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Numerous isoforms of plasmalemmal K-dependent (NCKX) and K-independent (NCX) Na+/Ca2+ exchangers are expressed in the brain. The physiological functions of each isoform are presently unknown. Therefore, in this study, we compared expression of NCKX and NCX transcripts between primary cultures of cerebellar granule cells, and astrocytes. Northern blot analysis showed that granule cells expressed NCKX2, NCKX3, NCKX4 and NCX3, whereas astrocytes expressed primarily NCX1. Consistent with this molecular characterization, a significant fraction of 45Ca2+ accumulation in Na-loaded granule cells, but not in astrocytes, depended on external K+. This is the first demonstration of native NCKX activity in neurons derived from the central nervous system. Our data suggest that NCKX isoform expression may correspond to the unique Ca2+ homeostasis requirements of neurons., (Copyright 2002 Lippincott Williams & Wilkins.)

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., (Copyright 2001 Wiley-Liss, Inc.)

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium-calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called 'simplified analogues', where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium-calcium exchanger was screened on both cell types by measuring their effect on 45Ca(2+) uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)-N'-(1-naphtylmethyl)guanidine (IC(50)=3.4 microM) was found more active than amiloride (IC(50)=690 microM) and 3,4-dichlorobenzamil (IC(50)=15.2 microM), the reference inhibitor.