Juan Carlos Saez - US grants

Gap junctions between pinealocytes provide a pathway for intercellular communication affecting electrical activity, metabolism, and possibly gene expression and secretion. We intend to combine techniques of electrophysiology, biochemistry, immunology and molecular biology to characterize these junctions and determine their physiology role. We will study the biophysical properties of junctional communication between pairs of acutely dissociated pinealocytes. We will measure macroscopic junctional conductance (gj), determine its voltage dependence and measure single channel conductances. The presence of other gating processes including closure by H+, Ca2+ and octanol will be tested for. [Permeability to Ca2+ will be assayed quantitatively as a indicator of permeability to second messengers, e.g. cAMP and cGMP]. We will study short term modulation of gJ by agents that affect melatonin secretion, e.g. norepinephrine (NE) and the effect of environmental factors including normal innervation, light/dark cycles, constant illumination, and constant darkness. Using immunological techniques and Northern blot analysis we found that rat pineals express both connexins 43 and 26 but not connexin 32. [Connexin 26 is restricted to pinealocytes and connexin 43 is found only in the astrocytes]. Levels of pinealocyte connexin 26 will be measured by Western blotting after treatments found to affect gj. If levels of connexin 26 change, the levels of the mRNA encoding it and the transcription rate will be measured. Turnover of the protein and of the mRNA will be determined in primary cultures of pairs and small groups of pinealocytes or in pineals in organ culture by means of [35S]-methionine and 3H-uridine pulse-chase experiments respectively. These measurements should show whether the expression of these gap junction proteins is regulated at the level of mRNA synthesis, mRNA stability, protein synthesis or protein degradation. Since it is thought that gap junctions participate in the regulation of secretion, we propose to study the relationship between pinealocyte coupling, gap junction permeability to Ca2+ and melatonin secretion. The effect on secretion of melatonin will be evaluated indirectly; secretion by isolated cells and by cell clusters in the presence and absence of an uncoupling agent such as octanol will be compared. The effect of octanol on secretion by isolated cells will control for its effect on junctions formed by connexin 26, which could be relevant to function of the pineal gland as well as to regulation of gap junctions in general. The pineal gap junction may also serve as a model of electrical synapses in the central nervous system.