Rate Of Intracellular Acidification Research Articles

The effect of bile acids on human pancreatic duct cells

We have previously shown that exposure of intact guinea pig pancreatic ducts to bile acids, which may occur e.g. in acute pancreatitis, induces intracellular acidification and a marked increase in intracellular Ca2+ concentration ([Ca2+]i). Moreover, a low dose of chenodeoxycholate (CDC) stimulates the luminal anion exchanger which suggests a possible role of cystic fibrosis transmembrane conductance regulator (CFTR) in this effect. There are no available data concerning the effect of bile acids on human pancreatic duct cells. The aim of our study was to examine the effects of CDC or glycochenodeoxycholate (GCDC) in human cystic fibrosis CFPAC-1 pancreatic duct cells which are homozygous for the most common delF508 cystic fibrosis transmembrane conductance regulator (CFTR) mutation or in CFPAC-1 cells which are corrected for the defective CFTR. Methods: We transduced polarized CFPAC-1 cells (n=4–6) with recombinant Sendai virus (SeV) constructs (multiplicity of infection=3) containing the CFTR or β-galactosidase (LacZ) cDNA. 48–72h after infection, the intracellular pH (pHi) and [Ca2+]i of the cells were recorded by microfluorimetry. Results: Both apical and basolateral administration of CDC or GCDC caused dose-dependent (100–1000µM) reversible intracellular acidification. Our results did not show significant differences in the rate of intracellular acidification caused by bile acids in SeV-CFTR and SeV-LacZ transduced cells. The exposure of cell lines to bile acids induced a marked increase in [Ca2+]i and CDC caused the largest response. Similarly to that observed in primary ducts, the effect of CDC administration from the apical membrane was greater than from the basolateral membrane. Furthermore, glycine conjugated bile acids also resulted in Ca2+ signalling when administered from either membrane of the cells. Conclusions: Human pancreatic duct cells respond similarly to administration of bile acids compared to that observed in guinea pigs. CFTR expression is not necessary for pHi changes and Ca2+ signalling caused by bile acids.

Contractile properties of skeletal muscle fibre bundles from mice deficient in carbonic anhydrase II

The function of cytosolic carbonic anhydrase (CA) isozyme II is largely unknown in skeletal muscle. Because of this, we compared the in vitro contractile properties of extensor digitorum longus (EDL) and soleus (SOL) fibre bundles from mice deficient in CA II (CAD) to litter mate controls (LM). Twitch rise, 1/2 relaxation time and peak twitch force at 22 degrees C of fibre bundles from CAD EDL [28.4+/-1.4 ms, 31.2+/-2.3 ms, 6.2+/-1.0 Newton/cm(2) (N/cm(2)), respectively] and CAD SOL (54.2+/-7.5 ms, 75.7+/-13.8 ms, 2.9+/-0.5 N/cm(2), respectively) were significantly higher compared to LM EDL (20.5+/-2.2 ms, 21.9+/-3.7 ms, 4.5+/-0.2 N/cm(2)) and LM SOL (42.8+/-3.5 ms, 51.4+/-2.4 ms, 2.1+/-0.4 N/cm(2)). However, in acidic Krebs-Henseleit solution, mimicking the pH, PCO(2), and HCO(3) (-) of arterial blood from CAD mice, twitch rise, 1/2 relaxation time, and peak twitch force of fibre bundles from CAD EDL (19.3+/-0.7 ms, 19.7+/-2.3 ms, 4.8+/-0.8 N/cm(2)) and CAD SOL (41.4+/-3.6 ms, 51.9+/-5.5 ms, 2.2+/-0.7 N/cm(2)) were not significantly different from LM fibre bundles in normal Krebs-Henseleit solution (EDL: 19.7+/-1.1 ms, 21.6+/-0.6 ms, 4.7+/-0.2 N/cm(2); SOL: 42.5+/-3.1 ms, 51.8+/-2.6 ms, 1.8+/-0.3 N/cm(2)). A higher pH(i) during exposure to acidic bathing solution was maintained by CAD EDL (7.37+/-0.02) and CAD SOL (7.33+/-0.05) compared to LM EDL (7.28+/-0.04) and LM SOL (7.22+/-0.02). This suggests that the skeletal muscle of CAD mice possesses an improved defense of pH(i) against elevated pCO(2). In support of this, apparent non-bicarbonate buffer capacity (in mequiv H(+) (pH unit)(-1) (kg cell H(2)O)(-1)) as determined by pH microelectrode was markedly increased in CAD EDL (75.7+/-4.1) and CAD SOL (85.9+/-3.3) compared to LM EDL (39.3+/-4.7) and LM SOL (37.5+/-3.8). Both latter phenomena may be related to the slowed rate of intracellular acidification seen in CAD SOL in comparison with LM SOL upon an increase in PCO(2) of the bath. In conclusion, skeletal muscle from mice deficient in CA II exhibits altered handling of acid-base challenges and shows normal contractile behavior at normal intracellular pH.

Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea-pig pancreatic ducts.

