INTERACTIONS BETWEEN OXOGLUTARATE OXIDATION AND ACID SECRETION IN ISOLATED RABBIT GASTRIC GLANDS

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1 Experimental Physiology (1 995), 80, Printed in Great Britain INTERACTIONS BETWEEN OXOGLUTARATE OXIDATION AND ACID SECRETION IN ISOLATED RABBIT GASTRIC GLANDS JESUS CHACIN AND ILEANA HERNANDEZ Laboratorio de Investigaciones Gastrointestinales, Instituto de Investigaciones Biol6gicas, Facultad de Medicina, Universidad del Zulia, Apartado Postal 526, Maracaibo, Venezuela (MANUSCRIPT RECEIVED 11 OCTOBER ACCEPTED 9 DECEMBER 1994) SUMMARY The effects of gastric secretagogues, and other agents that modify H+,K+-ATPase activity and cell calcium concentration, on the rate of oxoglutarate oxidation were investigated in isolated gastric glands. Oxoglutarate was oxidized in a dose-dependent manner by gastric glands, with an apparent Km for oxoglutarate of mm. Oxoglutarate progressively inhibited the rate of glucose oxidation. In the presence of 0 5 mm oxoglutarate plus 10 mm glucose, the latter substrate was preferentially oxidized and contributed most to oxygen uptake. With 10 mm oxoglutarate plus 10 mm glucose, the rate of glucose oxidation was greatly inhibited and oxoglutarate oxidation accounted for most of the oxygen consumption. Acid secretion (aminopyrine accumulation) was significantly increased by 0.1 mm histamine in glands oxidizing 10 mm oxoglutarate, although this stimulation was significantly lower than that observed in the presence of 0-5 mm oxoglutarate plus 10 mm glucose. Omeprazole, an inhibitor of the H+,K+-ATPase, significantly reduced the oxidation of oxoglutarate, whereas NH4+, an activator of the enzyme, stimulated the oxidation of a submaximal dose of oxoglutarate. Carbachol at 0.1 mm significantly increased the rate of oxidation of non-saturating concentrations of oxoglutarate. The calcium ionophore ionomycin at 10 /M produced a similar effect. Chelation of intracellular calcium by BAPTA AM caused a significant inhibition of oxoglutarate oxidation. The results provide further evidence that changes in the ATP: ADP ratio resulting from activation of the H+,K+-ATPase, and calcium ions are involved in the mechanisms of activation of oxidative metabolism in the parietal cell. INTRODUCTION The process of acid secretion by the oxyntic cell is highly dependent on the rate of oxidative metabolism that supplies the necessary energy (ATP) for an optimal functioning of the gastric H+,K+-ATPase, the final biochemical step of the H+ secretory process (Sachs, Munson, Hall & Hersey, 1990). The stimulation of the rate of H+ secretion by gastric secretagogues is followed by activation of oxidative metabolism to meet the enhanced energy demand. The mechanisms of this metabolic activation have not been clarified. Historically, this stimulation of energy metabolism was thought to be achieved mainly by passive means, whereby stimulation of the gastric ATPase and ATP utilization would lead to an increase in ADP concentration (or a fall in phosphorylation potential, ATP/(ADP + PF)) in the cytosol and subsequently in the mitochondria. Within mitochondria, a fall in the ATP: ADP ratio will result in an increased flow of electrons along the respiratory chain and increased oxygen consumption. However, recent evidence has suggested that alternative direct mechanisms may be involved, such as activation by second messengers of some metabolic enzymes, i.e. dehydrogenases (Subero, Lobo & Chacin, 1989; Hernindez & Chacin, 1994).

