Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Somogyi, Roland; Batzer, A.; Kolb, Hans-Peter

doi:10.1007/bf01871742

Cited by 32 publications

(11 citation statements)

References 47 publications

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“…After application of OAG for about 40 min a stationary current/voltage relationship was obtained. This time span is in the same range as that observed for changes of gap junction conductance in coupled cells by OAG-dependent phosphorylation [6]. The threshold potential of 55 mV, above which a significant voltage-dependent inactivation appeared, was similar to that observed during spontaneously occurring inactivation (Figs.…”

Section: Resultssupporting

confidence: 53%

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Ngezahayo¹,

Zeilinger²,

Todt³

et al. 1998

Pfl�gers Archiv European Journal of Physiology

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Hemichannels of rat connexin 46 (rCx46) were expressed in Xenopus laevis oocytes and analysed by two-electrode voltage-clamp experiments. It is established that rCx46 hemichannels can be activated at low external Ca2+ and positive membrane potentials. Upon larger depolarizations, the hemichannels of oocytes activate in a time-dependent manner, occasionally followed by a spontaneous inactivation. We found that, in the absence of inactivation, treatment of oocytes with 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of protein kinase C (PKC), reversibly reduced the amplitude of the rCx46-mediated current and, after an incubation time of about 30 min, induced inactivation of the voltage-dependent current. After wash-out of OAG the corresponding membrane conductance increased and the inactivation behaviour disappeared. The OAG-induced inactivation, as well as the spontaneous inactivation, could be removed by application of the specific PKC inhibitor calphostin C and also by phloretin. The data provide evidence that the activation and inhibition of PKC affect the rCx46-mediated membrane conductance as well as the voltage-dependent current inactivation in an inverse manner.

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Section: Resultssupporting

confidence: 53%

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Ngezahayo¹,

Zeilinger²,

Todt³

et al. 1998

Pfl�gers Archiv European Journal of Physiology

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“…Activation of PKC had either no effect or caused a slight increase in junctional conductance in rat pancreatic acinar cells [35] which express connexin 32 and connexin 26 [14]. In contrast, injection of PKC or 1 -oleoy1-2-acetyl-sn-glycerol into mouse pancreatic acinar cells decreased the junctional conductance [34]. Comparison of our results with those using other tissues are also complicated because different connexins are expressed in different tissues.…”

Section: Discussionmentioning

confidence: 77%

Phosphorylation of connexin 32, a hepatocyte gap‐junction protein, by cAMP‐dependent protein kinase, protein kinase C and Ca²⁺/calmodulin‐dependent protein kinase II

