Developmental changes in intracellular pH buffering power in smooth muscle

 Intracellular pH (pH_i) is known to modulate contraction. Neonatal tissues can differ from adult tissue in contractile response to stimuli known to alter pH_i e.g. hypoxia. Changes of pH are attenuated by buffering, thus any difference in buffering power (β) between tissues could affect their functional response to pH_i perturbation. Similarly the extent to which any extracellular pH (pH_o) alteration is transmitted into a pH_i change will also influence function. We have therefore determined the intrinsic β and effect of pH_o change on pH_i in neonatal and adult ureteric, uterine and gastric smooth muscles using the pH-sensitive fluorophore carboxy-SNARF. β was found to be similar in the three adult tissues, but there were significant differences between neonatal tissues. In contrast, we found little difference in the amount of pH_i change produced by pH_o change between neonatal and adult tissues from the same smooth muscle, but a difference between smooth muscles. These data highlight significant differences between smooth muscles and their developmental state, which may contribute to different degrees of protection when pH is perturbed. Received: 17 October 1997 / Received after revision: 27 November 1997 / Accepted: 28 November 1997     

pH regulation of voltage-dependent K+ channels in canine pulmonary arterial smooth muscle cells

 Voltage-dependent K⁺ currents (K_v) may play a role in hypoxic pulmonary vaso constriction. The effects of changes in extracellular pH (pH_o) and intracellular pH (pH_i) on K_v currents in smooth muscle cells isolated from canine pulmonary artery were studied using the amphotericin B perforated-patch technique for whole-cell recording. Under these conditions, cellular mechanisms for pH_i regulation remain intact, and the effects of pH_o were examined by directly changing the pH of external solutions and changes in pH_i were produced by external application of weak extracellular acids and bases and the cation/H⁺ ionophore, nigericin. Ca²⁺-free external solutions were used to isolate whole-cell K_v currents from contaminating Ca²⁺-activated K⁺ currents. Extracellular acidification (pH_o = 6.4–7.0) reduced K_v currents, produced a positive voltage shift in steady-state activation and reduced maximum K_v conductance (-g _K). Extracellular alkalinization (pH_o = 8.0–8.4) increased K_v currents, produced a small negative voltage shift in steady-state activation, and increased -g _K. Intracellular acidification produced by exposure of cells to external sodium butyrate (20 mM) or nigericin (5 μg/ml) increased K_v currents, produced a negative voltage shift in steady-state activation, and increased -g _K. Intracellular alkalinization produced by exposure of cells to external trimethylamine (20 mM) reduced K_v currents, produced a small positive voltage shift in steady-state activation and reduced -g _K. These results suggest that the effects of pH_o and pH_i on K_v currents are distinctly different, but are consistent with reported effects of pH_o and pH_i on hypoxic pulmonary vasoconstriction, suggesting that such modulation may be mediated in part by pH-induced alterations in K_v channel activity. Received: 1 November 1996 / Received after revision: 19 December 1996 / Accepted: 3 January 1997     

Modulation of HERG potassium channel properties by external pH

We expressed the human eag-related gene (HERG), known to encode for the cardiac potassium channel I_Kr, in Chinese hamster ovary (CHO) cells. This study investigated effects of external pH (pH_o) on HERG current properties using the whole-cell patch-clamp technique. We observed that current amplitude was decreased and kinetics of activation and deactivation were faster when pH_o was lowered from 7.4 to 6.0, while activation was accelerated and V _1/2 negatively shifted when pH_o was changed from 7.4 to 8.0. These effects can be explained by surface charge screening, amino acid group titration and/or changes in ionic atmosphere. We conclude that alterations of HERG channel by pH_o could have consequences for the onset of arrhythmias during cardiac ischemia. Received: 11 February 1999 / Received after revision: 19 April 1999 / Accepted: 26 April 1999     

Simultaneous measurement of intracellular pH and contraction in uterine smooth muscle

Changes in intracellular pH (pH_i) are thought to produce large changes in force production in the uterus. There have however, been no simultaneous measurements of pH_i and force in the uterus and therefore no direct information is available about the relation between the two. We have used carboxy-SNARF (a pH-sensitive fluorophore) in small strips of longitudinal myometrium and obtained simultaneous measurements of pH_i and force. SNARF did not alter contractile function, and continuous measurements of pH_i could be made for 2 hours. The mean resting pH_i (7.16) was similar to that reported previously. Application of weak bases rapidly raised pH_i, in a concentration-dependent manner, followed by a gradual restoration of pH_i to resting levels. Alkalinization greatly increased the frequency of contractions, often accompanied by a small increase in their amplitude. Removal of base produced a rebound acidification which transiently abolished contractions. Direct acidification of the cytoplasm, by application of weak acid, also abolished contractions. However the alkalinization which accompanied removal of acid, produced variable effects on force.Supported by the M.R.C.     

Roles of pH in control of cell proliferation

Precise spatiotemporal regulation of intracellular pH (pH_i) is a prerequisite for normal cell function, and changes in pH_i or pericellular pH (pH_e) exert important signalling functions. It is well established that proliferation of mammalian cells is dependent on a permissive pH_i in the slightly alkaline range (7.0‐7.2). It is also clear that mitogen signalling in nominal absence of is associated with an intracellular alkalinization (~0.3 pH unit above steady‐state pH_i), which is secondary to activation of Na⁺/H⁺ exchange. However, it remains controversial whether this increase in pH_i is part of the mitogenic signal cascade leading to cell cycle entry and progression, and whether it is relevant under physiological conditions. Furthermore, essentially all studies of pH_i in mammalian cell proliferation have focused on the mitogen‐induced G0‐G1 transition, and the regulation and roles of pH_i during the cell cycle remain poorly understood. The aim of this review is to summarize and critically discuss the possible roles of pH_i and pH_e in cell cycle progression. While the focus is on the mammalian cell cycle, important insights from studies in lower eukaryotes are also discussed. We summarize current evidence of links between cell cycle progression and pH_i and discuss possible pH_i‐ and pH_e sensors and signalling pathways relevant to mammalian proliferation control. The possibility that changes in pH_i during cell cycle progression may be an integral part of the checkpoint control machinery is explored. Finally, we discuss the relevance of links between pH and proliferation in the context of the perturbed pH homoeostasis and acidic microenvironment of solid tumours.     

pH aspects of transient changes in conduction velocity in isolated heart fibers after partial replacement of chloride with organic anions

Conduction velocity in isolated rabbit atrial fibers was continuously measured in solutions having a different anionic composition. When 20 mmol/l of chloride was replaced by 20 mmol/l lactate or other anions of weak organic acids at constant pH 6.8, biphasic initial transient changes in conduction velocity were observed. The produced transient changes had a greater amplitude with organic acids which have a greater pK and lipid/water partition ratio. The magnitude of the transients was also greater at pH 6.8 than at pH 7.5, and also when the buffering capacity of the superfusion solution was smaller. Measurements of intracellular pH (pH_i) in sheep Purkinje fibers and of pH at the surface (pH_s) of sheep Purkinje and rabbit atrial fibers with pH sensitive microelectrodes, showed a transient increase of pH_s and a sustained decrease of pH_i on replacement of 20 mmol/l chloride by organic anions of weak acids (at constant pH of the superfusion solution). A combined influence of the transient pH_s change and the sustained pH_i modification seems to be important in the explanation of the biphasic changes in conduction velocity.     

Alterations in proteolytic activity at low pH and its association with invasion: A theoretical model

The extracellular pH (pH_e) of solid tumours is often lower than in normal tissues, with median pH values of about 7.0 in tumours and 7.5 in normal tissue. Despite this more acidic tumour microenvironment, non-invasive measurements of intracellular pH (pH_i) have shown that the pH_i of solid tumours is neutral or slightly alkaline compared to normal tissue (pH_i 7.0–7.4). This gives rise to a reversed cellular pH gradient between tumours and normal tissue, which has been implicated in many aspects of tumour progression. One such area is tumour invasion: the incubation of tumour cells at low pH has been shown to induce more aggressive invasive behaviour in vitro. In this paper the authors use mathematical models to investigate whether altered proteolytic activity at low pH is responsible for the stimulation of a more metastatic phenotype. The authors examined the effect of culture pH on the secretion and activity of two different classes of proteinases: the metalloproteinases (MMPs), and the cysteine proteinases (such as cathepsin B). The modelling suggests that changes in MMP activity at low pH do not have significant effects on invasive behaviour. However, the model predicts that the levels of active-cathepsin B are significantly altered by acidic pH. This result suggests a critical role for the cysteine proteinases in tumour progression.     

The effect of metabolic inhibition on rat uterine intracellular pH and its role in contractile failure

Although intracelluar pH (pH_i) is expected to change during inhibition of oxidative phosphorylation and to affect force, there have been no simultaneous measurements of its effect on pH_i and force in smooth muscle. Therefore, we have investigated the relationship between force and pH_i in strips of longitudinal rat myometrium, loaded with the pH-sensitive indicator carboxy-SNARF and simultaneously measured tension. The application of cyanide produced an abolition of spontaneous contractions and a rapid initial fall in pH_i. In 9/19 preparations pH_i then started to return to resting values but there was no corresponding restoration of force. Cyanide and weak base were simultaneously added to uterine preparations to prevent any acidification; force still fell. Addition of cyanide to depolarized preparations also produced an acidification and a fall in force. Depolarization of preparations in which spontaneous force had been abolished by cyanide often produced a transient rise in force, despite further acidification of the cytoplasm. Cyanide produced an acidification in zero Ca²⁺-containing solution, similar to that in the presence of Ca²⁺ indicating little role for changes in [Ca²⁺] in producing the acidifiction. It is concluded that cyanide decreases pH_i and force in the uterus, but that there is not a simple relationship between the two.     

Effect of a peptide extract of fetal brain tissue on the intracellular pH of mouse peritoneal phagocytes

The intracellular pH (pH_i) of mouse peritoneal neutrophils, initially 0.2 U, drops after a 15-min incubation of these cells with a peptide extract of fetal brain tissue. Treatment with the preparation leads to appreciable changes in the distribution of neutrophils by the examined parameter. For macrophages, the acidifying effect of the agent and its effect on the pattern of cell distribution in terms of pH values are far less expressed. The effects of the agent in dilutions 1∶10² and 1∶10⁴ on the mean pH_i are virtually the same. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 12, pp. 626–630, December, 1995 Presented by V. I. Kulakov, Member of the Russian Academy of Medical Sciences     

Role of nonconserved charged residues of the AE2 transmembrane domain in regulation of anion exchange by pH

The ubiquitous AE2/SLC4A2 anion exchanger is acutely and independently regulated by intracellular (pH_i) and extracellular pH (pH_o), whereas the closely related AE1/SLC4A1 of the red cell and renal intercalated cell is relatively pH-insensitive. We have investigated the contribution of nonconserved charged residues within the C-terminal transmembrane domain (TMD) of AE2 to regulation by pH through mutation to the corresponding AE1 residues. AE2-mediated Cl⁻/Cl⁻ exchange was measured as 4,4′-di-isothiocyanatostilbene-2,2′-disulfonic acid-sensitive ³⁶Cl⁻ efflux from Xenopus oocytes by varying pH_i at constant pH_o, and by varying pH_o at near-constant pH_i. All mutations of nonconserved charged residues of the AE2 TMD yielded functional protein, but mutations of some conserved charged residues (R789E, R1056A, R1134C) reduced or abolished function. Individual mutation of AE2 TMD residues R921, F922, P1077, and R1107 exhibited reduced pH_i sensitivity compared to wt AE2, whereas TMD mutants K1153R, R1155K, R1202L displayed enhanced sensitivity to acidic pH_i. In addition, pH_o sensitivity was significantly acid- shifted when nonconserved AE2 TMD residues E981, K982, and D1075 were individually converted to the corresponding AE1 residues. These results demonstrate that multiple conserved charged residues are important for basal transport function of AE2 and that certain nonconserved charged residues of the AE2 TMD are essential for wild-type regulation of anion exchange by pH_i and pH_o. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. K. Stewart and C. E. Kurschat have contributed equally to this work.     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

Relaxation in ferret ventricular myocytes: role of the sarcolemmal Ca ATPase

In ferret ventricular myocytes the rate of intracellular Ca concentration [Ca]_i decline and relaxation is remarkably fast (compared with rabbit and rat) under conditions where both the sarcoplasmic reticulum Ca uptake and Na/Ca exchange are inhibited. Here we explore the possibility that this rapid [Ca]_i decline in ferret cells is attributable to the sarcolemmal Ca ATPase by using carboxyeosin (a potent inhibitor of the sarcolemmal Ca-ATPase). We compare the effects of carboxyeosin with those of elevated extracellular [Ca] ([Ca]_o) (a thermodynamic approach to limit Ca transport by the sarcolemmal Ca ATPase). In rabbit cells, carboxyeosin and high [Ca]_o slowed [Ca]_i decline similarly and both virtually abolished [Ca]_i decline when mitochondrial Ca uptake was also inhibited. In ferret cells, carboxyeosin treatment produced these same effects on [Ca]_i decline, but high [Ca]_o did not mimic them. Moreover, only in carboxyeosintreated ferret cells did additional inhibition of mitochondrial Ca uptake nearly abolish [Ca]_i decline. We conclude that, carboxyeosin loading can inhibit the sarcolemmal Ca-ATPase in intact myocytes; that this pump seems likely to be responsible for the much faster relaxation observed in ferret cells after block of SR Ca accumulation and Na/Ca exchange transport and that the sarcolemmal Ca pump apparently has different characteristics in rabbit and ferret ventricular myocytes. Present address: Centro de Engenharia Biomédica Caixa Postal 6040, Universidade Estadual de Campinas (UNICAMP), 13081 Campinas, SP, Brazil     

Force during stretch and shortening of frog sartorius muscle: Effects of intracellular acidification due to increased carbon dioxide

Summary The force—velocity relation of frog sartorius muscle was observed during slow stretch and during shortening in solutions with and without CO₂ at extracellular pH (pH_o) 6.9 and pH_o 7.5 (5° C). Less force was produced with CO₂ than without CO₂ during stretch, during shortening, and under isometric conditions. Compared with pH_o 7.5, the effects were greater at pH_o 6.9, where the concentration of CO₂, a permeant acid, was greater and would cause a greater acidification of intracellular pH (pH_i). The reduction of force caused by CO₂ was smaller during stretch than during shortening or isometric contraction. This result indicates that the crossbridge states specific to stretch retain their ability to produce force better under acidic conditions than those characteristic of shortening and isometric conditions. This difference between stretch and shortening suggests that there may be compensating changes in the pattern of motor unit activity during fatiguein vivo.     

In situ probing of intracellular pH by fluorescence from inorganic nanoparticles

Intracellular pH (pH_i) plays a critical role in the physiological processes of cells. Nanoscale sensors based on pH-sensitive fluorescent proteins attached on nanoparticles (NPs) have been designed but inorganic NP-dependent fluorescent nanosensors have not yet been explored. Herein we describe a pH sensitive inorganic semiconductor fluorescent probe based on ultrathin 3C–SiC NPs which can effectively monitor pH in the range of 5.6–7.4 by taking advantage of the linear dependence between the fluorescent intensity ratio of the surface OH⁻ and H⁺ bonding states to band-to-band recombination and pH. Detection of pH_i is demonstrated in living HeLa cells. In particular, pH_i measurements during apoptosis confirm the validity and sensitivity of this technique in monitoring real-time changes in the intracellular environment. Toxicity assessment and confocal laser scanning microscopy indicate that the 3C–SiC NPs have low cytotoxicity and are compatible with living cells.     

Intracellular pH in depolarized cardiac Purkinje strands

In isolated sheep cardiac Purkinje strands the effect of membrane depolarization on intracellular pH (pH_i) and on pH_i changes produced by addition and withdrawal of NH ₄ ⁺ and CO₂/HCO ₃ ^– was investigated. pH_i was continuously measured with double-barreled glass microelectrodes. Repetitive stimulation at high rate resulted in a moderate intracellular acidification (approximately 0.03 pH unit after a 3 Hz train of 2 min), whereafter pH_i returned toward its pre-stimulus level. Prolonged depolarization, evoked either by current injection or by superfusion with high K⁺ solutions, was accompanied by a small acid shift. In the depolarized cell, addition of NH ₄ ⁺ to the superfusate caused intracellular alkalinization followed by re-acidification which was slower than at normal membrane potential. Following intracellular acidification caused by withdrawal of NH ₄ ⁺ , pH_i recovery also was slightly slower than in the normally polarized cell. In the depolarized fiber, removal and readdition of CO₂/HCO ₃ ^– produced the expected intracellular alkalinization and acidification respectively. Recovery from CO₂-induced acidosis was slowed somewhat in high K⁺ (low Na⁺) superfused fibers, not in current depolarized fibers. In the depolarized cell, steady state pH_i in CO₂/HCO ₃ ^– containing and in CO₂/HCO ₃ ^– free solution tended to become identical. These experiments support the hypothesis that in the normally polarized Purkinje fiber passive shuttle movement of NH ₄ ⁺ /NH₃ and CO₂/HCO ₃ ^– occurs and could perhaps at least be partly responsible for the lower steady state pH_i as compared to that reached in NH ₄ ⁺ -free and CO₂/HCO ₃ ^– -free solutions respectively.     

The effect of neuronal morphology and membrane-permeant weak acid and base on the dissipation of depolarization-induced pH gradients in snail neurons

Neuronal depolarization causes larger intracellular pH (pH_i) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pH_i measurements using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pH_i microdomains. In the absence of added mobile buffers, facilitated H⁺ diffusion down the length of the axon plays a critical role in determining pH_i microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membrane-permeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pH_i dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pH_i signals without perturbing steady-state pH_i. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pH_i are likely to be dominated by local pH_i-regulating mechanisms rather than simply following the soma pH_i.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH

We analyzed the pH dependence of K⁺ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than ?50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pH_o), being increased at alkaline, and decreased at acidic pH_o. The strongest effect of a pH_o change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K⁺ currents. Analysis of the time course of current activation at different pH_o values led to the conclusion that the pH-sensitivity of K⁺ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pH_o. This observation and the finding that even a strong acidification to a pH_o value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pH_o changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K⁺ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K⁺.     

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH

We analyzed the pH dependence of K⁺ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than –50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pH_o), being increased at alkaline, and decreased at acidic pH_o. The strongest effect of a pH_o change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K⁺ currents. Analysis of the time course of current activation at different pH_o values led to the conclusion that the pH-sensitivity of K⁺ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pH_o. This observation and the finding that even a strong acidification to a pH_o value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pH_o changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K⁺ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K⁺.     

A quantitative study of the relation between intracellular pH and force in rat mesenteric vascular smooth muscle

Strips of rat mesenteric artery were loaded with carboxy-seminaphthorhodafluor (SNARF) to measure intracellular pH (pH_i) and force simultaneously. pH_i was altered by using weak acids and bases. Alkalinization produced an increase in force. For equal elevations of pH_i a greater and faster increase of force was obtained in depolarized (high K⁺) than in non-depolarised preparations. Acidification produced little change in force unless the tissue was contracted (high-K⁺), in which case it elicited relaxation. Examination of the relationship between pH_i and force in depolarized preparations showed that acidification produced a greater change in force than alkalinization. Removal of weak bases produced a transient acidification that was accompanied by a fall in force in all preparations. This was followed by a secondary contraction in depolarized preparations during the period over which pH_i was acidic and being restored to resting values. Some preparations demonstrated a hysteresis in the relation between pH_i and force. It is concluded that the relationship between pH_i and force in mesenteric vascular smooth muscle is not constant but depends on the previous history of the preparation, and may involve differences in the interactions between H⁺, Ca²⁺ and the contractile machinery.