2 442 J. CHACiN AND 1. HERNANDEZ It is currently accepted that calcium acts as a second messenger in the mechanism of parietal cell activation induced by some gastric secretagogues, such as cholinergic agonists and gastrin (Negulescu & Machen, 1988; Delvalle, Tsunoda, Williams & Yamada, 1992). Stimulation by these secretagogues is accompanied by an increase in cytosolic calcium concentration ([Ca2+]e) (Negulescu & Machen, 1988; Delvalle et al. 1992). Some recent findings have led to the hypothesis that the increases in [Ca2+]0 could be relayed into the mitochondria, resulting in the activation of several key dehydrogenases, and probably other steps, within the process of oxidative phosphorylation (Hernandez & Chacin, 1994). There is now a considerable body of evidence in favour of this mechanism in different mammalian cells that are activated by hormones and other agents to produce increases in [Ca2+]0 (Denton & McCormack, 1990). The Krebs cycle is a major source of energy in animal cells and represents a common final pathway where different substrates (carbohydrates, fatty acids and amino acids) are finally oxidized. Previous findings have suggested that Krebs cycle activity plays a central role in the regulation of oxidative metabolism and acid secretion in the gastric mucosa (Chacin, Rinc6n, Inciarte, Cafiizales, Martinez & Alonso, 1979). The oxoglutarate dehydrogenase (OGDH) enzyme that catalyses the oxidation of oxoglutarate is one of the key dehydrogenases of the Krebs cycle. This pathway is one of the key sites of production of NADH and is considered to be an important regulatory site of oxidative metabolism. OGDHis a complex enzyme subjected to regulation by changes in the ratios of NADH: NAD+, ATP: ADP and succinyl coenzyme A: coenzyme A. In particular, OGDH is one of the mitochondrial dehydrogenases that are activated by physiological calcium concentrations in a variety of tissues (Denton & McCormack, 1986, 1990; Hernandez & Chacin, 1994). All of the above leads us to study the oxidation of oxoglutarate in isolated rabbit gastric glands and, in particular, to investigate the influence of gastric secretagogues and other agents that affect cell calcium concentration. METHODS Chemicals All chemicals were of high reagent grade and purchased from Sigma (USA), unless otherwise indicated. The acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA AM) was obtained from Molecular Probes (USA). Omeprazole was synthesized at Hiissle (Molndal, Sweden). ['4C]Glucose (uniformly labelled), [1-14C]oxoglutarate and ['4C]aminopyrine were purchased from New England Nuclear (USA). Animals Male albino rabbits weighing kg were used. All experiments and surgical procedures using rabbits conformed to guidelines established by legal requirements in the UK for the proper care and use of laboratory animals. Isolation of gastric glands Gastric glands were isolated from the fundus of the rabbit stomach as previously described by Berglindh & Obrink (1976). Briefly, rabbits were killed by cervical fracture/dislocation, and the stomachs perfused under high pressure with phosphate-buffered saline (PBS). After perfusion, the stomachs were rapidly removed, cut open along the lesser curvature and rinsed with PBS. Minced mucosa from the fundus of the stomach was then subjected to digestion with collagenase. Gastric glands were finally suspended (2-6 mg ml-') in incubation medium containing (mm): NaCl, 5 4 KCl, 5 Na2HPO4, 1 NaH2P04, 1.2 MgSO4, 1 CaCl2. 05 dithiothreitol, 10 glucose, 2 mg ml-' bovine serum albumin, ph 7 4.

3 OXOGLUTARATE METABOLISM IN GASTRIC GLANDS 443 Oxygen uptake The rate of 02 consumption was measured in a Gilson respirometer using 1-2 ml of gland suspension, as previously described (Chacin, Martinez & Severin, 1980). Oxidation of glucose and oxoglutarate The rates of oxidation of glucose and oxoglutarate were estimated by measuring the production of '4C02 from [14C]glucose and ['4C]oxoglutarate, respectively, as previously reported (Chacin et al. 1980). Acid secretion This parameter was indirectly estimated by measuring the accumulation of the weak base [14C]aminopyrine (AP), as previously described (Berglindh, Helander & Obrink, 1976). This accumulation is expressed as the ratio (AP ratio) of label in the glands divided by that remaining in the medium. Therefore, an increase in the AP ratio is interpreted as an increase in acid secretion by the glands. Statistical analysis Results are expressed as means + S.E.M. Statistical significance was determined by Student's paired t test. Differences were considered statistically significant when P < RESULTS Effect of oxoglutarate concentration on its rate of oxidation Oxoglutarate was oxidized by isolated rabbit gastric glands in a dose-dependent fashion. The concentration-response curve (Fig. 1) was complex, tending to be sigmoidal and to reach a maximum at an oxoglutarate concentration of about 10 mm. The half-maximal rate of ;,100 X 80- = 60- r0 0, 40- o [Oxoglutarate] (mm) Fig. 1. Oxidation of oxoglutarate plotted vs. oxoglutarate concentration in isolated rabbit gastric glands. Values are means of seven experiments and vertical bars represent one S.E.M. Oxoglutarate oxidation was estimated by measuring the production of 14CO2 from [L4C]oxoglutarate as described in the Methods.

