Browsing by Author "MORENO, AP"

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    HUMAN CONNEXIN43 GAP JUNCTION CHANNELS - REGULATION OF UNITARY CONDUCTANCES BY PHOSPHORYLATION
    (1994) MORENO, AP; SAEZ, JC; FISHMAN, GI; SPRAY, DC
    Connexin43 is the major gap protein in the heart and cardiovascular system. Single channel recordings of human connexin43 gap junction channels exogenously expressed in transfected SKHep1 cells demonstrate two discrete classes of channel events, with unitary conductances of predominantly 60 to 70 and 90 to 100 pS when recorded with an internal solution containing CsCl as the major current-carrying ionic species and at moderate transjunctional voltages (<60 mV). Human connexin43 expressed in SKHep1 cells displays multiple electrophoretic mobilities (apparent M(r), approximate to 41 to 45 kD) when resolved in Western blots. Treatment of connexin43 from these cells with alkaline phosphatase collapses the bands into a single 41-kD species; application of alkaline phosphatase to the cell interior through patch pipettes yields channels that are predominantly of the larger unitary conductance. The smaller 60- to 70-pS unitary conductance values correspond to the most common channel size seen in cultured rat cardiac myocytes; these channels were more frequently observed after treatment with the phosphatase inhibitor okadaic acid, which was shown to increase phosphorylation of human connexin43 in these cells under similar conditions. Exposure to the protein kinase inhibitor staurosporine shifted the proportion of events toward the largest unitary conductance and resulted in decreased phosphorylation of human connexin43 in seryl residues in these cells. Thus, the unitary conductance of human connexin43 gap junction channels covaries with the phosphorylation state of the protein. This change in unitary conductance appears to be a unique effect of phosphorylation on gap junction channels, since it has not been observed for other ion channels that have thus far been evaluated.
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    NOREPINEPHRINE INDUCES CA2+ RELEASE FROM INTRACELLULAR STORES IN RAT PINEALOCYTES
    (1994) SAEZ, JC; MORENO, AP; SPRAY, DC
    In rat pinealocytes, an increase in intracellular Ca2+ concentration ([Ca2+](i)) due to Ca2+ influx in response to norepinephrine (NE) is a well recognized event involved in regulating several metabolic functions. Since NE also stimulates the metabolism of phosphatidyl inositols in rat pineal gland, it is conceivable that Ca2+ release from intracellular stores also contributes to the NE-induced increase in [Ca2+](i). In this communication, we report that in rat pinealocytes loaded with fura-2, a Ca2+ indicator, NE induced a transient increase in [Ca2+](i) that preceded the known Ca2+ influx. This novel [Ca2+](i) response to NE was detected in pinealocytes bathed with Ca2+-free saline and prevented by TMB-8, a blocker of Ca2+ release from intracellular stores, supporting the notion that the transient NE-induced Ca2+ response was due to Ca2+ release from intracellular stores. In addition, after an extended exposure to NE a new addition of this neurotransmitter did not elicit the phasic Ca2+ response, and application of increasing amounts of NE induced a Ca2+ response that was progressively smaller, suggesting desensitization. Thus, NE is proposed to increase [Ca2+](i) in rat pinealocytes by two mechanisms: (1) phasic release from intracellular stores and (2) tonic influx through a mechanism activated by larger applications of NE than required to evoke the phasic release.