Transport of (22)Na(+) and( 45)Ca(2+) by Xenopus laevis oocytes expressing mRNA from lobster hepatopancreas.
This paper describes the development of a functional assay system to express crustacean epithelial electrogenic 2Na(+)/1H(+) antiporters in Xenopus laevis oocytes. Subsequent publications will use this assay method to establish nucleotide and amino acid sequence information about this transporter by functionally screening an hepatopancreatic cDNA library. In this method, oocytes were injected with hepatopancreatic mRNA (50 ng) isolated from Homarus americanus, while control oocytes received injections of an equivalent volume of distilled water. Three to five days post-injection, oocytes were incubated in media containing either (22)Na(+) or (45)Ca(2+) for specific time intervals and the rates of ion transfer into the oocytes were monitored under a variety of experimental conditions. Uptakes of both radiolabelled cations were stimulated by mRNA injection. mRNA-stimulated (22)Na(+) uptake was significantly (P < 0.05) inhibited by addition of calcium, amiloride, or by an antiporter-specific monoclonal antibody to the external medium. mRNA-stimulated (45)Ca(2+) uptake was significantly (P < 0.05) inhibited by addition of sodium, amiloride, cadmium, zinc, or by the antiporter-specific monoclonal antibody (also inhibitory for (22)Na(+) transport) to the external medium. The kinetics of (22)Na(+) influx in mRNA-injected oocytes were sigmoidal functions of external sodium concentration, exhibiting a Hill Coefficient (n) of approximately 3.0. Both calcium and amiloride significantly (P < 0.05) reduced sigmoidal sodium influx kinetics by alterations in the J(max) (amiloride) or K(Na) (calcium) of the transporter. Size fractionation of hepatopancreatic mRNA resulted in a single fraction that was most stimulatory for sodium and calcium transport and which likely contains the antiporter transcript. The results of this study provide the basis for using (22)Na(+) and (45)Ca(2+) transport assays of lobster mRNA-injected oocytes to functionally screen an hepatopancreatic cDNA library for clones that will provide full length nucleotide and amino acid sequences of the invertebrate electrogenic 2Na(+)/1H(+) antiporter protein.
Mandal
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Department of Zoology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Mandal
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P
Ahearn
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D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay / Q000379:methods; D002132:Calcium Radioisotopes / Q000493:pharmacokinetics; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D004064:Digestive System; D004232:Diuretics / Q000494:pharmacology; D005786:Gene Expression Regulation; D015723:Gene Library; D007700:Kinetics; D008121:Nephropidae / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000235:genetics*; D014982:Xenopus laevis / Q000502:physiology
D000911:Antibodies, Monoclonal; D002132:Calcium Radioisotopes; D017931:DNA Primers; D018076:DNA, Complementary; D004232:Diuretics; D019831:Sodium-Calcium Exchanger; D000584:Amiloride; D012964:Sodium