4 444 J. CHACIN AND I. HERNANDEZ Table 1. Effect of oxoglutarate on the rate of glucose oxidation in isolated rabbit gastric glands Condition Glucose oxidation (nmol h-' (mg dry wt)-') Control Oxoglutarate (0.5 mm) * Oxoglutarate (10 mm) * Values are means + S.E.M. of five different preparations. The rate of glucose (10 mm) oxidation was estimated by measuring the production of 14Co2 from [14C]glucose, as described in the Methods, in the absence and presence of two different concentrations of oxoglutarate. * P < 0.05 vs. controls by Student's paired t test. Table 2. Effect of histamine on acid secretion in the presence of oxoglutarate in isolated rabbit gastric glands AP ratio Oxoglutarate Oxoglutarate Condition (0.5 mm) (10 mm) Control 14.6± Histamine (0. 1 mm) * *t Values are means + S.E.M. of six different preparations. Acid secretion was estimated by measuring the accumulation of ['4C]aminopyrine (AP ratio) as described in the Methods. Glucose was present at 10 mm. * P < 0 05 vs. respective controls. t P < 0-05 vs. histamine with 0.5 mm oxoglutarate. oxidation (Vmax/2) occurred approximately with an oxoglutarate concentration of mm (Km). The contribution of oxoglutarate oxidation to the total 02 consumption by gastric glands was dependent on the concentration of oxoglutarate. In the presence of 10 mm glucose and 0 5 mm oxoglutarate, glucose was preferentially oxidized and accounted for a major fraction of the total 02 uptake (8 + 1 sl h-1 (mg dry wt)-1, n = 7), while the contribution of oxoglutarate oxidation was < 10%. Oxoglutarate progressively inhibited glucose oxidation. In the presence of equimolar concentrations (10 mm glucose and 10 mm oxoglutarate), glucose oxidation was drastically inhibited and most (> 80 %) of the total 02 consumption (12 + 3,ul h- (mg dry wt)-', n = 7) was accounted for by the oxidation of oxoglutarate. The effect of oxoglutarate concentration on the rate of glucose oxidation is shown in Table 1. Oxoglutarate as an energy source for acid secretion The efficiency of oxoglutarate as an energy source for acid secretion was investigated by studying the effect of a maximal dose of histamine on acid secretion (AP ratio) in tissue oxidizing mostly glucose and in tissue oxidizing mostly oxoglutarate (Table 2). Basal AP ratio was lower in the presence of a saturating concentration of oxoglutarate (10 mm) compared with that measured in the presence of a low oxoglutarate concentration (0-5 mm) and 10 mm glucose. Addition of 0.1 mm histamine significantly increased AP ratio under both conditions, although the magnitude of the increment was significantly lower in the presence of 10 compared with 0 5 mm oxoglutarate. These results suggest that glucose is

5 OXOGLUTARATE METABOLISM IN GASTRIC GLANDS 445 Table 3. Effects of omeprazole and NH4+ on oxoglutarate oxidation in isolated rabbit gastric glands Oxoglutarate oxidation (% of control) Oxoglutarate Oxoglutarate Condition (0-5 mm) (10 mm) Control Omeprazole (0.1 mm) * * NH4+ (30 mm) * (n.s.) Values are means + S.E.M. of 5-7 different preparations. The rate of oxoglutarate oxidation was estimated by measuring the production of "4CO2 from ['4C]oxoglutarate, as described in the Methods. Glucose was present at 10 mm. Control values (100%) were: nmol h-' (mg dry wt)-' for 0-5 mm oxoglutarate and nmol h-' (mg dry wt)-' for 10 mm oxoglutarate. * P < 0-05 vs. respective controls by Student's paired t test. n.s., not significant. much more efficient than oxoglutarate in supporting the rate of acid secretion in isolated rabbit gastric glands. Possibly, the rate of energy supply to the acid secretory machinery in the presence of 10 mm oxoglutarate is not enough to support an optimal rate of H+ secretion. This view is in accordance with the observation that the rate of 02 consumption induced by 0-1 mm histamine in the presence of 10mm glucose plus 05 mm oxoglutarate ( #l h-' (mg dry wt)-1, n = 7) was significantly higher than that obtained in the presence of 10 mm glucose plus 10 mm oxoglutarate ( ,ul h-' (mg dry wt)-1, n = 7). Interactions between H+,K+-ATPase activity and oxoglutarate oxidation Since the oxidation of oxoglutarate by OGDH may be affected by