Sáez

Nairn

Czernik

et al. 1990

European Journal of Biochemistry

184

100

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Phosphorylation of connexin 32, the major liver gap-junction protein, was studied in purified liver gap junction and in hepatocytes. In isolated gap junctions, connexin 32 was phosphorylated by CAMP-dependent protein kinasc (CAMP-PK), by protein kinase C (PKC) and by Ca2+/calmodulin-dependent protein kinase I1 (Ca2+/CaM-PK 11) Connexin 26 was not phosphorylated by these three protein kinases. Phosphopeptide mapping of connexin 3; demonstrated that CAMP-PK and PKC primarily phosphorylated a seryl residue in a peptide termed peptide 1 PKC also phosphorylated seryl residues in additional peptides. Ca2+/CaM-PK I1 phosphorylated serine and tc a lesser extent, threonine, at sites different from those phosphorylated by the other two protein kinases. A synthetic peptide PSRKGSGFGHRL-amine (residues 228 -239 based on the deduced amino acid sequence of rat connexiI 32) was phosphorylated by CAMP-PK and by PKC, with kinetic properties being similar to those for othei physiological substrates phosphorylated by these enzymes. Ca2+/CaM-PK I1 did not phosphorylate the peptide Phosphopeptide mapping and amino acid sequencing of the phosphorylated synthetic peptide indicated thai Ser233 of connexin 32 was present in peptide 1 and was phosphorylated by CAMP-PK or by PKC. In hepatocyte: labeled with [32P]~rthopho~phoric acid, treatment with forskolin or 20-deoxy-20-oxophorbol 12,13-dibutyratc (PDBt) resulted in increased 32P-incorporation into connexin 32. Phosphopeptide mapping and phosphoamino acid analysis showed that a seryl residue in peptide 1 was most prominently phosphorylated under basal conditions. Treatment with forskolin or PDBt stimulated the phosphorylation of peptide 1. PDBt treatment also increased the phosphorylation of seryl residues in several other peptides. PDBt did not affect the CAMP-PK activity in hepatocytes. It has previously been shown that phorbol ester reduces dye coupling in several cell types, however in rat hepatocytes, dye coupling was not reduced by treatment with PDBt. Thus, activation of PKC may have differential effects on junctional permeability in different cell types; one source of this variability may be differences in the sites of phosphorylation in different gap-junction proteins.Gap junctions provide a pathway for cytoplasmic communication between adjacent cells [I, 21. These channels are permeable to ions and small molecules such as second messengers [3, 41. Hepatocyte gap junctions can be isolated by subcellular fractionation following detergent or alkaline extraction [5 -91. These preparations are highly enriched in proteins that have mobilities in the range of 26-28 kDa as determined by SDSjPAGE [7 -1 I]. Rat and human liver cDNAs have been isolated [lo, 111 which encode proteins having molecular masses of approximately 32 kDa. The rat or human liver proteins have been termed connexin 32 [12]. Rat liver also expresses a homologous protein of apparent molecular mass 21 kDa for which a cDNA has also been sequenced. Its predicted molecular mass is 26 kDa and it has been termed connexin ...

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“…Several potential PKC modification sites are present in the carboxy terminus of the a1 protein (20). Further, addition of PKC to electrically coupled cell pairs resulted in loss of junctional conductance (48).…”

Section: Resultsmentioning

confidence: 99%

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

et al. 1991

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Gap junctional intercellular communication is inhibited in response to tumor promoters and oncogene transformation, suggesting that loss of this function is an important step in tumor formation. To elucidate the molecular mechanisms responsible for this inhibition, we examined the expression of gap junction proteins and mRNA in mouse primary keratinocytes after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or ras transformation. During normal cell growth, keratinocytes express the a, (connexin 43) and P (connexin 26) proteins. Within 5 min of TPA treatment, the al protein became rapidly phosphorylated on serine residues and its expression was dramatically reduced by 24 h. The 12 protein, after an initial increase in expression, was also significantly reduced 24 h after treatment with TPA. ras transformation caused changes similar to those induced by TPA. The al protein underwent an increase in serine phosphorylation, although its expression declined only slightly, while 012 expression was greatly reduced.The effects of TPA and ras on a, expression were additive; treatment of ras-transformed cells with TPA resulted in increased a, phosphorylation, with greatly decreased protein levels, much lower than those generated by either agent alone. These data provide a likely explanation for the similar and synergistic inhibition of gap junctional intercellular communication by phorbol esters and ras.

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Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Cited by 32 publications

References 47 publications

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Phosphorylation of connexin 32, a hepatocyte gap‐junction protein, by cAMP‐dependent protein kinase, protein kinase C and Ca²⁺/calmodulin‐dependent protein kinase II

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

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Inhibition of electrical coupling in pairs of murine pancreatic acinar cells by OAG and isolated protein kinase C

Cited by 32 publications

References 47 publications

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Inactivation of expressed and conducting rCx46 hemichannels by phosphorylation

Phosphorylation of connexin 32, a hepatocyte gap‐junction protein, by cAMP‐dependent protein kinase, protein kinase C and Ca2+/calmodulin‐dependent protein kinase II

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins.

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Phosphorylation of connexin 32, a hepatocyte gap‐junction protein, by cAMP‐dependent protein kinase, protein kinase C and Ca²⁺/calmodulin‐dependent protein kinase II