Browsing by Author "SAEZ, JC"

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    ALTERED EXPRESSION AND FUNCTION OF HEPATOCYTE GAP-JUNCTIONS AFTER COMMON BILE-DUCT LIGATION IN THE RAT
    (1995) FALLON, MB; NATHANSON, MH; MENNONE, A; SAEZ, JC; BURGSTAHLER, AD; ANDERSON, JM
    Gap junction channels allow intercellular exchange of ions and small molecules between adjacent cells. Such communication coordinates cellular and organ function in tissues, although it is unclear if altered gap junction expression and communication contribute to organ dysfunction in pathological states. We examined the immunofluorescent (IF) localization and mRNA and protein levels of the two hepatocyte gap junction proteins connexin 32 and connexin 26, after hepatic injury induced by common bile duct ligation (CBDL) in the rat. Intercellular communication was measured by comparing gap junction-mediated coordination of hormone-induced Ca2+ signals in isolated rat hepatocyte couplets from control and CBDL animals. Connexin 32 plasma membrane IF, protein, and mRNA levels decreased markedly early after CBDL and remained low at 14 days. Connexin 26 plasma membrane IF and protein levels also decreased markedly after CBDL, but mRNA levels rose, and a partial return in membrane IF and protein levels was noted at 9 and 14 days. Coordination of vasopressin-induced Ca2+ signals between cells in isolated rat hepatocyte couplets 1 day after CBDL was significantly impaired compared with controls. These results demonstrate that hepatocyte gap junction communication is impaired early after CBDL because of decreased connexin protein levels. Disruption of gap junctions after CBDL may contribute to loss of hepatic functions that depend on gap junction communication.
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    CHANGES IN CONNEXIN43, THE GAP JUNCTION PROTEIN OF ASTROCYTES, DURING DEVELOPMENT OF THE RAT PINEAL-GLAND
    (1993) BERTHOUD, VM; SAEZ, JC
    The abundance of gap junctions between rat pineal astrocytes formed by connexin43 (Cx43) was studied during development. Levels and distribution of Cx43 were measured by immunoblotting and indirect immunofluorescence, respectively. The amount of Cx43 in cells located within the gland was low until about the 7th postnatal day and increased to adult values between the 14th and 21 st days postpartum. Although astrocytes, recognized by their vimentin immunoreactivity, were scarce before birth, they were abundant by the 7th postnatal day suggesting that the low levels of Cx43 found at this age corresponded to a low expression of this protein. Localization of the immunoreactivity to Cx43 and vimentin showed a close correlation, indicating that mature or immature pineal astrocytes form -ap junctions made of Cx43. Since Cx43 levels attained their adult values at about the time the innervation and the functional state of the gland reached maturity weeks after birth), it is proposed that astrocyte gap functions are involved in the function of the adult rat pineal gland.
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    EFFECTS OF CGMP DEPENDENT PHOSPHORYLATION ON RAT AND HUMAN CONNEXIN43 GAP JUNCTION CHANNELS
    (1995) KWAK, BR; SAEZ, JC; WILDERS, R; CHANSON, M; FISHMAN, GI; HERTZBERG, EL; SPRAY, DC; JONGSMA, HJ
    The effects of 8-bromoguanosine 3': 5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g(j)) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas g(j) was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g(j) in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gamma(j)) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gamma(j) distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of[P-32] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of P-32] into human Cx43 was not affected. We conclude that 8Br-cCMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g(j), which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.
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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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    LEUKOCYTES EXPRESS CONNEXIN-43 AFTER ACTIVATION WITH LIPOPOLYSACCHARIDE AND APPEAR TO FORM GAP-JUNCTIONS WITH ENDOTHELIAL-CELLS AFTER ISCHEMIA-REPERFUSION
    (1995) JARA, PI; BORIC, MP; SAEZ, JC
    Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 mu g/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions, In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43, Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication, Gap junctions may play a role in leukocyte extravasation.
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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.
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    ON THE MECHANISMS OF CELL UNCOUPLING INDUCED BY A TUMOR PROMOTER PHORBOL ESTER IN CLONE-9 CELLS, A RAT-LIVER EPITHELIAL-CELL LINE
    (1993) BERTHOUD, VM; ROOK, MB; TRAUB, O; HERTZBERG, EL; SAEZ, JC
    It is known that in Clone 9 (C9) cells, intercellular gap junctional communication (IGJC) is rapidly blocked by the tumor promoter phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), but it recovers spontaneously a few hours later and becomes refractory to TPA (Yada et al., J. Membr. Biol. 88, 217-232 (1985)). We now report that gap junctions between C9 cells contain at least two junctional proteins, connexin26 (Cx26) and connexin43 (Cx43), and that the TPA-induced changes in IGJC correlate temporally to changes in the state of phosphorylation of Cx43. The latter changes were prevented by inhibition of protein kinase C. Phosphoamino acid analysis and two-dimensional tryptic peptide maps of P-32-labeled Cx43 showed that during the TPA-induced phosphorylation at least two of the phosphorylated forms of Cx43 were differentially phosphorylated in seryl residues as compared to control. TPA induced a drastic reduction in junctional conductance as well as a redistribution of unitary gap junction channel event sizes seen in control cells. These changes were associated with retrieval of Cxs from the plasma membrane. Reappearance of gap junctions formed by Cx43 but not by Cx26 accounted for the spontaneous recovery in IGJC. It is proposed that gap junctions between C9 cells contain two types of channels each formed by Cx43 or Cx26 and that they are differentially affected during the action of TPA.