Gap junctions between pinealocytes provide a pathway for intercellular communication affecting electrical activity, metabolism, gene expression and cell secretion. We intend to combine techniques of electrophysiology, biochemistry, immunology and molecular biology to characterize these junctions and determine their physiological 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. 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 21 but not connexin 27. 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 21 change, the levels of the mRNA encoding the gap junction protein and its 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 [(35)S]-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. Permeability to Ca2+ will be assayed using fura-2 and ratio image processing. The effect of secretion of melatonin will be evaluated indirectly by comparing secretion by isolated cells and cell clusters and by determining the effect on secretion by isolated cells and cell cluster of uncoupling concentration of octanol. We expect to obtain insight into the regulation of gap junctions formed by connexin 21, 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.
