Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells.
In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca(2+)](i)) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca(2+)](I) and the implication of Ca(2+) on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca(2+)](i) in a dose-dependent manner (1 pM-100 nM). In cells from aged rats, hormone concentrations higher than physiological (>/=1 nM) were required to observe significant increases in [Ca(2+)](i). Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca(2+)](i) biphasic response to PTH of young enterocytes while in old cells, no effects were observed. The voltage-dependent calcium-channel blocker (VDCC), nitrendipine, suppressed PTH-dependent changes of the sustained [Ca(2+)](i) phase in young and aged animals. In this study, we analysed, for the first time, alterations in phosphatidylinositol 3-kinase (PI3K) activity and response to PTH in rat enterocytes with ageing. Basal PI3K activity was significantly modified by ageing. Acute treatment with 10(-8) M PTH increased enzyme activity, with a maximun at 2 min (+3-fold) in young rats and only elevated by less than 1-fold basal PI3K activity in aged animals. Hormone-induced tyrosine phosphorylation of p85alpha, the regulatory subunit of PI3K, as well as the phosphorylation on Thr(308) of its downstream effector Akt/PKB was evident in enterocytes from 3-month-old rats, whereas it was greatly reduced in the cells from 24-month-old animals. Intracellular Ca(2+) chelation (BAPTA-AM, 5 microM) affected the tyrosine phosphorylation of p85alpha and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca(2+) is required for full activation of PI3K in enterocytes stimulated with PTH. The Thr phosphorylation of PI3K downeffector, Akt/PKB, was also fully dependent on Ca(2+). Taken together, these results suggest that PTH regulation of enterocyte [Ca(2+)](i) involves Ca(2+) mobilization from IP(3)-sensitive stores and the influx of the cation from the extracellular milieu, the former pathway being blunted during ageing. The data also indicates a positive role for intracellular calcium in one of the early signals of PTH in rat enterocytes, the activation of PI3K, and that hormone regulation of PI3K activity and Akt/PKB phosphorylation on Thr(308) is impaired with ageing.
Gentili
Claudia
C
Dpto. Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, San Juan 670, 8000, Bahía Blanca, Argentina.
Picotto
Gabriela
G
Morelli
Susana
S
Boland
Ricardo
R
de Boland
Ana Russo
AR
D000375:Aging; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents; D004533:Egtazic Acid / Q000031:analogs & derivatives*; D007421:Intestine, Small / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010281:Parathyroid Hormone / Q000494:pharmacology* / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D002118:Calcium; D002614:Chelating Agents; D004533:Egtazic Acid; D010281:Parathyroid Hormone; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D010738:Type C Phospholipases; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; C494920:Akt1 protein, rat