Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3 ',5 '-cyclic AMP and Ca2+

in a previous study, we demonstrated that parathyroid hormone (PTH) stimula tes in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) iso forms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipa se C and increases intracellular Ca2+ in these cells, in the present study we evaluated the involvement of cAMP, Ca2+ and protein kinase C (PKC) on PT H-induced MAPK activation. We found that MAPK phosphorylation by the hormon e did not depend on PKC activation. PTH response could, however, be mimicke d by addition of forskolin (5-15 muM), an AC activator, or Sp-cAMP (50-100 muM), a cAMP agonist. and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2-0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). R emoval of external Ca2+ (EGTA 0.5 mM), chelation of intracellular Ca (2+) w ith BAPTA (5 muM), or blockade of L-type Ca (2+)-channels with verapamil (1 0 muM) significantly decreased PTH-activation of MAPK. Furthermore, a simil ar degree of phosphorylation of MAPK was elicited by the Ca 2+ mobilizing a gent thapsigargin, the Ca 2+ ionophore A23187, ionomycin and membrane depol arization with high K+. Inclusion of the calmodulin inhibitor fluphenazine (50 muM) did not prevent hormone effects on MAPK. Taken together, these res ults indicate that cAMP and Ca2+ play a role upstream in the signaling mech anism leading to MAPK activation by PTH in rat enterocytes. As Ca 2+ and cA MP antagonists did not block totally PTH-induced MAPK phosphorylation, it i s possible that linking of the hormone signal to the MAPK pathway may addit ionally involve Src, which has been previously shown to be rapidly activate d by PTH. Of physiological significance, in agreement with the mitogenic ro le of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this ef fect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, i ndicating that hormone modulation of MAPK through these messenger systems s timulates duodenal cell proliferation. (C) 2001 Elsevier Science B.V. All r ights reserved.