changes in the ATP: ADP ratio, it was relevant to study the relationship between oxoglutarate oxidation and gastric ATPase function. This was done by examining the effects of agents that directly affect the activity of the H+,K+-ATPase on the rate of oxoglutarate oxidation. Incubation with the ATPase inhibitor omeprazole at 0.1 mm caused a significant reduction in the rate of oxoglutarate oxidation, which was clearly evident in tissue oxidizing 10 mm oxoglutarate (Table 3). On the other hand, incubation with 30 mm NH4, a direct activator of the H+,K+- ATPase (Lorentzon, Sachs & Wallmark, 1988), caused a significant stimulation of the rate of oxidation of submaximal doses of oxoglutarate. This effect of NH + was not seen in the presence of saturating concentrations of oxoglutarate (Table 3). The above results seem to suggest that the rate of oxoglutarate oxidation is coupled to the function of the H+,K+- ATPase. This interaction is most likely to be mediated through changes in the ATP: ADP ratio that may affect mitochondrial respiration and OGDH activity. Effects of agents that alter [Ca2+], on oxoglutarate oxidation Carbachol (0.1 mm), a gastric secretagogue that acts through an increase in [Ca2+], (Negulescu & Machen, 1988), produced a significant stimulation of the rate of oxoglutarate oxidation. This effect was only observed when using non-saturating concentrations of oxoglutarate (Table 4). A similar stimulation of the rate of oxoglutarate oxidation was observed with the addition of 10 /LM ionomycin, a calcium ionophore that also acts by increasing [Ca2+]1 (not shown). On the other hand, incubation of gastric glands with the intracellular calcium chelator BAPTA AM at 50 /tm caused a significant reduction in the rate of oxidation of 10 mm oxoglutarate, from to nmol h-' (mg dry wt)-1

6 446 J. CHACIN AND I. HERNANDEZ Table 4. Effect of 0.1 mm carbachol on the rate of oxoglutarate oxidation in isolated rabbit gastric glands Oxoglutarate oxidation (nmol h-' (mg dry wt)-') [Oxoglutarate] (mm) Control Carbachol * * 05 45± * * n.s. Values are means S.E.M. of 3-8 different preparations. The rate of oxoglutarate oxidation was estimated by measuring the production of 4CO2 from ['4C]oxoglutarate using different substrate concentrations. Glucose was present at 10 mm. * P < 0 05 vs. respective controls by Student's paired t test. n.s., not significant. (n = 6, P < 0.05). Incubation with the extracellular calcium chelator EGTA (1 mm) did not significantly affect the rate of oxoglutarate oxidation. DISCUSSION The present work shows that oxoglutarate is efficiently oxidized by isolated rabbit gastric glands and that the rate of this oxidation is coupled to the function of the H+,K+-ATPase and hence to the process of HCl secretion. These results are in agreement with previous findings showing that the production of 14Co2 is a function of the rate of turnover of the gastric H+,K+-ATPase (Fryklund, Gedda, Scott, Sachs & Wallmark, 1990). The kinetics of oxoglutarate oxidation in intact gastric glands (Fig. 1) was somewhat different from that obtained in isolated mitochondria, where a kinetics close to hyperbolic has been observed (Denton, McCormack & Edgell, 1980). However, the apparent Km for oxoglutarate in isolated gastric glands, 3.9 mm, was close to that obtained in isolated mitochondria (-2-3 mm) (Denton et al. 1980). The differences in the kinetics of oxoglutarate oxidation might be related to different permeability rates in the process of penetration of oxoglutarate into the cells. In the isolated toad gastric mucosa, cell permeability is a ratelimiting factor for oxoglutarate oxidation, and lowering the nutrient ph from 7.4 to 5 0 facilitates penetration and oxidation (Chacin et al. 1979). Oxoglutarate progressively reduced the rate of glucose oxidation (Table 1), so that in the presence of equimolar concentrations (10 mm), oxoglutarate was preferentially oxidized and accounted for most of the 02 uptake, while the rate of glucose oxidation was drastically reduced. This inhibitory effect of oxoglutarate on glucose oxidation could be explained by changes in certain regulatory factors of carbohydrate metabolism, such as citrate concentration, coenzyme A concentration, NADH: NAD* and ATP: ADP ratios. The finding that histamine significantly stimulated acid secretion (AP ratio) in glands oxidizing mostly oxoglutarate (Table 2) suggests that this substrate may serve as an energy source for the process of H+ secretion. However, the stimulating effect of histamine on AP ratio was significantly greater in glands oxidizing mostly glucose (10 mm glucose plus 0-5 mm oxoglutarate), indicating that glucose is much more efficient than oxoglutarate in supporting the rate of acid secretion in isolated rabbit gastric glands. Presumably, the rate of

