P S Haddock1. 1. Rayne Institute, St. Thomas' Hospital, London, UK.
Abstract
OBJECTIVES: Concentrative transport systems for nucleobases have been identified in renal epithelial cells and jejunal tissue rings. However, the existence and role of nucleobase transport has yet to be described in the heart. The aims of this study were, therefore, to use isolated guinea pig myocytes to: (i) identify whether such a mechanism is present in the heart and (ii) characterise and compare its activity to that already described in other tissues. METHODS: The Na(+)-dependent and Na(+)-independent uptake of [3H]hypoxanthine was quantified in ventricular myocytes. These were isolated from the hearts of adult guinea pigs using a collagenase-based enzymatic digestion technique. The specific incorporation of cellular radioactivity, in the presence and absence of extracellular sodium and other pharmacological agents, was determined by liquid scintillation counting. RESULTS: A system that accumulates hypoxanthine against a concentration gradient in the presence of extracellular sodium was identified. Furthermore, a sodium-independent component of hypoxanthine uptake was also characterised, a proportion (approximately 50%) of which was inhibited by adenine (3 mmol/l). In the absence of sodium and the presence of adenine, the remaining fraction of hypoxanthine uptake was assumed to occur via diffusion. The sodium-dependent uptake of hypoxanthine was saturable at 25 degrees C with a Km of 8.1 +/- 2.1 mumol/l and a Vmax of 50.1 +/- 5.2 pmol/mg protein/min. Dipyridamole inhibited sodium-dependent uptake but had no effect on sodium-independent flux. The Na(+)-independent system was saturable with a Km of 157.3 +/- 6.2 mumol/l and a Vmax of 233.6 +/- 5.6 pmol/mg protein/min. Extracellular Ni2+ stimulated sodium-dependent uptake in a concentration-dependent manner (0-100 mEq/l). Another divalent cation (Ba2+, 100 mEq/l) had no effect on the either Na(+)-dependent or the Na(+)-independent component. CONCLUSIONS: These data provide the first evidence for sodium-dependent nucleobase co-transport in guinea pig myocytes. The sensitivity of this transporter to Ni2+ has not been described previously and may provide a pharmacological tool to study this uptake mechanism further.
OBJECTIVES: Concentrative transport systems for nucleobases have been identified in renal epithelial cells and jejunal tissue rings. However, the existence and role of nucleobase transport has yet to be described in the heart. The aims of this study were, therefore, to use isolated guinea pig myocytes to: (i) identify whether such a mechanism is present in the heart and (ii) characterise and compare its activity to that already described in other tissues. METHODS: The Na(+)-dependent and Na(+)-independent uptake of [3H]hypoxanthine was quantified in ventricular myocytes. These were isolated from the hearts of adult guinea pigs using a collagenase-based enzymatic digestion technique. The specific incorporation of cellular radioactivity, in the presence and absence of extracellular sodium and other pharmacological agents, was determined by liquid scintillation counting. RESULTS: A system that accumulates hypoxanthine against a concentration gradient in the presence of extracellular sodium was identified. Furthermore, a sodium-independent component of hypoxanthine uptake was also characterised, a proportion (approximately 50%) of which was inhibited by adenine (3 mmol/l). In the absence of sodium and the presence of adenine, the remaining fraction of hypoxanthine uptake was assumed to occur via diffusion. The sodium-dependent uptake of hypoxanthine was saturable at 25 degrees C with a Km of 8.1 +/- 2.1 mumol/l and a Vmax of 50.1 +/- 5.2 pmol/mg protein/min. Dipyridamole inhibited sodium-dependent uptake but had no effect on sodium-independent flux. The Na(+)-independent system was saturable with a Km of 157.3 +/- 6.2 mumol/l and a Vmax of 233.6 +/- 5.6 pmol/mg protein/min. Extracellular Ni2+ stimulated sodium-dependent uptake in a concentration-dependent manner (0-100 mEq/l). Another divalent cation (Ba2+, 100 mEq/l) had no effect on the either Na(+)-dependent or the Na(+)-independent component. CONCLUSIONS: These data provide the first evidence for sodium-dependent nucleobase co-transport in guinea pig myocytes. The sensitivity of this transporter to Ni2+ has not been described previously and may provide a pharmacological tool to study this uptake mechanism further.