Muscle Cell pH Research Articles

Transgenic expression of carbonic anhydrase III in cardiac muscle demonstrates a mechanism to tolerate acidosis.

Carbonic anhydrase III (CAIII) is abundant in liver, adipocytes, and skeletal muscles, but not heart. A cytosolic enzyme that catalyzes conversions between CO2 and in the regulation of intracellular pH, its physiological role in myocytes is not fully understood. Mouse skeletal muscles lacking CAIII showed lower intracellular pH during fatigue, suggesting its function in stress tolerance. We created transgenic mice expressing CAIII in cardiomyocytes that lack endogenous CAIII. The transgenic mice showed normal cardiac development and life span under nonstress conditions. Studies of ex vivo working hearts under normal and acidotic conditions demonstrated that the transgenic and wild-type mouse hearts had similar pumping functions under normal pH. At acidotic pH, however, CAIII transgenic mouse hearts showed significantly less decrease in cardiac function than that of wild-type control as shown by higher ventricular pressure development, systolic and diastolic velocities, and stroke volume via elongating the time of diastolic ejection. In addition to the effect of introducing CAIII into cardiomyocytes on maintaining homeostasis to counter acidotic stress, the results demonstrate the role of carbonic anhydrases in maintaining intracellular pH in muscle cells as a potential mechanism to treat heart failure.

Sulfur Dioxide Activates Cl-/HCO3 - Exchanger via Sulphenylating AE2 to Reduce Intracellular pH in Vascular Smooth Muscle Cells.

Sulfur dioxide (SO2) is a colorless and irritating gas. Recent studies indicate that SO2 acts as the gas signal molecule and inhibits vascular smooth muscle cell (VSMC) proliferation. Cell proliferation depends on intracellular pH (pHi). Transmembrane cystein mutation of Na+- independent Cl-/HCO3- exchanger (anion exchanger, AE) affects pHi. However, whether SO2 inhibits VSMC proliferation by reducing pHi is still unknown. Here, we investigated whether SO2 reduced pHi to inhibit the proliferation of VSMCs and explore its molecular mechanisms. Within a range of 50–200 μM, SO2 was found to lower the pHi in VSMCs. Concurrently, NH4Cl pre-perfusion showed that SO2 significantly activated AE, whereas the AE inhibitor 4,4′-diisothiocyanatostilbene- 2,20-disulfonic acid (DIDS) significantly attenuated the effect of SO2 on pHi in VSMCs. While 200 μM SO2 sulphenylated AE2, while dithiothreitol (DTT) blocked the sulphenylation of AE2 and subsequent AE activation by SO2, thereby restoring the pHi in VSMCs. Furthermore, DIDS pretreatment eliminated SO2-induced inhibition of PDGF-BB-stimulated VSMC proliferation. We report for the first time that SO2 inhibits VSMC proliferation in part by direct activation of the AE via posttranslational sulphenylation and induction of intracellular acidification.

Increased NBCn1 expression, Na+/HCO3- co-transport and intracellular pH in human vascular smooth muscle cells with a risk allele for hypertension.

Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/ co-transporter NBCn1 which regulates intracellular pH (pHi). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype-dependent differences for rs13082711 in DNA-nuclear protein interactions, where the risk allele is associated with increased SLC4A7 expression level, NBCn1 availability and function as reflected in elevated steady-state pHi and accelerated recovery from intracellular acidosis. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady-state pHi persisted only in vascular smooth muscle cells but not endothelial cells. We found no discernable effect of the missense polymorphism resulting in the amino acid substitution Glu326Lys. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provides an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.

