HYPOTHALAMIC HYPOPHYSEAL INHIBITORY FACTOR (HHIF) INCREASES INTRASYNAPTOSOMAL FREE CALCIUM-CONCENTRATION

We have isolated from bovine hypothalamic and pituitary tissues a sodi um pump inhibitor that is structurally different from ouabain. By mass spectrometric analysis, this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra dif ferent from that of ouabain. It has been previously shown that this fa ctor inhibits the Ca2+,Mg2+-ATPase of the plasma membrane of synaptoso mes. Because Ca2+ plays a major role in cellular excitability, we carr ied out a systematic study of the effects of this inhibitor on the Ca2 + transport processes across the plasma membrane of synaptosomes: We m easured ATP-dependent calcium uptake, Na+-Ca2+ exchange, and passive p ermeability using Ca-45(2+) and Millipore filtration, chlortetracyclin e fluorescence, and light-scattering, respectively. This factor inhibi ts the Na+,K+-ATPase activity of the synaptosomal plasma membrane vesi cles in the same range of concentrations that produced an increase of intrasynaptosomal free calcium, with nearly the same K-0.5 value. In a ddition, in this concentration range, this factor stimulated 10- to 11 -fold the passive flux of Ca2+ and 2.5- to 3-fold the Ca2+ influx via the Na+-Ca2+ exchange in these membranes with respect to control value s. Measurements of fluorescence anisotropy showed that in this concent ration range, the inhibitor did not significantly change the order par ameter (fluidity) of these membranes. These results suggest that besid es its known inhibition of the sodium pump, this factor could play a r ole in the control of Ca2+ homeostasis by direct modulation of transpo rt systems implicated in the control of intracellular calcium.