³H-Substance P binding to salivary gland membranes. Regulation by guanyl nucleotides and divalent cations

With appropriate measures to protect ³H-substance P (³H-SP) from proteolytic degradation and from nonspecific adsorption to glass-fiber filters, we have been able to demonstrate reliably a high-affinity specific binding of ³H-SP to rat submaxillary/sublingual gland membranes with a K(D) of 1 nM and B(max) of 6 pmoles/g of tissue. The relative potencies of various SP fragments and related analogues in reducing ³H-SP binding parallel their potencies in stimulating phosphatidylinositol turnover, amylase release, and salivation, thus supporting an association of the observed ³H-SP binding site with the physiological SP receptors in this tissue. This binding is selectively stimulated by some divalent cations (Mn²⁺ > Mg²⁺ > Ca²⁺) but inhibited by several guanyl nucleotides, suggesting a possible linkage to adenylate cyclase. However, no effect of SP on either the basal or the norepinephrine-stimulated adenylate cyclase activity could be demonstrated in salivary gland homogenates.