7 OXOGLUTARATE METABOLISM IN GASTRIC GLANDS energy supply (ATP) to the acid secretory machinery in the presence of 10 mm oxoglutarate is not enough to support an optimal rate of H+ secretion. In a number of animal species, carbohydrates and fatty acids seem to be the main energy sources for acid secretion (Alonso, Nigon, Dorr & Harris, 1967; Chacin, Prieto & Cardenas, 1985). The following findings suggest that the rate of oxoglutarate oxidation may be coupled to the function of the H+,K+-ATPase and the process of H+ secretion. First, omeprazole, a specific inhibitor of the proton pump, caused a significant reduction in the rate of oxoglutarate oxidation (Table 3); and second, addition of NH4', a direct activator of the gastric ATPase (Lorentzon et al. 1988), produced a significant stimulation of the rate of oxidation of submaximal concentrations of oxoglutarate (Table 3). It is likely that the interaction between H+,K+-ATPase activity and oxoglutarate oxidation is accomplished through changes in the ATP: ADP ratio, which is a known regulatory factor of the mitochondrial respiratory chain and OGDH activity. This mechanism of coupling would ensure an adequate rate of oxidative metabolism to satisfy the enhanced energy demand. The following results also indicate that the oxidation of oxoglutarate may be affected by changes in [Ca2"],. First, the addition of carbachol, a gastric secretagogue that acts through an increase in [Ca2t]0 (Negulescu & Machen, 1988; Delvalle et al. 1992), stimulated the rate of oxoglutarate oxidation (Table 4). This effect was only observed with non-saturating concentrations of oxoglutarate, which is in accordance with the known fact that calcium activates OGDH by lowering the apparent Km for oxoglutarate without altering the Vmax (Denton & McCormack, 1986, 1990; McCormack, Halestrap & Denton, 1990). Second, ionomycin, a calcium ionophore that also acts by increasing [Ca2t]c. stimulated the oxidation of submaximal concentrations of oxoglutarate. Third, chelation of intracellular calcium by incubation with BAPTA AM caused a significant reduction in the rate of oxoglutarate oxidation. All these findings support the view that calcium plays a role in the regulation of oxidative metabolism in gastric glands. Our results are in agreement with those previously found in the isolated amphibian gastric mucosa (Subero et al. 1989) and isolated gastric glands (Hernandez & Chacin, 1994) that showed that carbohydrate oxidation is dependent on calcium concentration. An important question, which remains unanswered, is that of the mechanisms whereby calcium may activate oxoglutarate oxidation in isolated gastric glands. On the basis of present and other recent findings (Hernandez & Chacin, 1994), two possibilities should be considered: (1) calcium may have an indirect effect through the activation of the H+,K+- ATPase and the resulting increase in ATP utilization. A decrease in the ATP: ADP ratio will result in stimulation of the mitochondrial respiratory chain and OGDH activity; and (2) the increase in [Ca2t]c could be relayed into the mitochondria, resulting in activation of Ca2t_ sensitive dehydrogenases, such as OGDH. There is now substantial evidence from a variety of sources suggesting that many hormones and other agents which activate energy-requiring processes by causing increases in [Ca2t]c may stimulate oxidative metabolism through parallel increases in mitochondrial calcium and activation of Ca2t-sensitive mitochondrial dehydrogenases (Denton & McCormack, 1986, 1990; Hansford, 1987; McCormack et al. 1990; Duchen, Peuchen & Nowicky, 1993). The above-mentioned mechanisms may be cooperative and may act synergistically to ensure an efficient stimulus-metabolism-secretion coupling, so that the rate of energy supply will meet the enhanced energy demand. In conclusion, our present results provide further evidence that changes in the ATP: ADP ratio resulting from activation of the Ht,K+-ATPase, and calcium ions are key factors in regulating the rate of oxidative metabolism in the parietal cell. 447

