REGULATORY MECHANISMS OF GAP JUNCTION CHANNELS (CELL DEATH, FREE RADICALS)
SAEZ, JUAN C.
MetadataShow full item record
High intracellular cAMP, cAMP (,i), levels cause a rapid (minutes) increase in junctional conductance (g(,j)), which can be prevented by blocking the cAMP-dependent kinase. The increase in g(,j) is paralleled by an increase in ('32)P incorporation into the main gap junctional protein component (MP27). Since both changes are temporally correlated, phosphorylation of MP27 is presumably responsible for the change in g(,j). High cAMP (,i) also causes a longterm (hours) effect on g(,j) by delaying the disappearance of functional channels in primary cultures. The mechanism of this effect is not yet clear. A third effect of high cAMP levels is to induce the reexpression of functional channels after 4 days of culture. The reappearance of coupling is dramatically enhanced when the cultures are supplemented with certain proteoglycans or glycosaminoglycans. The reexpression requires de novo synthesis of gap junctions. A similar promotion of coupling by high cAMP (,i) was seen in superior cervical ganglion neurons cultured in collagen-coated culture dishes. These results show that the liver cell has at least two target sites for cAMP effects on regulation of gap junctions: the transcriptional machinery and the junctional membrane.;A secondary goal of this thesis was to study the initial events leading to cell death following ischemia, since one important function of controls on junctional conductance appears to be in the prevention of propagation of cell death from an injured cell to its coupled neighbors (a phenomenon termed healing over in cardiac muscle). The generation of free radicals was visualized with novel techniques; it occurred in parallel with cell death induced in an in vitro model of neural ischemia (substrate deprivation in sympathetic neurons). Extension of these studies to hepatocytes revealed that treatment with CCl(,4) (which rapidly generates free radicals) caused junctional uncoupling.;This thesis thus identifies and clarifies conditions under which gap junctional communication can be regulated in response to physiological and pathological conditions. Hormonal stimulation that increases cAMP levels increases junctional conductance within sec to min, retards turnover of gap junctions within hours, and stimulates de novo expression within hours to days. Among the consequences are propagation of the hormonal signal and synchronization of tissue response. In contrast, closure of gap junctions following injury prevents communication of dying cells with healthy ones to avoid the propagation of cell death. (Abstract shortened with permission of author.).