1. HCO3- secretion was investigated in interlobular duct segments isolated from guinea-pig pancreas using a semi-quantitative fluorometric method. Secretagogue-induced decreases in intracellular pH, following blockade of basolateral HCO3- uptake with a combination of amiloride and DIDS, were measured using the pH-sensitive fluoroprobe BCECF. Apparent secretory HCO3- fluxes were calculated from the initial rate of intracellular acidification. 2. In the presence of HCO3-, stimulation with secretin (10 nM) or forskolin (5 microM) more than doubled the rate of intracellular acidification. This effect was abolished in the absence of HCO3-. It was also abolished in the presence of HCO3- when DIDS and NPPB were applied to the luminal membrane by microperfusion. We therefore conclude that the increase in acidification rate is a useful index of secretagogue-induced HCO3- secretion across the luminal membrane. 3. Secretin, cholecystokinin (CCK) and bombesin each stimulated HCO3- secretion in a dose-dependent fashion. They evoked comparable maximal responses at about 10 nM and the EC50 values were 0.5 nM for secretin, 0.2 nM for CCK and 30 pM for bombesin. Acetylcholine (ACh) was also effective, with a maximum effect at 10 microM. 4. The stimulatory effect of CCK was blocked completely by the CCK1 receptor antagonist devazepide but not by the CCK2 receptor antagonist L365,260. The CCK analogue JMV-180 (Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-phenylethyl ester), which is an agonist of the high-affinity CCK1 receptor but an antagonist of the low-affinity receptor, also stimulated HCO3- secretion but with a smaller maximal effect than CCK. JMV-180 partially inhibited the response to a high concentration of CCK but not to a lower concentration, suggesting that both high- and low-affinity states of the CCK1 receptor evoke HCO3- secretion. 5. The stimulatory effect of bombesin was blocked completely by the gastrin-releasing peptide (GRP) receptor antagonist D-Phe6-bombesin(6-13)-methyl ester (BME) but not by the neuromedin B (NMB) receptor antagonist D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH2 (BIM-23127). 6. Secretagogue-evoked fluid secretion was also examined using video microscopy to measure the rate of swelling of ducts whose ends had sealed during overnight culture. Secretin, CCK, bombesin and ACh all evoked fluid secretion with maximal rates of approximately 0.6 nl x min(-1) x mm(-2), and with concentration dependences similar to those obtained for HCO3- secretion. 7. We conclude that CCK, bombesin and ACh stimulate the secretion of a HCO3--rich fluid by direct actions on the interlobular ducts of the guinea-pig pancreas and that these responses are mediated by CCK1 receptors, GRP receptors and muscarinic cholinoceptors, respectively.

Carbon Dioxide Permeability of Aquaporin-1 Measured in Erythrocytes and Lung of Aquaporin-1 Null Mice and in Reconstituted Proteoliposomes

Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.

COMPARISON OF IN VITRO ENZYMATIC ASSAYS WITH IN VIVO 31P MRS OF MUSCLE METABOLISM

1160 The aim of this study was to examine the relationship between noninvasive measures of muscle metabolism, derived with magnetic resonance spectroscopy (31P-MRS), and in vitro measures. 8 healthy subjects underwent 31P-MRS measurements and needle biopsies of the medial gastrocnemius. Spectra were acquired using an oblong surface coil at rest, during plantar flexion and recovery. The PCr recovery rate constant (kPCr = 2.07±0.23 min−1) was determined following submaximal exercise (PCr=48.2±4.0 of initial; pH=6.85±0.03) as an index of the in vivo oxidative capacity. In vivo indices of glycolytic potential were derived based on intracellular pH changes during 60 s of maximal rate exercise (PCr=16.8±2.2 of initial; pH=6.58±0.04; dpH/dt=1.38±0.12 pH units/min). Both quantitative histochemistry and whole muscle homogenate biochemical analysis were used to determine in vitro indices of oxidative (SDH, CS, Type I fibers) and glycolytic (GPD, PFK, GAPDH) capacities. Regression analysis between the PCr resynthesis rate constant, CS, SDH activity and fiber type composition demonstrated only a poor to moderate relationship between in vivo and in vitro measures of oxidative metabolism. The highest correlation was found between kPCr and fiber type composition (r=0.59; p=0.06). In contrast, a strong relationship was found between in vitro glycolytic markers and in vivo pH kinetics during maximal exercise. In particular the PFK activity (r=0.92-0.94; p=0.01 − p<0.001) and ratio of SDH-to-GPD (r=−0.86; p=0.03) showed a strong relationship with the level and rate of intracellular acidification during high intensity exercise. In conclusion, our results indicate a strong relationship between in vitro and in vivo measures of glycolysis and a relatively moderate relationship between in vivo and in vitro markers of oxidative phosphorylation. This information is essential if muscle metabolic data is desired in populations where needle biopsies are contraindicated. This work was supported by NIH HD 33738

Electrogenic sodium-dependent bicarbonate secretion by glial cells of the leech central nervous system.

