular and molecular studies.

	Effects of pH on Myofilament Ca2+ Sensitivity

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
Although changes in [Ca²⁺]_i are clearly the most important pathway by which force may be modulated in vascular smooth muscle, changes in the Ca²⁺ sensitivity of the myofilaments, which may be influenced by pH, may also alter tone. Peng et al²⁷ concluded that although a reduction in [Ca²⁺]_i was responsible for the reduction in force seen with acidosis, with severe acidosis there was also a reduced sensitivity of the myofilaments to Ca²⁺. Changes in sensitivity were also suggested by Nagesetty and Paul¹⁵ from their work with coronary vessels, in which reciprocal changes in [Ca²⁺]_i and force were reported (Table).

Effects of H⁺ on Ca²⁺ sensitivity is best studied in permeabilized tissues in which the myofilament environment can be controlled. When -toxin–permeabilized human umbilical artery and rat portal vein were used, no effect of acidosis on Ca²⁺ sensitivity was found, although it was significantly depressed by alkaline pH.⁷⁵ The effects observed may also depend on the type of vascular tissues studied; whereas maximum Ca²⁺-activated force in the portal vein was potentiated by acidic pH, no effect was seen in the umbilical artery.⁷⁵ Because experimental differences cannot underlie these observations, differences between the interactions between Ca²⁺ and H⁺ ions in tonic and phasic vascular tissues may be present. Both artery size and developmental state may also influence sensitivity.^{26 76}

The direct effects of pH on the contractile apparatus of vascular smooth muscle is poorly understood and has been little investigated; however, a number of reports have suggested that the individual components may be sensitive to [H⁺],⁷⁷ resulting in a modification of tone. The available data concerning modification of these components by acidosis and the effect these interactions may have on tone are summarized in Figure 2 and have been reviewed elsewhere.⁷⁷

View larger version (13K): [in this window] [in a new window]   Figure 2. Summary of the available evidence indicating how acidification may alter myofilament Ca2+ sensitivity. Note that by interacting with different components of the pathway, H+ may increase or decrease tone. MLC indicates myosin light chain; MLCK, MLC kinase; and MLC.P, MLC phosphorylation. *Reference 78; Reference 79.

	pH Regulatory Mechanisms

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
pH regulatory mechanisms are important in both the maintenance of resting pH_i and the recovery of pH_i after perturbation. These mechanisms have been extensively reviewed^{77 80} and so will only be considered briefly in the present review. The major mechanisms present in vascular smooth muscle involved in the regulation of pH_i after an acid load are Na⁺-H⁺ exchange, Na⁺-HCO₃^- cotransport, which may or may not be linked to Cl^- efflux, and Cl^--HCO₃^- exchange. The exchangers involved in recovery from an alkaline load have yet to be fully identified but involve the Na⁺-independent Cl^--HCO₃^- exchanger and possibly a Na⁺-dependent Cl^--HCO₃^- exchanger. The activity of these Na⁺-dependent regulatory mechanisms may, in turn, influence the activity of other Na⁺-dependent transporters, cotransporters, or countertransporters, eg, the Na⁺-Ca²⁺ exchanger, which could, in turn, alter contractility. The activity of these pH-regulating mechanisms, especially the Na⁺-H⁺ exchanger, has been linked to disease states, including diabetes and hypertension,^{81 82} although the mechanisms responsible are still unresolved.

Ca²⁺ and H⁺ may both rise in vascular smooth muscle after the activation of the Na⁺-H⁺ exchanger by vasoconstrictors and growth factors (see recent review⁸² ). The rise in pH_i required a rise in Ca²⁺ but persisted in the absence of external Ca²⁺, suggesting that SR Ca²⁺ release by agonists is sufficient to cause the activation of the exchanger. However, the detailed mechanism of second-messenger activation of Na⁺-H⁺ exchanger and its importance in intact vessels (as opposed to cultured cells) and in pathophysiological conditions, such as hypertension and proliferation, remain to be established.

Thus, when agonists bind to the vascular smooth muscle cell membrane, not only do they cause a rise in [Ca²⁺]_i to activate the contractile process, but they may also stimulate pH regulatory mechanisms to alkalinize the cell. Because contraction itself produces acidification that may be due to ATP hydrolysis or activation of the surface membrane Ca²⁺- and H⁺-ATPase, activation of the Na⁺-H⁺ exchanger by agonists may be a control mechanism to decrease the expected acidification. This, in turn, could affect force production and underlie vasoconstriction responses seen after acidification.

	Buffering Power

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
In the short term, the cell is able to buffer protons, thus limiting the magnitude of pH changes. Anything that limits the extent of a change in pH_i may alter, either qualitatively or quantitatively, interactions with Ca²⁺ and, thus, the effects on tone. Some buffering is mediated by HCO₃^-/CO₂, although much is independent (intrinsic) and due to intracellular proteins. The intrinsic buffering power of vascular smooth muscle has been shown (at resting values of pH_i) to be between 30 and 60 mmol/L per pH unit,^{4 83} although this may change with development⁸⁴ and with pH itself.⁸³ An increase in intrinsic buffering power with acidic values of pH_i may be important physiologically in helping protect the cells during periods of acidity, such as may occur during ischemia or hypoxia.⁸³

	Summary

Top Abstract Introduction Vascular Tone Effects of pH on... Mechanisms That Alter [Ca2+]i... Effects of pH on... pH Regulatory Mechanisms Buffering Power Summary References

 
Interactions between H⁺ and Ca²⁺ ions are occurring at almost every point in the stimulatory pathways leading to vascular contraction. Some interactions are simple competition mechanisms, eg, one ion replacing the other from a common binding site. Some of their interactions are direct, eg, H⁺ decreasing Ca²⁺ entry via Ca²⁺ channels or H⁺ entry on the Ca²⁺- and H⁺-ATPase stimulated by a rise in [Ca²⁺]_i. Others are more complex, eg, stimulation of pH regulation after agonist binding and elevation of Ca²⁺ or the relation between pH and IP₃ production and Ca²⁺ release from the SR. We consider that in most preparations, the changes pH produces in [Ca²⁺]_i explain the observed effect on tone and that modulation of myofilament Ca²⁺ sensitivity by H⁺ may be a less general mechanism. We suggest that the effects of pH on Ca²⁺ are variable because of the level of excitation and that this variation reflects differences in ion channel expression and the role of SR, along with their sensitivity to pH. Thus, at the first level, if acidification has a more potent inhibitory effect on K⁺ than Ca²⁺ channels, then force may increase, but if Ca²⁺ channels are significantly inhibited, force will decrease. At a second level, different subtypes of channels have different pH sensitivities. Thus, acidification will inhibit some and activate others, and different vessels can encompass a range of responses to pH.

	Acknowledgments

 
We are grateful to the Wellcome Trust and British Heart Foundation for grant support.

	Footnotes

 
This MiniReview is part of a thematic series on Calcium Cycling in Cardiovascular Cells, which includes the following articles:

Ca²⁺ Release Mechanisms, Ca²⁺ Sparks, and Local Control of Excitation-Contraction Coupling in Normal Heart Muscle

Interactions Between Ca²⁺ and H⁺ and Functional Consequences in Vascular Smooth Muscle

Intracellular Calcium Release Channels

Calcium Fluxes Involved in Control of Cardiac Myocyte Contraction

C. William Balke, Guest Editor

Received August 3, 1999; accepted December 15, 1999.

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