Biochimica et Biophysica Acta, 1145 (1993) 199-204 199 © 1993 Elsevier Science Publishers B.V. All rights reserved 0005-2736/93/$06.00 BBAMEM 75859 Effects of alkaline pH on the stimulation of glucose transport in rat skeletal muscle Jian-Ming Ren, Jang H. Youn, Eric A. Gulve, Erik J. Henriksen and John O. Holloszy Department of Internal Medicine, Washington Uniuersity School of Medicine, St. Louis, MO (USA)" (Received 7 October 1992) Key words: Epitrochlearis muscle; Hypoxia; Insulin; Muscle contraction; 3-O-Methylglucose Alkaline pH has been reported to cause release of Ca e+ from skeletal muscle sarcoplasmic reticulum (SR). Elevation of sarcoplasmic Ca 2÷ concentration is thought to stimulate glucose transport in skeletal muscle. In this context, we examined the effect of alkaline pH (extracellular pH of 8.6) on 3-O-methylglucose transport in skeletal muscle. Incubation of rat epitrochlearis muscles at pH 8.6 for 45 min resulted in an approx. 3-fold increase in glucose transport activity, which was not affected by reducing Ca z+ concentration in the incubation medium and essentially completely blocked by 25/zM dantrolene, an inhibitor of SR Ca 2÷ release. In addition to stimulating glucose transport by itself, alkaline pH may partially inhibit the stimulation of sugar transport by insulin hypoxia and contractions, as the combined effect of alkaline pH and the maximal effect of insulin, contractions, or hypoxia on glucose transport are not different from the maximal effects of insulin, hypoxia, or contractions alone. The maximal effects of insulin and contractions, and of insulin and hypoxia, on glucose transport are normally additive in muscle. Alkaline pH completely prevented this additivity. In summary, our results show that alkaline pH stimulates glucose transport activity in skeletal muscle and provide evidence suggesting that this effect is mediated by Ca 2÷. They further show that alkaline pH blocks the additivity of the maximal effects of insulin and contractions or hypoxia suggesting that alkaline pH may partially inhibit the stimulation of glucose transport by insulin, contraction and hypoxia. Introduction In skeletal muscle, glucose transport can be acti- vated by insulin, contractile activity, and hypoxia. The maximal effects on glucose transport activity of insulin and contractions [1-4], and of insulin and hypoxia [5] are additive, while the maximal effects of contractions and hypoxia are not additive [5]. These findings have been taken as evidence that contractions and hypoxia stimulate glucose transport by the same pathway, and that insulin and contractile activity/hypoxia stimulate glucose transport by different pathways [1,2,5]. Evidence has accumulated suggesting that an in- crease in cytoplasmic Ca 2+ concentration stimulates glucose transport in skeletal muscle and that this is the mechanism by which muscle contractions and hypoxia induce an increase in glucose transport activity [5-14]. Alkaline pH triggers Ca 2+ release from skeletal muscle sarcoplasmic reticulum [15], and stimulates glucose transport in adipocytes and some cell lines [16-18]. Correspondence to: J.-M. Ren, Department of Internal Medicine, Campus Box 8113, Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110, USA. However, we have not been able to find any informa- tion regarding the effect of alkaline pH on glucose transport in muscle. In this context, the present study was undertaken to determine whether or not alkaline pH stimulates glucose transport in skeletal muscle. Our results show that alkaline pH does increase glucose transport activity in skeletal muscle and sug- gest that this effect is mediated by an increase in cytoplasmic Ca 2+. Methods Animals and muscle preparation Male Wistar rats weighing 100-120 g were obtained from SASCO (Omaha, NE) and fed Purina Chow and water. After an overnight fast, rats were anesthetized with 5 mg/100 g body wt. of pentobarbital sodium injected intraperitoneally, and the epitrochlearis mus- cles [19] were dissected out. Muscle incubations The muscles were incubated in 2 ml of Krebs- Henseleit bicarbonate buffer (KHB) [20] containing 8 mM glucose, 32 mM mannitol, 0.1% bovine serum albumin and the additions indicated for each experi-