The ability to move acid/base equivalents across the membrane of identified glial cells was investigated in isolated segmental ganglia of the leech Hirudo medicinalis. The intracellular pH (pHi) of the glial cells was measured with double-barreled, neutral-ligand, ion-sensitive microelectrodes during step changes of the external pH (pHo 7.4-7.0). The rate of intracellular acidification after the decrease in extracellular pH (pHo) was taken as a measure of the rate of acid/base transport across the glial membrane. Taking into account the total intracellular buffering power, the maximum rate of acid/base flux was 0.4 mM/min in CO2/HCO3-free saline, and 3.92 mM/min in the presence of 5% CO2/10 mM HCO-3, suggesting that the acid/base flux was dependent upon HCO3-. The rate of acid influx/base efflux increased both with the external HCO3- concentration and with increasing pHi (and hence HCO3-i). This suggested that the decrease in pHi was due to HCO3- efflux. The rapid decrease of pHi was accompanied by a HCO3--dependent depolarization of the glial membrane from -74 +/- 5 mV (n = 20) to -54 +/- 7 mV (n = 13). Both this depolarization and the rate of intracellular acidification were greatly reduced by the anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.3-0.5 mM), but were not affected by the removal of external Cl-. Reduction of the external Na+ concentration to one-tenth normal affected the rate of intracellular acidification only in the presence of CO2/HCO3-: the rate increased within the first 3-5 min after lowering external Na+; after longer exposures in low external Na+ the rate decreased, presumably due to depletion of intracellular Na+. Amiloride (1 mM), which inhibits the Na+-H+ exchange in these cells, had no effect on the rate of intracellular acidification. The intracellular Na activity (aNai) of the glial cells was measured to be 5.2 +/- 1.0 mM (n = 8) in CO2/HCO3-free saline; aNai increased to 7.3 +/- 2.2 mM (n = 8) after the addition of 5% CO2/24 mM HCO3-. Upon a change in pHo to 7.0 in the presence of CO2/HCO3-, aNai decreased by an average of 2 +/- 1.1 mM (n = 5); in CO2/HCO3--free saline external acidification produced a transient increase in aNai. It is concluded that, in the presence of CO2/HCO3-, the rate of intracellular acidification in glial cells is dominated by an outwardly directed, electrogenic Na+-HCO3-cotransport. Neurons, which do not possess this cotransporter, acidify at much lower rates under similar conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular acidosis of identified leech neurones produced by substitution of external sodium

The intracellular pH, pH i, of identified neurones of the central nervous system of the leech Hirudo medicinalis L. was measured with double-barrelled neutral carrier pH-sensitive microelectrodes. The active regulation of pH i of these neurons is due to amiloride-sensitive Na H exchange and hence requires extracellular Na, Na o. We have measured a decrease of pH i following the removal of Na o. The rate of intracellular acidification in Na-free saline was similar to that in the presence of 2 mM amiloride suggesting that the acidification was due to inhibition of the Na H exchange. The rate of intracellular acidification depended on the Na substitute chosen; it was 0.02 ± 0.005 pH units/min (±S.D., n = 17) when Na was replaced by N-methyl- d-glucamine. A similar rate of acidification occurred with tris-hydroxymethyl-aminomethane (Tris) while the rate of acidification was higher with bis-2-hydroxymethyl-dimethyl-ammonium (BDA, 0.033 ± 0.016 pH units/min (±S.D., n = 7) and tetramethylammonium TMA, 0.046 ± 0.017 pH units/min ( n = 3) as Na substitutes. A high, non-linear rate of intracellular acidification was observed, when Li, K or choline were used as Na substitute. The recovery of pH i from acidification upon readdition of Na o was fast, only when Li had replaced Na was the pH i recovery considerably delayed. In conclusion, in all experiments using different Na substitutes the removal of Na o caused a ssubstantial intracellular acidification presumably due to inhibition of Na H exchange. These changes in pH i might be relevant for results obtained by experiments in which Na-free solutions are used.

Influence of organic acids on intracellular pH.

By use of double-barreled pH-sensitive microelectrodes, intracellular pH was measured in isolated sheep cardiac Purkinje strands. After equimolar substitution of 20 mmol/l Cl- by several organic anions at constant extracellular pH 6.8, the rate of induced intracellular acidification was measured. For many organic acids tested, a relation was found between the rate of intracellular acidification and the product of their dissociation constant (pK'a) and diisopropylether-to-water partition ratio (p'). L-Lactate and pyruvate, and also cyanoacetate and alpha-ketobutyrate, caused faster acidifications than anticipated from their pK'a and p'. The rate of intracellular acidification, induced by L-lactate and pyruvate, was markedly depressed in the presence of 4 mmol/l alpha-cyano-4-hydroxycinnamate, a known inhibitor of the carrier-mediated pyruvate transport. The drug also had an effect on the acidification produced by cyanoacetate, alpha-ketobutyrate, glycolate, alpha-hydroxybutyrate, and alpha-chloropropionate, but not on that produced by propionate and acetate. L-Lactate caused a faster acidification than D-lactate. Our results suggest the existence of a facilitated diffusion for L-lactate, pyruvate, and some other organic acids in sheep Purkinje cells.