モノカルボン酸トランスポータ4の役割に着目した疾患と副作用に関する研究

Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, are the most widely used cholesterol-lowering agents for prevention of obstructive cardiovascular events. However, statins can cause a variety of skeletal muscle problems including myopathy and rhabdomyolysis, which is characterized by an elevation of creatine kinase (CK). The occurrence of myopathy has been estimated to range from 1% to 5%. The prevalence of muscular symptoms is as high as 25% among statin users who exercise. However, the mechanism by which statins cause myopathy is not precisely known. Since exercise induces the expression of monocarboxylate transporter (MCT) 4, we focused on the association between MCT4 and statin-induced muscle injury.This review summarizes our findings:1) MCT4 is a pH-dependent transporter and contributes to lactate release in RD cells as a model of in vitro skeletal muscle. AMP-activated protein kinase (AMPK) activated by exercise, increased MCT4 expression level and lactate efflux by MCT4 in RD cells and rat skeletal muscle.2) Statins reduce the number of viable cells and up-regulate MCT4 in RD cells. MCT4 knockdown suppresses statin-induced reduction of cell viability and apoptosis compared with negative control-treated RD cells.The cytotoxicity of cerivastatin, one of the statins, is associated with intracellular acidification and caspase-3/7 activation.3) Bicarbonate prevents cerivastatin-induced pH alteration, cytotoxicity in RD cells and muscle damage in an in vivo study.These results suggest that both increased expression of MCT4 and disturbance of skeletal muscle cell pH homeostasis may play an important role in statin-induced cytotoxicity.

Transgenic Over-Expression of Carbonic Anhydrase III in Cardiac Muscle Demonstrates a Mechanism to Resist Acidosis

Carbonic anhydrase III (CAIII) is an abundant protein in skeletal muscle, liver and adipose cells. A cytosolic enzyme that catalyzes conversions between CO2 and HCO3- in regulating intracellular pH, its physiological function in muscle is unclear. Mice lacking CAIII showed lower than wild type intracellular pH in skeletal muscle cells during fatigue treatment. To further understand the role of CAIII in muscle functions and stress adaptation, we developed transgenic mice overexpressing CAIII in the heart under the control of a cloned myosin heavy chain promoter for phenotype comparisons with wild type mouse hearts that are CAIII negative. Three months old transgenic mice showed normal cardiac phenotypes under non-stress conditions. Cardiac function was examined using ex vivo working heart preparations under normal and low pH conditions to investigate CAIII function in pH regulation of cardiac muscle. With equilibration of 5% CO2 generating pH 7.4 in normal Kreb's perfusion buffer, 10% CO2 was used to lower pH to 7.0. Functional data showed that transgenic and wild type hearts had similar pumping functions under normal pH. Perfused with low pH buffer, heart functions of both groups were decreased. In comparison with wild type controls at low pH, CAIII transgenic mouse hearts showed higher left ventricular pressure development and systolic and diastolic velocities under both baseline conditions and increased afterload stress, indicating a better tolerance to acidosis. The results suggest that CAIII may function in compensating for intracellular pH under acidotic conditions, a tractive novel approach to develop new treatment of chronic congestive heart failure.

Maintained intracellular acidification reduces myogenic tone and vasomotion in mice cerebral arteries (1098.3)

The effect of smooth muscle (VSMC) and endothelial cell pH (pHi) on myogenic tone is unknown. Here the role of Na+,HCO3‐‐cotransporter NBCn1 for pHi in these cells, and the influence of low pHi on myogenic tone and vasomotion were determined in intact mouse middle cerebral arteries. Na+,HCO3‐‐cotransport contributed to extrusion of acid from wildtype arteries but not from arteries where NBCn1 was knocked out. pHi of VSMCs was approximately 0.3 units lower in arteries from NBCn1 knockout than wildtype mice. Substantial myogenic tone developed after inhibition of NO‐synthase with L‐NAME in wildtype arteries. Less tone developed in arteries from NBCn1 knockout mice. This myogenic tone was in both type of arteries abolished by the rho‐kinase inhibitor Y‐27632. The arteries displayed vasomotion, which was attenuated in arteries from NBCn1 knockout mice. No differences in membrane potential or [Ca2+]i were seen between VSMC in arteries from NBCn1 knockout and wildtype mice. We propose that NO production and rho‐kinase‐dependent VSMC Ca2+‐sensitivity are reduced at low pHi in mouse middle cerebral arteries, which likely impedes the ability of these arteries to adjust to changes in perfusion pressure.

