PTH stimulates PLCbeta and PLCgamma isoenzymes in rat enterocytes: influence of ageing.
We previously reported that in rat duodenal cells (enterocytes), parathyroid hormone (PTH [1-34]: PTH) stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP(3)) and diacylglycerol (DAG) and that this mechanism is severely altered in old animals. In the present study, we show that PTH [1-34]-dependent IP(3) release in young rats was blocked to a great extent by an antibody against guanine nucleotide binding protein Galphaq/11, indicating that the hormone activates a beta isoform of PLC coupled to the alpha subunit of Gq/11. In addition, PTH rapidly (within 30 s, with maximal effects at 1 min) stimulated tyrosine phosphorylation of PLCgamma in a dose-dependent fashion (10(-10)-10(-7) M). The hormone response was specific as PTH [7-34] was without effects. The tyrosine kinase inhibitors, genistein (100 microM) and herbimycin (2 microM), suppressed PTH-dependent PLCgamma tyrosine phosphorylation. Stimulation of PLCgamma tyrosine phosphorylation by PTH [1-34] greatly decreased with ageing. PP1 (10 microM), a specific inhibitor of the Src family of tyrosine kinases, completely abolished PLCgamma phosphorylation. The hormone-induced Src tyrosine dephosphorylation, a major mechanism of Src activation, an effect that was blunted in old animals. These results indicate that in rat enterocytes PTH generates IP(3) mainly through G-protein-coupled PLCbeta and stimulates PLCgamma phosphorylation via the nonreceptor tyrosine kinase Src. Impairment of PTH activation of both PLC isoforms upon ageing may result in abnormal hormone regulation of cell Ca(2+) and proliferation in the duodenum.
Gentili
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Departamento de Biologia, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000, Bahia Blanca, Argentina.
Boland
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de Boland
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AR
D000375:Aging; D000818:Animals; D016227:Benzoquinones; D015153:Blotting, Western; D002478:Cells, Cultured; D004386:Duodenum / Q000166:cytology; D004591:Electrophoresis, Polyacrylamide Gel; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism*; D019833:Genistein / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D047029:Lactams, Macrocyclic; D008297:Male; D010281:Parathyroid Hormone / Q000378:metabolism*; D054799:Phospholipase C beta; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011233:Precipitin Tests; D020033:Protein Isoforms; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011809:Quinones / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017828:Rifabutin / Q000031:analogs & derivatives; D013347:Subcellular Fractions; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D016227:Benzoquinones; D007295:Inositol Phosphates; D007527:Isoenzymes; D047029:Lactams, Macrocyclic; D010281:Parathyroid Hormone; D020033:Protein Isoforms; D011809:Quinones; D017828:Rifabutin; D014443:Tyrosine; C020754:herbimycin; D019833:Genistein; D016392:Proto-Oncogene Proteins pp60(c-src); D010738:Type C Phospholipases; D054799:Phospholipase C beta; D051966:Phospholipase C gamma