Hypothalamic hypophyseal inhibitory factor (HHIF) increases intrasynaptosomal free calcium concentration

We have isolated from bovine hypothalamic and pituitary tissues a sodium 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 different from that of ouabain. It has been previously shown that this factor inhibits the Ca2+, Mg(2+)-ATPase of the plasma membrane of synaptosomes. Because Ca2+ plays a major role in cellular excitability, we carried out a systematic study of the effects of this inhibitor on the Ca2+ transport processes across the plasma membrane of synaptosomes: We measured ATP-dependent calcium uptake, Na(+)-Ca2+ exchange, and passive permeability using 45Ca2+ and Millipore filtration, chlortetracycline fluorescence, and light-scattering, respectively. This factor inhibits the Na+, K(+)-ATPase activity of the synaptosomal plasma membrane vesicles in the same range of concentrations that produced an increase of intrasynaptosomal free calcium, with nearly the same K0.5 value. In addition, 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 values. Measurements of fluorescence anisotropy showed that in this concentration range, the inhibitor did not significantly change the order parameter (fluidity) of these membranes. These results suggest that besides its known inhibition of the sodium pump, this factor could play a role in the control of Ca2+ homeostasis by direct modulation of transport systems implicated in the control of intracellular calcium.