The Effect of Extracellular pH Changes on Intracellular pH and Nitric Oxide Concentration in Endothelial and Smooth Muscle Cells from Rat Aorta

AimsIt has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pHo) changes on intracellular pH (pHi) and intracellular nitric oxide concentration ([NO]i) in freshly isolated endothelial cells and cross sections from rat aorta.Main MethodsThe HCl was used to reduce the pHo from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pHo from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pHi and [NO]i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy.Key FindingsThe endothelial and vascular smooth muscle pHi was increased at pHo 8.5. The extracellular acidification did not change the endothelial pHi, but the smooth muscle pHi was reduced at pHo 7.0. At pHo 8.5 and pHo 6.5, the endothelial [NO]i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]i.SignificanceNot all changes in pHo did result in pHi changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

NHE1 knockout reduces blood pressure and arterial media/lumen ratio with no effect on resting pHi in the vascular wall

Acid–base transport in the vascular wall remains incompletely understood. Here, we investigated (a) implications of Na(+)/H(+) exchanger NHE1 knockout for vascular smooth muscle (VSMC) and endothelial cell (EC) pH(i) regulation, mesenteric artery morphology, vasomotor function and blood pressure regulation, and (b) consequences of sustained EC and VSMC acidification for vasomotor function. Na(+)/H(+) exchange activity was abolished in VSMCs and ECs from NHE1 knockout mice, but with CO(2)/HCO(3)(−) present, steady-state pH(i) was unaffected. Active tension was 30% smaller in arteries from NHE1 knockout than wild-type mice, and media thickness equally reduced. Number of VSMCs per unit artery length was unchanged whereas volume and cross-sectional area of individual VSMCs were reduced. Media stress, force production per VSMC cross-sectional area and VSMC Ca(2+) responses were unaffected. Blood pressure was 25 mmHg lower in NHE1 knockout than wild-type mice. Omission of CO(2)/HCO(3)(−) caused VSMCs and ECs to acidify substantially more in NHE1 knockout (0.3–0.6 pH-units) than wild-type (0.02–0.1 pH units) mice. Removing CO(2)/HCO(3)(−) inhibited acetylcholine-induced NO-mediated relaxations in arteries from NHE1 knockout but not wild-type mice. Without CO(2)/HCO(3)(−), effects of NO synthase and rho kinase inhibition on noradrenaline-induced contractions were smaller in arteries from NHE1 knockout than wild-type mice whereas the EC Ca(2+) response to acetylcholine, VSMC Ca(2+) response to noradrenaline and vasorelaxation to S-nitroso-N-acetylpenicillamine were unaffected. In conclusion, NHE1 mediates the Na(+)/H(+) exchange in ECs and VSMCs. Under physiological conditions, CO(2)/HCO(3)(−)-dependent mechanisms mask the pH(i)-regulatory function of NHE1. NHE1 knockout causes hypotrophy of VSMCs, reduced artery tension and lower blood pressure. At acidic pH(i), NO-mediated vasorelaxation and rho kinase-dependent VSMC Ca(2+) sensitivity are reduced.

Ovarian cancer G protein-coupled receptor 1-dependent and -independent vascular actions to acidic pH in human aortic smooth muscle cells

Atherosclerosis is a chronic inflammation disease characterized by acidic micromilieu and the accumulation of numerous bioactive lipid mediators, such as lysophosphatidic acid (LPA) and prostaglandins, in the atherosclerotic lesion. Chronic acidification induced various effects on vascular smooth muscle cells, but the molecular mechanisms underlying these effects remain unknown. In this study, we examine the role of proton-sensing ovarian cancer G protein-coupled receptor 1 (OGR1) in extracellular acidification-induced regulation of cyclooxygenase (COX)-2 induction, PGI(2) production, MAPK phosphatase (MKP)-1 expression, and plasminogen activator inhibitor (PAI)-1 expression and proliferation in human aortic smooth muscle cells (AoSMCs). Experiments with knockdown with small interfering RNA specific to OGR1 and specific inhibitors for G proteins showed that acidification-induced COX-2 expression, PGI(2) production, and MKP-1 expression, but not PAI-1 expression and inhibition of proliferation, were dependent on OGR1 and mainly mediated by G(q/11) protein. LPA remarkably enhanced, through the LPA(1) receptor/G(i) protein, the OGR1-mediated vascular actions to acidic pH. In conclusion, acidic pH-induced vascular actions of AoSMCs can be dissected to OGR1-dependent and -independent pathways: COX-2 expression, PGI(2) production, and MKP-1 expression are mediated by OGR1, but PAI-1 expression and inhibition of proliferation are not. LPA, which is usually thought to be a proatherogenic lipid mediator, may exert antiatherogenic actions under acidic micromilieu through cross-talk between LPA(1)/G(i) protein and OGR1/G(q/11) protein.

