Effect of ageing in the early biochemical signals elicited by PTH in intestinal cells

CLAUDIA GENTILI; GABRIELA PICOTTO; SUSANA MORELLI; RICARDO BOLAND; ANA RUSSO DE BOLAND

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS

ELSEVIER SCIENCE BV

Año: 2003 vol. 1593 p. 169 - 178

0304-4165

In previous work, we have demonstrated that rPTH(1-34) increases cytoplasmic calcium concentration ([Ca2 +]i) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca2 +]I and the implication of Ca2 + on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca2 +]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 [Ca2 +]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2 +]i2 +]i) in isolated rat enterocytes. In the present study, we have identified the sources of PTH-mediated increase in [Ca2 +]I and the implication of Ca2 + on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca2 +]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 [Ca2 +]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2 +]i2 +]I and the implication of Ca2 + on hormone early signals in enterocytes isolated from young (3-month-old) and aged (24-month-old) rats. In young enterocytes, PTH raised [Ca2 +]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 [Ca2 +]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2 +]i2 +]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 [Ca2 +]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2 +]i2 +]i. Phospholipase C (PLC) inhibitors blocked the initial acute elevation of the [Ca2 +]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 [Ca2 +]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 p85a, the regulatory subunit of PI3K, as well as the phosphorylation on Thr308 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 Ca2 +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 p85a, the regulatory subunit of PI3K, as well as the phosphorylation on Thr308 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 Ca2 +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 p85a, the regulatory subunit of PI3K, as well as the phosphorylation on Thr308 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 Ca2 +a, the regulatory subunit of PI3K, as well as the phosphorylation on Thr308 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 Ca2 +2 + chelation (BAPTA-AM, 5 AM) affected the tyrosine phosphorylation of p85a and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca2 + 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 Ca2 +. Taken together, these results suggest that PTH regulation of enterocyte [Ca2 +]i involves Ca2 + mobilization from IP3-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 Thr308 is impaired with ageing.AM) affected the tyrosine phosphorylation of p85a and inhibited PTH-dependent PI3K activation by 75% in young rats and completely abolished the enzyme activity in aged animals, demonstrating that Ca2 + 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 Ca2 +. Taken together, these results suggest that PTH regulation of enterocyte [Ca2 +]i involves Ca2 + mobilization from IP3-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 Thr308 is impaired with ageing.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 Ca2 +. Taken together, these results suggest that PTH regulation of enterocyte [Ca2 +]i involves Ca2 + mobilization from IP3-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 Thr308 is impaired with ageing.2 +. Taken together, these results suggest that PTH regulation of enterocyte [Ca2 +]i involves Ca2 + mobilization from IP3-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 Thr308 is impaired with ageing.2 +]i involves Ca2 + mobilization from IP3-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 Thr308 is impaired with ageing.308 is impaired with ageing.