physalaemin and sodium-sulfate

[3H]Substance P ([3H]SP), in a high ionic strength incubation medium, binds to a single class of saturable, noninteracting binding sites on rat submaxillary gland membranes with a KD = 2.8 +/- 0.34 nM and maximum binding (Bmax) = 220 +/- 31 fmol/mg of protein. The rank order of potency of various tachykinins, SP fragments and analogs to compete against [3H]SP is correlated with their potency to induce salivation. These findings indicate that, under the conditions described, [3H]SP binds to a physiologically relevant tachykinin receptor of the SP-P subtype. [3H]SP binding increases by 35% in the presence of optimal concentrations of Mn++ and Mg++ whereas guanine nucleotides reduce [3H]SP binding. The effect produced by either divalent cations or guanine nucleotides is due to increasing or decreasing the Bmax, respectively, without changing the affinity of [3H]SP. Guanine nucleotides reduce the Bmax of [3H]SP to the same level in the presence or absence of divalent cations, indicating that divalent cations increase the population of SP receptors that are sensitive to guanine nucleotides. In low ionic strength media, and when the nonspecific binding is defined by 1 microM SP, [3H]SP binds to two sites: a high affinity site with a KD of 0.14 nM and a Bmax of 370 fmol/mg of protein and a low affinity high capacity site. When the nonspecific binding is defined by 1 microM physalaemin, the high affinity is the only detectable site. However, in low ionic strength media, physalaemin has about one-fiftieth the potency of SP in competing with [3H]SP. These results prove that increasing the ionic strength of the media reduces the affinity of SP and some of its fragments and allows the determination of physiologically relevant SP-P binding sites.

Topics: Animals; Binding, Competitive; Cations, Divalent; GTP-Binding Proteins; Guanine Nucleotides; HEPES; In Vitro Techniques; Kinetics; Osmolar Concentration; Peptide Fragments; Physalaemin; Rats; Receptors, Neurokinin-1; Receptors, Neurotransmitter; Submandibular Gland; Substance P; Sucrose; Sulfates; Tritium