Steady state decrease of smooth muscle cell and endothelial cell pH prevents hypertension induced by angiotensin II or NO‐synthase inhibition

The role of steady state changes in smooth muscle and endothelial cell pH for vascular function is not known. In this study we knocked out the Na, HCO3‐cotransporter NBCn1 (slc4a7) in mice which caused a reduced pH in mesenteric artery smooth muscle and endothelial cells. Isolated arteries had reduced acetylcholine induced, NO‐mediated relaxations and rho‐kinase dependent smooth muscle Ca‐sensitivity. NBCn1 knockout mice were hypertensive, displayed attenuated blood pressure increase to NO‐synthase inhibition and blood pressure decrease to rho‐kinase inhibition and were resistant to developing hypertension during angiotensin II infusion. The increase of extracellular NO in the presence of acetylcholine was reduced in arteries from NBCn1 knockout mice while dilation to the NO‐donor S‐nitroso‐N‐acetylpenicillamine, acetylcholine‐induced endothelial Ca‐increase and endothelial NO‐synthase expression were unaffected. Rho‐kinase activity was reduced at low pH. In the absence of CO2/HCO3−, no differences were observed between isolated arteries from NBCn1 knockout and wild type mice. Using a knockout mouse to induce sustained intracellular acidification, we show a novel interaction between smooth muscle cell and endothelial cell steady state intracellular pH, vascular function and blood pressure regulation.

Effect of Digoxin on Hypoxic Pulmonary Hypertension (HPH)

Many lung diseases induce HPH, leading to increased mortality. HPH, caused by vascular contraction and remodeling, is associated with increased intracellular pH (pHi) and calcium concentration ([Ca2+]i) in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia‐inducible factor 1 (HIF‐1), a transcription factor, plays a critical role in the development of HPH. Recently, digoxin (Dig) was found to inhibit HIF‐1. We hypothesized that Dig could attenuate the development of HPH. Mice were weighed and injected daily with saline or Dig (low = 0.2 mg/kg or high = 1.0 mg/kg; i.p.) and kept in hypoxia (10% O2) or room air. After 21 days, right ventricular (RV) pressures were measured via closed‐chest puncture. Hypoxia increased RV pressures in mice receiving saline. Low Dig reduced, and high Dig normalized, RV pressure in hypoxic mice. RV hypertrophy (RVH) increased in hypoxic mice treated with saline or low Dig, but was reduced in hypoxic mice receiving high Dig. Hypoxia increased hematocrit in mice treated with saline or low Dig, whereas high Dig reduced the hypoxia‐induced increase in hematocrit. Both pHi and [Ca2+]i increased in PASMCs from saline‐treated hypoxic mice, and treatment with low or high Dig prevented these changes in ion homeostasis. From these results, we conclude that digoxin attenuates pulmonary vascular responses to chronic hypoxia and may be a possible candidate for treatment of HPH.HL67191, GM008074

The p38 mitogen‐activated protein kinase pathway is involved in the regulation of heme oxygenase‐1 by acidic extracellular pH in aortic smooth muscle cells

