Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation.
The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [32P] orthophosphate. An increased (4-6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent Mr of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [3H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed beta-adrenergic receptor antagonist but not with physiological concentrations of alpha- or beta 1-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the beta 2-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.
Purushotham
K R
KR
Department of Oral Biology, University of Florida, Gainesville 32610.
Zelles
T
T
Humphreys-Beher
M G
MG
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D005700:Galactosyltransferases / Q000378:metabolism*; D007294:Inositol / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D010306:Parotid Gland / Q000187:drug effects / Q000502:physiology*; D010643:Phenoxybenzamine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011433:Propranolol / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D015398:Signal Transduction / Q000502:physiology; D013936:Thymidine / Q000378:metabolism
D002147:Calmodulin; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D010643:Phenoxybenzamine; D007294:Inositol; D011433:Propranolol; D005700:Galactosyltransferases; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D007545:Isoproterenol; D013936:Thymidine