Presynaptic Localization of Sodium/Calcium Exchangers in Neuromuscular Preparations

Calcium ions play a critical role in neurotransmitter release. The cytosolic Ca 2+ concentration ([Ca2+]cyt) at nerve terminals must therefore be carefully controlled. Several different mechanisms, including a plasmalemmal Na/Ca exchanger, are involved in regulating [Ca2+]cyta We employed immunofluorescence microscopy with polyclonal antiserum raised against dog cardiac sarcolemmal Na/Ca exchanger to determine the distribution of the exchanger in vertebrate neuromuscular preparations. Our data indicate that the Na/Ca exchanger is concentrated at the neuromuscular junctions of the rat diaphragm. The exchanger is also present in the nonjunctional sarcolemma, but at a much lower concentration than in the junctional regions. Denervation markedly lowers the concentration of the exchanger in the junctional regions; this implies that the Na/Ca exchanger is concentrated in the presynaptic nerve terminals. In Xenopus laevis nerve and muscle cell cocultures, high concentrations of the exchanger are observed along the neurites as well as at the nerve terminals. The high concentrations of Na/Ca exchanger at presynaptic nerve terminals in vertebrate neuromuscular preparations suggest that the exchanger may participate in the Ca-dependent regulation of neurotransmitter release. The Na/Ca exchanger is also abundant in developing neurites and growth cones, where it may also be important for Ca2+ homeostasis

Different effects of low and high dose cardiotonic steroids on cytosolic calcium in spontaneously active hippocampal neurons and in co-cultured glia

The Na+ pump is crucial for the regulation of [Na+](i) (the intracellular Na+ concentration) in all cells. Three Na+ pump alpha subunit isoforms, alpha 1, alpha 2 and alpha 3, are expressed in rat hippocampal neurons, and alpha 1 and alpha 2 are expressed in glia, but the significance of these isoforms is not understood. We exploited the different ouabain affinities of the Na+ pump alpha subunit isoforms in rat (alpha 1, low ouabain affinity; alpha 2 and alpha 3, high ouabain affinity) to probe their possible physiological roles. Low and intermediate doses (1-10 mu M) of ouabain and its readily reversible analog, dihydroouabain, altered the spontaneous elevations of [Ca2+](i) (the intracellular Ca2+ concentration) in neurons and induced [Ca2+](i) transients in glia. Complete inhibition of all Na+ pump isoforms (greater than or equal to 100 mu M ouabain) caused sustained increases in global neuronal [Ca2+](i) in rat neuronal/glial hippocampal co-cultures and transient [Ca2+](i) increases in surrounding glia. High dose ouabain was also associated with increased [Na+](i) and [H+](i) in neurons and glia. In contrast, 1 mu M ouabain (a concentration that completely inhibits only alpha 2 and alpha 3) was not associated with sustained increases in global neuronal [Ca2+](i) or the sustained derangements in [Na+](i) and [H+](i) observed with high dose ouabain. Reduction of [K+](o) to 1 mM suppressed the spontaneous [Ca2+](i) oscillations in neurons and induced Ca2+ transients in some glia; removal of external K+ induced sustained elevation of neuronal [Ca2+](i). These studies indicate that the alpha 1 isoform is the 'housekeeper' required for maintenance of the global Na+ gradient. As suggested by their restricted plasmalemmal distribution, the high ouabain-affinity Na+ pump isoforms may have more specific roles in neurons and glia. (C) 1998 Elsevier Science B.V. All rights reserved