Extracellular acidosis (EA) regulates Heme Oxygenase-1 (HO-1) expression in vascular smooth muscle cells via transcriptional and posttranscriptional mechanisms but the signaling pathways involved are not known. We examined the role of Mitogen-Activated Protein Kinase (MAPK) pathways in HO-1 regulation by EA. Primary rat aortic smooth muscle cells were exposed to EA or physiologic pH. Levels of the total and phosphorylated forms of p38, extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and HO-1 protein were assessed by Western analysis and HO-1 mRNA levels were assessed by quantitative PCR. Inhibition of p38 MAPK was achieved with the chemical inhibitor SB203580, or adenoviral infection of a dominant-negative form of p38alpha. Phospho p38 MAPK activity was evaluated with an in vitro kinase activity assay. Binding of Activator Protein-1 (AP-1), a known target of MAPK pathways, was assessed by Electromobility shift assay (EMSA). EA induced phosphorylation of p38 MAPK in a biphasic manner while total p38 was unchanged. EA did not alter levels of phospho ERK 1/2 and phospho JNK 1/2. There was increased phospho p38 MAPK activity in the setting of EA which preceded the induction of HO-1. Inhibition of phospho p38 activity with either SB20358 or a dominant negative p38alpha oligonucleotide abrogated the induction of HO-1 by EA. Increased specific binding of AP-1 in the setting of EA was shown by EMSA. Increased phospho p38 activity precedes and likely mediates HO-1 induction by EA. Increased AP-1 binding may underlie the transcriptional regulation of HO-1 by EA.

Safety evaluation of chicken breast extract containing carnosine and anserine

Chicken breast extract (CBEX™) is obtained via hot water extraction of chicken breast and contains among its primary constituents carnosine and anserine, which are histidine-containing dipeptides present in the muscle tissues of most vertebrate species. Dietary intake of CBEX™ has been previously shown to buffer hydrogen ions formed during high-intensity exercise in human skeletal muscle cells, thereby inhibiting a decrease in muscle cell pH and subsequent muscle fatigue. The objective of this paper is to report the results of safety studies completed on CBEX™. CBEX™ was determined to have an oral LD 50 value of more than 6000 mg/kg body weight in rats. Gavage doses of 500 or 2000 mg CBEX™/kg body weight/day administered to rats for 90 days produced no toxicologically significant, dose-related, differences between control and treated animals with respect to body weight gain, food consumption, behavioral effects, hematological and clinical chemistry parameters, absolute and relative organ weights, or gross and microscopic findings. In the presence or absence of metabolic activation, CBEX™ exerted no mutagenic activity in the Ames assay conducted in various strains of Salmonella typhimurium and Escherichia coli. The results of these studies support the safety of CBEX™ as a potential dietary source of carnosine and anserine.

Muscle cell volume and pH changes due to glycolytic ATP synthesis

This letter deals with a theoretical question raised by a recent paper (Usher-Smith et al. 2006) which, as well as experimental data, presents a kinetic simulation and a steady-state analysis of glycolytic cell swelling in muscle. Here I focus on the role of cellular buffer capacity, extending the steady-state analysis and connecting it with other work on cellular acid–base balance in order to show that one of the authors' main findings, that simulated cell swelling decreases with increasing cellular buffer capacity (Fig. 9B in Usher-Smith et al. (2006), hereinafter called simply Fig. 9B), must be due to complex changes in transmembrane fluxes rather than directly to altered buffering. I also briefly extend the analysis to include phosphocreatine (PCr) breakdown, an important feature of exercising muscle.

Hydrogen sulphide regulates intracellular pH in vascular smooth muscle cells

We investigated the role of hydrogen sulphide (H 2S) in intracellular pH (pH i) regulation in vascular smooth muscle cells and its contribution on vasodilation. NaHS, a H 2S donor, decreased pH i in a concentration-dependent manner ranging from 10 μM to 1 mM. Neither inhibition of the Na +/H + exchanger with 5-( N-ethyl- N-isopropyl) amiloride, (EIPA, 10 μM), nor plasmalemmal Ca 2+-ATPase with CdCl 2 (20 nM) alters the effect of NaHS on pH i. Blockade of the Cl - / HCO 3 - exchanger with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) significantly attenuated the pH i lowering effect of NaHS. Moreover, NaHS significantly increased the activity of Cl - / HCO 3 - exchanger when measured with NH 4Cl prepulse method. DIDS attenuated the vasorelaxation induced by NaHS whereas EIPA and CdCl 2 did not cause any change. In conclusion, H 2S induced intracellular acidification via activation of Cl - / HCO 3 - exchanger, which is, at least partially, responsible for H 2S-mediated vasorelaxation.

Mechanoreceptors and central command

static exercise increases mean arterial pressure, heart rate, and ventilation. Central command and the exercise pressor reflex are the two neural mechanisms causing these responses to static exercise. Central command is defined as the parallel activation of the locomotor and autonomic circuits in

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.

Point: The muscle metaboreflex does restore blood flow to contracting muscles

Imposed reductions in oxygen delivery to active skeletal muscle during dynamic exercise in conscious dogs evoke a powerful pressor response, termed the muscle metaboreflex. During submaximal work rates, the major mechanism used by this reflex is to increase cardiac output (CO), which generates a

Stimulation of protein degradation by low pH in L6G8C5 skeletal muscle cells is independent of apoptosis but dependent on differentiation state.

In chronic renal failure, metabolic acidosis increases protein degradation (PD) in skeletal muscle, an effect which in vivo requires glucocorticoid (GC). This disorder is poorly understood, but can be studied in vitro using L6G8C5 rat skeletal muscle cells. Two potential confounding factors in studies of PD in culture are apoptosis and dedifferentiation, both of which resemble catabolic states. The aim of this study was to determine the extent to which these factors contribute to the observed effects of acid and GC on PD. PD was measured in intact cells by pre-labelling cell protein with [(14)C]phenylalanine. Apoptosis was assessed morphologically by staining DNA with Hoechst 33342, by terminal deoxynucleotide transferase-mediated nick-end labelling and by cell-surface binding of Annexin V. Differentiation was assessed morphologically from myotube fusion and from activity of the marker enzyme creatine phosphokinase (CPK). In undifferentiated myoblasts, pH had no detectable effect on apoptosis provided that serum was present and GC (dexamethasone; 5 nmol/l) decreased apoptosis. In spontaneously fused cultures in 2% serum, inhibition of apoptosis with caspase-3 inhibitor (C3I; Ac-Asp-Met-Gln-Asp-CHO; 50 micro mol/l) only decreased PD by 9% at pH 7.4. In contrast, the proteasome inhibitor MG132 decreased PD by 79%. Acid (pH 7.1) increased PD, with no requirement for GC, and this effect was blocked by MG132, but not by C3I. Differentiation was unaffected by 1-4 days of exposure to acid or GC. However, differentiation to myotubes led to decreased sensitivity of PD to acid. This effect of acid was lost completely in highly fused myotubes, but was partly restored by 500 nmol/l dexamethasone. Stimulation of PD in these cells by acid and GC is not an artefact of apoptosis or dedifferentiation, but differentiation state does determine whether PD responds spontaneously to acid or (as in vivo) only does so in the presence of GC.

Enhanced inactivation and pH sensitivity of Na(+) channel mutations causing hypokalaemic periodic paralysis type II.

Hypokalaemic periodic paralysis (hypoPP) is a dominantly inherited muscle disorder characterized by episodes of flaccid weakness. Previous genetic studies revealed mutations in the voltage-gated calcium channel alpha1-subunit (CACNA1S gene) in families with hypoPP (type I). Electrophysiological studies on these mutants in different expression systems could not explain the pathophysiology of the disease. In addition, several mutations (Arg669His, Arg672His, Arg672Gly and Arg672Ser) in the voltage sensor of the skeletal muscle sodium channel alpha-subunit (SCN4A gene) have been found in families with hypoPP (type II). For Arg672Gly/His a fast inactivation defect was described, and for Arg669His an impairment of slow inactivation was reported. Except for the substitution for serine, we have now expressed all mutants in a human cell-line and studied them electrophysiologically. Patch-clamp recordings show an enhanced fast inactivation for all three mutations, whereas two of them reveal enhanced slow inactivation. This may reduce the number of functional sodium channels at resting membrane potential and contribute to the long-lasting periods of paralysis experienced by hypoPP patients. The gating of both histidine mutants (Arg669His, Arg672His) can be modulated by changes of extra- or intracellular pH. The inactivation defects of Arg669His and Arg672His can be alleviated by low pH to a significant degree, suggesting that the decrease of pH in muscle cells (e.g. during muscle work) might lead to an auto-compensation of functional defects. This may explain a delay or prevention of paralytic attacks in patients by slight physical activity. Moreover, the histidine residues may be the target for a potential therapeutic action by acetazolamide.