Adenylate cyclase was stimulated by prostaglandin E2 (PGE2) and parathyroid hormone-related protein (PTHrP) in both these types of fibroblast and by calcitonin gene-related protein (CGRP) in the human fibroblasts in vitro. PGE2 (1 microM), CGRP (1 microM), and PTHrP (1 microM) stimulated adenylate cyclase up to 50-fold, 10-fold and 9-fold, respectively. Calcitonin (CT), substance P (SP), interleukin-1 beta (IL-1 beta), and transforming growth factor-beta 1 (TGF beta 1) had no effect on adenylate cyclase in either fibroblast. Intracellular Ca2+ (iCa2+) was measured in individual fibroblasts from the periodontal ligament using Indo-1 and an adherent cell analysis and sorting interactive laser cytometer. Ionomycin (3 microM) caused a transient rise of iCa2+ in all human and canine fibroblasts tested. The mean percentage increase in iCa2+ in response to ionomycin was 820 and 840% for human and canine fibroblasts, respectively. The human fibroblasts responded to PGE2 (1 microM) by an increased iCa2+ concentration; the mean percentage increase in iCa2+ was 187%. SP caused a less pronounced increase in iCa2+ in the human fibroblasts (56%). CGRP and SP caused a similar response in the canine fibroblasts. The mean percentage increase in iCa2+ in response to SP and CGRP was 95 and 78%, respectively. PTH, PTHrP, platelet-activating factor, CT, and IL-1 beta had no effect on iCa2+ in either type of fibroblast. The data indicate that cAMP and calcium have roles as intracellular secondary messengers in the action of PGE2, SP, CGRP, and PTHrP in fibroblasts of human and canine periodontal ligament.