Substance P and related tachykinins induce receptor-mediated hydrolysis of polyphosphoinositides in the rat anterior pituitary.
In the present study characterization of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIP2-PLC) activity and receptor-mediated hydrolysis of PIP2 in rat anterior pituitary membranes were investigated. Incubation of the membrane fraction of anterior pituitary homogenate with [3H]inositol-labeled PIP2 in the presence of calcium increased the concentration of the water-soluble degradation product inositol trisphosphate (IP3) in a time-dependent manner. PIP2-PLC in the rat anterior pituitary had a pH optimum at 5.5 and a requirement for cations. Ca2+ and Mg2+ could activate the enzyme. Activity was maximal at a total magnesium concentration of 1 mM and at a free Ca2+ concentration of 100 microM. The addition of the detergent Triton X-100 (0.05% w/v) to the membrane fraction resulted in a 50% decrease of PIP2-PLC activity, whereas the presence of sodium deoxycholate (1 mg/ml) in the membrane fraction increased the PIP2-PLC activity by 100%. The tachykinins substance P, 8-Tyr-substance P, physalaemin, neurokinin A, eledoisin, kassinin and neurokinin B induced receptor-mediated breakdown of [3H]inositol-labeled PIP2 in the membrane fraction in a concentration-dependent manner, but with different potencies. The tachykinins displayed the following rank order of potencies: substance P greater than 8-Tyr-substance P greater than physalaemin greater than neurokinin A greater than eledoisin greater than kassinin greater than neurokinin B, which is consistent with the involvement of a NK-1 receptor. Combined treatment of anterior pituitary membranes by substance P and thyrotropin-releasing hormone (TRH) resulted in an additional increase in PIP2-PLC activity compared to stimulation with TRH alone.
Protein Laboratory, University of Copenhagen, Denmark.
S E
SE
Mau
P J
PJ
Larsen
J A
JA
Mikkelsen
T
T
Saermark
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D017830:Octoxynol; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols / Q000378:metabolism*; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism; D013373:Substance P / Q000378:metabolism / Q000494:pharmacology*; D015320:Tachykinins / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000378:metabolism*
D003902:Detergents; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D015320:Tachykinins; D013373:Substance P; D011092:Polyethylene Glycols; D017830:Octoxynol; D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium