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.     

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.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Paradoxical decrease in cytosolic calcium with increasing depolarization by potassium in guinea-pig mesotubarium smooth muscle

The free intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. [Ca²⁺]_i and force were maximal at a relatively low (30 mM) concentration of extracellular K⁺ ([K⁺]_o), and declined at 90 and 140 mM K⁺. Plateau values of both [Ca²⁺]_i and force were higher in the presence of 5 · 10^–6 M ryanodine, indicating that the sarcoplasmic reticulum (SR) contributes to the decline with depolarization. Force and [Ca²⁺]_i at 90 mM K⁺ were both lower then the high-K⁺ solution was applied after a period in 30 mM K⁺ than after a period in normal solution (5.9 mM K⁺), consistent with inactivation of Ca²⁺ channels during prolonged depolarization. Addition of carbachol to the depolarized muscle caused a maintained increase in force without maintained increase in [Ca²⁺]_i. We conclude that the decrease in force at increased [K⁺]_o (the calcium-potassium paradox) is due to a membrane-potential-mediated decrease in [Ca²⁺]_i and, to a lesser extent, to desensitization of the contractile-regulatory apparatus to Ca²⁺.     

Different effects of depolarization and muscarinic stimulation on the Ca2+/force relationship during the contraction-relaxation cycle in the guinea pig ileum

The effects of K⁺ depolarization and of the muscarinic agonist carbachol on [Ca²⁺]_i and force were investigated in smooth muscle sheets of the longitudinal layer of the ileum loaded with Fura-2. K⁺ -rich solutions increased [Ca²⁺]_i and force to an initial peak value, which was determined by the concentration of [K⁺]_o. Thereafter, [Ca²⁺]_i and force declined to a lower maintained level. The Ca²⁺/force relationship observed during this contraction-relaxation cycle is represented by a clockwise hysteresis loop. At 140 mM [K⁺]_o, this loop consisted of three components while at lower [K⁺]_o a two-component loop was observed. The stimulation with 0.1 mM carbachol resulted in a transient increase of [Ca²⁺]_i and force followed by a continuous decline of these parameters despite the presence of the drug. Its EC₅₀ of relaxation was around 270 nM [Ca²⁺]_i. The Ca²⁺/force relationship proceeded along a counterclockwise hysteresis loop during the contraction-relaxation cycle. The extent of this loop decreased but remained unaltered in its direction during repeated stimulation with carbachol. These results suggest that (a) both agonists increase force and [Ca²⁺]_i during stimulation; (b) during depolarization with K⁺, desensitization to Ca²⁺ occurs resulting in a clockwise hysteresis loop; (c) during carbachol stimulation, a counterclockwise hysteresis is observed. This could be due to an increased sensitivity to Ca²⁺ mainly in tonic smooth muscle. These observations might be explained by a modulation of the Ca²⁺ sensitivity by sensitizing and desensitizing mechanisms. These modulations during different stimuli could be due to different myosin light-chain kinase/myosin light-chain phosphatase ratios.     

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.     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.     

The effects of metabolic inhibition on force, Ca2+ and pHi in guinea-pig ureteric smooth muscle

 The effect of metabolic inhibition on the contractile function of adult guinea-pig ureter has been investigated. Strips of ureteric smooth muscle were loaded with Indo-1 or SNARF to measure intracellular [Ca²⁺] ([Ca²⁺]_i) or pH (pH_i) simultaneously with force. Inhibiting oxidative phosphorylation with cyanide rapidly reduced phasic contractions and the associated Ca²⁺ transients, after initial transient increases. The effects of cyanide were reversible and related to the amount of contractile activity undertaken. Inhibition of glycolysis with iodoacetate abolished all force. In high-K⁺-depolarised preparations, cyanide reduced the tonic contraction, but this was not accompanied by a reduction in [Ca²⁺]_i, suggesting a desensitisation of the myofilaments. Cyanide produced a fall in pH_i, which may underlie the initial transient increase in force. These data suggest that metabolic inhibition reduces force in the ureter by affecting both excitation and hence the Ca²⁺ transient, and at the myofilaments to reduce their sensitivity to Ca²⁺. Thus when oxidative metabolism is impaired contractile dysfunction may arise in the ureter. Received: 22 July 1997 / Received after revision and accepted: 12 September 1997     

Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells

 In this study we examined the influence of intracellular pH (pH_i) on agonist-induced changes of intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. pH_i and [Ca²⁺]_i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH₃/NH₄ ⁺ and acetate were used to clamp pH_i to defined values. The magnitudes of the peak and plateau of [Ca²⁺]_i transients induced by carbachol (CCH, 10^–6 mol/l) were greatly enhanced by an acidic pH_i and nearly abolished by an alkaline pH_i. The relationship between pH_i and the [Ca²⁺]_i peak was nearly linear from pH_i 7.0 to 7.8. This effect of pH_i was also observed at higher CCH concentrations (10^–4 and 10^–5 mol/l), at which the inhibitory effect of an alkaline pH_i was more pronounced than the stimulatory effect of an acidic pH_i. An acidic pH_i shifted the CCH concentration/response curve to the left, whereas an alkaline pH_i led to a rightward shift. The influence of pH_i on [Ca²⁺]_i transients induced by neurotensin (10^–8 mol/l) or ATP (5 × 10^–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP ₃) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP ₃ increase induced by CCH was unaltered at an acidic pH_i, but was augmented at an alkaline pH_i. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V _m) measured after TriMA or acetate application. We conclude that pH_i is a physiological modulator of hormonal effects in HT₂₉ cells, as the [Ca²⁺]_i responses to agonists were significantly changed at already slightly altered pH_i. The measurements of InsP ₃, cell volume and V _m show that pH_i must act distally to the InsP ₃ production, and not via changes of cell volume or V _m. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Influence of acid-base changes on the intracellular calcium concentration of neurons in primary culture

The influence of changes in intra- and extracellular pH (pH_i and pH_e, respectively) on the cytosolic, free calcium concentration ([Ca²⁺]_i) of neocortical neurons was studied by microspectrofluorometric techniques and the fluorophore fura-2. When, at constant pH_e, pH_i was lowered with the NH₄Cl prepulse technique, or by a transient increase in CO₂ tension, [Ca²⁺]_i invariably increased, the magnitude of the rise being proportional to pH_i. Since similar results were obtained in Ca²⁺-free solutions, the results suggest that the rise in [Ca²⁺]_i was due to calcium release from intracellular stores. The initial alkaline transient during NH₄Cl exposure was associated with a rise in [Ca²⁺]_i. However, this rise seemed to reflect influx of Ca²⁺ from the external solution. Thus, in Ca²⁺-free solution NH₄Cl exposure led to a decrease in [Ca²⁺]_i. This result and others suggest that, at constant pH_e, intracellular alkalosis reduces [Ca²⁺]_i, probably by enhancing sequestration of calcium. When cells were exposed to a CO₂ transient at reduced pH_e, Ca²⁺ rose initially but then fell, often below basal values. Similar results were obtained when extracellular HCO ₃ ^- concentration was reduced at constant CO₂ tension. Unexpectedly, such results were obtained only in Ca²⁺-containing solutions. In Ca²⁺-free solutions, acidosis always raised [Ca²⁺]_i. It is suggested that a lowering of pH_e stimulates extrusion of Ca²⁺ by ATP-driven Ca²⁺/2H⁺ antiport.     

Effects of acidic stimuli on intracellular calcium in isolated type I cells of the neonatal rat carotid body

We have investigated the effects of acidic stimuli upon [Ca²⁺]_i in isolated carotid body type I cells from the neonatal rat using indo-1 (AM-loaded). Under normocapnic, non-hypoxic conditions (23 mM HCO₃ ^–, 5% CO₂ in air, pH_o=7.4), the mean [Ca²⁺]_i for single cells was 102±5.0 nM (SEM, n=55) with 58% of cells showing sporadic [Ca²⁺]_i fluctuations. A hypercapnic acidosis (increase in CO₂ to 10%–20% at constant HCO₃ ^–, pH_o 7.15–6.85), an isohydric hypercapnia (increase in CO₂ to 10% at constant pH_o=7.4) and an isocapnic acidosis (pH_o=7.0, constant CO₂) all increased [Ca²⁺]_i in single cells and cell clusters. The averaged [Ca²⁺]_i response to both hypercapnic acidosis and isohydric hypercapnia displayed a rapid rise followed by a secondary decline. The averaged [Ca²⁺]_i response to isocapnic acidosis displayed a slower rise and little secondary decline. The rise of [Ca²⁺]_i in response to all the above stimuli can be attributed to no single factor other than to a fall of pH_i. The hypercapnia-induced rise of [Ca²⁺]_i was almost completely abolished in Ca²⁺-free solution, suggesting a role for Ca²⁺ influx in triggering and/or sustaining the [Ca²⁺]_i response. These results are consistent with a role for type I cell [Ca²⁺]_i in mediating pH/PCO₂ chemoreception.     

Effect of intracellular and extracellular ion changes on E–C coupling and skeletal muscle fatigue

The causative factors in muscle fatigue are multiple, and vary depending on the intensity and duration of the exercise, the fibre type composition of the muscle, and the individual's degree of fitness. Regardless of the aetiology, fatigue is characterized by the inability to maintain the required power output, and the decline in power can be attributed to a reduced force and velocity. Following high-intensity exercise, peak force has been shown to recover biphasically with an initial rapid (2 min) recovery followed by a slower (50 min) return to the pre-fatigued condition. The resting membrane potential depolarizes by 10–15 mV, while the action potential overshoot declines by a similar magnitude. Following high-frequency stimulation of the frog semitendinous muscle, we observed intracellular potassium [K⁺]_i decrease from 142±5 to 97±8 mm , while sodium [Na⁺]_i rose from 16±1 to 49±6 mm . The [K⁺]_i loss was similar to that observed in fatigued mouse and human skeletal muscle, which suggests that there may be a limit to which [K⁺]_i can decrease before the associated depolarization begins to limit the action potential frequency. Fibre depolarization to -60 mV (a value observed in some cells) caused a significant reduction in the t-tubular charge movement, and the extent of the decline was inversely related to the concentration of extracellular Ca²⁺. A decrease in intracellular pH (pH_i) to 6.0 was observed, and it has been suggested by some that low pH may disrupt E–C coupling by directly inhibiting the SR Ca²⁺ release channel. However, Lamb et al. (1992) observed that low pH had no effect on Ca²⁺ release, and we found low pH_i to have no effect on t-tubular charge movement (Q) or the Q vs. V_m relationship. The Ca²⁺ released from the SR plays three important roles in the regulation of E–C coupling. As Ca²⁺ rises, it binds to the inner surface of the t-tubular charge sensor to increase charge (Q_γ) and thus Ca²⁺ release, it opens SR Ca²⁺ channels that are not voltage-regulated, and as [Ca²⁺]_i increases further it feeds back to close the same channels. The late stages of fatigue have been shown to be in part caused by a reduced SR Ca²⁺ release. The exact cause of the reduced release is unknown, but the mechanism appears to involve a direct inhibition of the SR Ca²⁺ channel.     

Secretagogue effects on intracellular calcium in pancreatic duct cells

Regulation of intracellular free calcium ([Ca²⁺]_i) in single epithelial duct cells of isolated rat and guinea pig pancreatic interlobular ducts by secretin, carbachol and cholecystokinin was studied by microspectrofluorometry using the Ca²⁺-sensitive, fluorescent probe Fura-2. Rat and guinea pig duct cells exhibited mean resting [Ca²⁺]_i of 84 nM and 61 nM, respectively, which increased by 50%–100% in response to carbachol stimulation, thus demonstrating the presence of physiologically responsive cholinergic receptors in pancreatic ducts of both species. The carbachol-induced increase in [Ca²⁺]_i involved both mobilization of Ca²⁺ from intracellular stores and stimulation of influx of extracellular Ca²⁺. In contrast, neither cholecystokinin nor secretin showed reproducible or sizeable increses in [Ca²⁺]_i. Both rat and guinea pig duct cells showed considerable resting Ca²⁺ permeability. Lowering or raising the extracellular [Ca²⁺]_i led, respectively, to a decrease or increase in the resting [Ca²⁺]_i. Application of Mn²⁺ resulted in a quenching of the fluorescence signal indicating its entry into the cell. The resting Ca²⁺ and Mn²⁺ permeability could be blocked by La³⁺ suggesting that it is mediated by a Ca²⁺ channel.     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

Physiological increases in lactate inhibit intracellular calcium transients,acidify myocytes and decrease force in term pregnant rat myometrium

Key points

• Lactate is increased in myometrial capillary blood from women in slow or non‐progressive labour (dystocia), suggesting that it is detrimental to uterine contractions.
• There are no studies of the effect of lactate on the myometrium. In the present study, we have investigated its effects and mechanism of action on myometrial strips from term pregnant rats.
• We show that lactate significantly decreased spontaneous contractility. Lactatedecreased pH_i and inhibited Ca²⁺ transients. Nulling the decrease in pH_i abolished the effects of lactate effects. If Ca²⁺ entry was enhanced, the effects of lactate were abolished.
• The present study suggests that the accumulation of extracellular lactate could contribute to labour dystocia.

Abstract

Lactate is increased in myometrial capillary blood from women in slow or non‐progressive labour (dystocia), suggesting that it is detrimental to uterine contractions. There are, however, no studies of the effect of lactate on the myometrium. We therefore investigated its effects and mechanism of action on myometrial strips from term pregnant rats. The effects on spontaneous and oxytocin‐induced contractility in response to sodium lactate and other weak acids (1–20 mM) were studied. In some experiments, simultaneous force and intracellular Ca²⁺ or pH (pH_i) were measured with Indo‐1 or Carboxy‐SNARF, respectively. Statistical differences were tested using non‐parametric tests. Lactate significantly decreased spontaneous contractility with an EC₅₀ of 3.9 mM. Propionate, butyrate and pyruvate also reduced contractions with similar potency. The effects of lactate were reduced in the presence of oxytocin but remained significant. Lactate decreased pH_i and nulling the decrease in pH_i abolished its effects. We also show that lactate inhibited Ca²⁺ transients, with these changes mirroring those produced on force. If Ca²⁺ entry was enhanced by depolarization (high KCl) or applying the Ca²⁺ channel agonist, Bay K 4644, the effects of lactate were abolished. Taken together, these data show that lactate in the physiological range potently decreases myometrial contractility as a result of its inhibition of Ca²⁺ transients, which can be attributed to the induced acidification. The present study suggests that the accumulation of extracellular lactate will reduce myometrial contractions and could therefore contribute to labour dystocia.

Abbreviations

AUC

area under the curve

pH_i

intracellular pH

PMCA

plasma membrane Ca²⁺‐ATPase

PSS

physiological saline solution

     

Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse

We compared the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), as measured with the fluorescent Ca²⁺ indicator fura-2, and the force development in intact smooth muscle of the tonic anococcygeus (AC) and the phasic vas deferens (VD) of the mouse, during activation by K²⁺ depolarization and by agonists. Resting [Ca²⁺]_i was observed to be 33% lower in AC (80 nM) than in VD (115 nM), while the Ca²⁺ threshold for contraction was found to be about 120 nM in AC and 160 nM in VD. For a similar [Ca²⁺]_i increase, the agonist stimulation induced a higher force development than the K⁺ depolarization in both muscle types. During prolonged depolarization, the force/calcium ratio increased in AC but strongly declined in VD. This decline of the force/calcium ratio in VD during depolarization was partially reversed by lowering [Ca²⁺]_o. Our results indicate that the Ca²⁺ threshold for force development was about 150% of the resting [Ca²⁺]_i in both cell types. The resting [Ca²⁺]_i was lower in the tonic AC than in the phasic VD. Agonist-induced sensitization to Ca²⁺ occurred in both muscle types. The tonic and phasic smooth muscles essentially differed in the respective modulation of their Ca²⁺ sensitivity during contraction. The desensitization to Ca²⁺ was specific for phasic muscle, in which it occurred as an early, timeand Ca²⁺-dependent process that was partially reversible.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle

The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca²⁺]_i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca²⁺]_i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K⁺ (21 mM), both LC20 phosphorylation and [Ca²⁺]_i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca²⁺]_i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha₁-mediated activation by 10^–5 M phenylephrine whereas a higher force was developed at a shorter time compared to K⁺. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10^–6 M) induced a slow contraction of similar magnitude to that induced by K⁺ without significantly changing either [Ca²⁺]_i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca²⁺]_i in all three types of activation. The initial levels of [Ca²⁺]_i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca²⁺]_i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca²⁺]_i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.     

Evidence for agonist-induced export of intracellular Ca2+ in epithelial cells

There is increasing evidence that some agonists not only induce intracellular Ca²⁺ increases, due to store release and transmembranous influx, but also that they stimulate Ca²⁺ efflux. We have investigated the agonist-stimulated response on the intracellular Ca²⁺ activity ([Ca²⁺]_i) in the presence of thapsigargin (10^–8 mol/l, TG) in HT₂₉ and CFPAC-1 cells. For CFPAC-1 the agonists ATP (10^–7–10^–3 mol/l, n=9), carbachol (10^–6–10^–3 mol/l, n=5) and neurotensin (10^–10–10^–7 mol/l, n=6) all induced a concentration-dependent decrease in [Ca²⁺]_i in the presence of TG. Similar results were obtained with HT₂₉ cells. This decrease of [Ca²⁺]_i could be caused by a reduced Ca²⁺ influx, either due to a reduced driving force for Ca²⁺ in the presence of depolarizing agonists or due to agonist-regulated decrease in Ca²⁺ permeability. Using the fura-2 Mn²⁺ quenching technique we demonstrated that ATP did not slow the TG-induced Mn²⁺ quench. This indicates that the agonist-induced [Ca²⁺]_i decrease in the presence of TG was not due to a reduced influx of Ca²⁺ into the cell, but rather due to stimulation of Ca²⁺ export. We used the cell attached nystatin patch clamp technique in CFPAC-1 cells to examine whether, in the presence of TG, the above agonists still led to the previously described electrical changes. The cells had a mean membrane voltage of –49±3.6 mV (n=9). Within the first 3 min ATP was still able to induce a depolarization which could be attributed to an increase in Cl^– conductance. This was expected, since at this time after TG stimulation all Ca²⁺ agonists still liberated some [Ca²⁺]_i. When TG incubation was prolonged, agonist application led to strongly attenuated or to no electrical responses. Therefore, the agonist-stimulated [Ca²⁺]_i decrease cannot be explained by the reduction of the driving force for Ca²⁺ into the cell. In the same cells hypotonic swelling (160 mosmol/l, n=15) still induced a further [Ca²⁺]_i increase in the presence of TG and concomitantly induced Cl^– and K⁺ conductances. We conclude that the agonist-induced decrease of [Ca²⁺]_i in the presence of TG probably unmasks a stimulation of [Ca²⁺]_i export.     

The distribution of intracellular calcium concentration in isolated single fibres of mouse skeletal muscle during fatiguing stimulation

Fluorescence microscopy has been used to examine the distribution of intracellular calcium concentration ([Ca²⁺]_i) in isolated single fibres from the mouse flexor brevis muscle during fatiguing stimulation. Under control conditions there was a virtually uniform distribution of [Ca²⁺]_i in fura-2 loaded fibres either at rest or during short (0.35 s, 100 Hz) tetani. Fatigue produced by repeated short tetani was accompanied by an early rise, followed by a marked fall, in tetanic [Ca²⁺]_i. Throughout the period of fatiguing stimulation the distribution of [Ca²⁺]_i remained uniform with no detectable gradients observed. In contrast, when fatigue was produced by continuous 100 Hz stimulation, a small gradient of [Ca²⁺]_i developed across the fibre with the [Ca²⁺]_i in the centre of the fibre lower than that at the edge of the fibre. This gradient was apparent after 1.7 s, persisted for at least 11 s and was superimposed on a rise followed by a fall in spatially averaged [Ca²⁺]_i. Reduction of the extracellular Na⁺ to 50% caused reduced force production and a reduced [Ca²⁺]_i in the centre of the fibre. To assess the contribution of reduced response of the myofibrillar proteins to [Ca²⁺]_i during continuous tetani, the relation between [Ca²⁺]_i and force throughout the long tetanus was compared with that obtained in short, unfatigued tetani. These results show that in long tetani, reduced tetanic [Ca²⁺]_i and reduced responsiveness of the myofibrillar proteins to [Ca²⁺]_i each make important contributions to the decline of force, whereas the gradients of [Ca²⁺]_i make only a small contribution.