Cyclic nucleotide crosstalk in salivary glands from partially fed Dermacentor variabilis (Say)

Enzyme immunosorbent assays were used to measure cyclic nucleotide concentrations in homogenates of salivary glands from partially fed female Dermacentor variabilis. The adenylyl cyclase activator forskolin (100 microM) increased homogenate cGMP concentrations greater than three-fold over controls. Competitive inhibition of nitric oxide synthase with 1mM l-NMMA, an l-arginine analog, demonstrated that crosstalk occurs downstream of nitric oxide synthesis. Forskolin-stimulated synthesis of cGMP was diminished 58% by the soluble guanylyl cyclase inhibitor ODQ (2 microM). The protein kinase A selective inhibitor Rp-cAMPS (50 microM) inhibited forskolin-stimulated cGMP by 49%. Whole glands treated with 10 microM dopamine increased cGMP levels two-fold in the presence of 1mM IBMX. Treatment of whole salivary glands with equimolar concentrations of 8-Br-cAMP and 8-Br-cGMP produced no greater fluid uptake than in glands treated with 8-Br-cGMP alone, suggesting that cAMP and cGMP share a downstream target. The protein kinase G-selective inhibitor Rp-8-pCPT-cGMPS (100 microM) impeded 10mM 8-Bromo-cGMP-stimulated gland weight increases. Pretreatment with verapamil, a Ca(2+) channel blocker, attenuated cyclic nucleotide-stimulated fluid uptake indicating that whole gland fluid changes are dependent on extracellular Ca(2+). Together, our data suggest that cGMP production is mediated in part by cAMP-dependent activation of soluble guanylyl cyclase. Experiments measuring changes in whole salivary gland weight support the hypothesis that cAMP and cGMP signaling cascades have a common target and that cyclic nucleotide-stimulated fluid movement is dependent on Ca(2+) influx.