Browsing by Author "BERTHOUD, VM"

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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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    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.
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    SPECIFICITY OF THE AMIDASE AND KININOGENASE METHODS FOR THE DETERMINATION OF RAT URINARY KALLIKREIN
    (1987) BERTHOUD, VM; CORTHORN, JL
    The specificity of the amidase and kininogenase methods for determining rat urinary kallikrein was studied. Male and female rat urine was employed. Esterase A1, A2 and kallikrein were separated by DEAE-Sephadex A-50 chromatography. Esterase A1 showed no amidase activity towards the substrate H-D-Val-Leu-Arg-p-nitroanilide. In contrast, esterase A2 and kallikrein attacked the substrate, and the activity of kallikrein was especially inhibited by aprotinin, while esterase A2 was more sensitive to soybean trypsin inhibitor. Esterase A1 did not show kininogenase activity, whereas esterase A2 showed this activity, but only towards the dog plasma substrate. Kallikrein possessed kininogenase activity towards both dog and rat plasma kininogen. We believe that the most specific method for measuring rat urinary kallikrein activity is the kininogenase method using partially purified rat plasma kininogen.