The second messenger signaling mechanisms of parathyroid hormone (PTH)- and PTH-related peptide (PTHRP)-stimulated osteoclast-like cell formation were investigated in mouse hemopoietic blast cells that possessed PTH binding sites. Human (h) PTH-(1-34) or hPTHRP-(1-34) resulted in a dose-dependent stimulation of tartrate-resistant acid phosphatase-positive multinucleated cells (MNC) formation. Pretreatment with [Nle8,18Tyr34]hPTH-(3-34) significantly blocked hPTH-(1-34)- and hPTHRP-(1-34)-stimulated MNC formation. Dibutyryladenosine 3',5'-cyclic monophosphate (10(-4) M) and forskolin (10(-5) M) as well as the stimulatory diastereoisomer of adenosine 3',5'-cyclic phosphorothioate (Sp-cAMPS), a direct activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) (10(-4) M), stimulated MNC formation, and Rp-cAMPS, an inhibitor of PKA activation (10(-4) M), almost completely inhibited MNC formation stimulated by the aforementioned agents but not by 1,25-dihydroxyvitamin D3. Moreover, Rp-cAMPS significantly blocked PTH- and PTHRP-stimulated MNC formation. Treatment with calcium ionophores (10(-8) and 10(-7) M) and phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator (10(-8) to 10(-6) M), but not 4 alpha-phorbol 12,13-didecanoate, a phorbol incapable of activating PKC, stimulated MNC formation. Two PKC inhibitors [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride and staurosporine] equally blocked PTH- and PTHRP-stimulated MNC formation. The combined pretreatment with Rp-cAMPS and PKC inhibitors completely blocked PTH- and PTHRP-stimulated MNC formation. Present findings indicate that the activation of PKA and PKC is directly linked to PTH- and PTHRP-stimulated osteoclast-like cell formation from hemopoietic blast cells.