8 448 J. CHACIN AND 1. HERNANDEZ We wish to thank M. Molero and M. Giuffrida for their collaboration in this work. We thank P. Cardenas and P. Lobo for their technical assistance, and G. Sandoval, R. Caspersen, A. Casanova and M. Pires for their assistance in preparing this manuscript. This investigation was supported in part by Consejo de Desarrollo Cientifico y Humanistico de la Universidad del Zulia and CONICIT (S1-2408), Venezuela. REFERENCES ALONSO, D., NIGON, K., DORR, I. & HARRIS, J. B. (1967). Energy sources for gastric secretion: Substrates. American Journal of Physiology 212, BERGLINDH, T., HELANDER, H. F. & OBRINK, K. J. (1976). Effects of secretagogues on oxygen consumption, aminopyrine accumulation, and morphology in isolated gastric glands. Acta Physiologica Scandinavica 97, BERGLINDH, T. & OBRINK, K. J. (1976). A method for preparing isolated gastric glands from rabbit gastric mucosa. Acta Physiologica Scandinavica 96, CHACiN, J., MARTiNEZ, G. & SEVERIN, E. (1980). Role of fatty acid oxidation in mechanism of action of gastric secretagogues. American Journal of Physiology 238, G CHACiN, J., PRIETO, A. & CA&RDENAS, P. (1985). Substrate-level energy dependence of acid secretion in the isolated human gastric mucosa. Gastroenterology 89, CHACIN, J., RINc6N, R., INCIARTE, I., CANIZALES, A., MARTiNEZ, G. & ALONSO, D. (1979). Effect of Krebs cycle intermediates and inhibitors on toad gastric mucosa. American Journal of Physiology 236,E DELVALLE, J., TSUNODA, Y., WILLIAMS, J. A. & YAMADA, T. (1992). Regulation of [Ca+ ]i by secretagogue stimulation of canine gastric parietal cells. American Journal of Physiology 262, G DENTON, R. M. & MCCORMACK, J. G. (1986). The calcium sensitive dehydrogenases of vertebrate mitochondria. Cell Calcium 7, DENTON, R. M. & MCCORMACK, J. G. (1990). Ca2' as a second messenger within mitochondria of the heart and other tissues. Annual Review of Physiology 52, DENTON, R. M., MCCORMACK, J. G. & EDGELL, N. J. (1980). Role of calcium ions in the regulation of intramitochondrial metabolism. Effects of Na+, Mg2+ and ruthenium red on the Ca2 -stimulated oxidation of oxoglutarate and on pyruvate dehydrogenase activity in intact rat heart mitochondria. Biochemical Journal 190, DUCHEN, M. R., PEUCHEN, S. & NowICKY, A. (1993). Changes in mitochondrial function in response to changes in cytosolic Ca2+ concentration. Biomedical Research 14, suppl. 2, FRYKLUND, J., GEDDA, K., Scorr, D., SACHS, G. & WALLMARK, B. (1990). Coupling of H',K -ATPase activity and glucose oxidation in gastric glands. American Journal of Physiology 258, G HANSFORD, R. G. (1987). Relation between cytosolic free Ca2+ concentration and the control of pyruvate dehydrogenase in isolated cardiac myocytes. Biochemical Journal 241, HERNANDEZ, I. & CHACiN, J. (1994). Mechanism of cholinergic stimulation of glucose oxidation in isolated gastric glands. American Journal of Physiology 267, G LORENTZON, P., SACHS, G. & WALLMARK, B. (1988). Inhibitory effects of cations on the gastric H,K - ATPase. Journal of Biological Chemistry 263, MCCORMACK, J. G., HALESTRAP, A. P. & DENTON, R. M. (1990). Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiological Reviews 70, NEGULESCU, P. A. & MACHEN, T. E. (1988). Intracellular Ca regulation during secretagogue stimulation of the parietal cell. American Journal of Physiology 254, C SACHS, G., MUNSON, K., HALL, K. & HERSEY, S. J. (1990). Gastric H+,K+-ATPase as a therapeutic target in peptic ulcer disease. Digestive Diseases and Sciences 35, SUBERO, O., LOBO, P. & CHACIN, J. (1989). Ca2+ requirement for metabolic effects of secretagogues in the amphibian gastric mucosa. American Journal of Physiology 257, G