ryanodine and 9-(tetrahydro-2-furyl)-adenine

The aim of this study was to investigate the signal transduction mechanisms underlying the inhibitory effect induced by pituitary adenylate cyclase activating peptide (PACAP-27) on the spontaneous contractile activity of longitudinal muscle of mouse ileum. Mechanical activity of ileal segments was recorded isometrically in vitro. PACAP-27 produced apamin-sensitive reduction of the amplitude of the spontaneous contractions. 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), adenylate cyclase inhibitor, or genistein and tyrphostin 25, tyrosine kinase inhibitors, had negligible effects on PACAP-27-induced inhibition. PACAP-27 effects were significantly inhibited by U-73122, phopholipase C (PLC) inhibitor, by 2-aminoethoxy-diphenylborate (2-APB), permeable blocker of inositol 1,4,5-triphosphate (IP3) receptors and by depletion of Ca2+ stores with cyclopiazonic acid or thapsigargin. Ryanodine did not reduce PACAP-27-inhibitory responses. We suggest that, in mouse ileum, the inhibitory responses to PACAP-27 involve stimulation of PLC, increased production of IP3 and localised Ca2+ release from intracellular stores, which could provide the opening of apamin-sensitive Ca2+-dependent K+ channels.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Apamin; Boron Compounds; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Genistein; Ileum; In Vitro Techniques; Indoles; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein-Tyrosine Kinases; Pyrrolidinones; Ryanodine; Thapsigargin; Tyrphostins; Vasodilator Agents

Bitter substances are a structurally diverse group of compounds that appear to act via several transduction mechanisms. The bitter-tasting denatonium ion has been proposed to act via two different G-protein-regulated pathways, one involving inositol 1,4, 5-trisphosphate and raised intracellular calcium levels, the other involving phosphodiesterase and membrane depolarization via a cyclic nucleotide-suppressible cation channel. The aim of the present study was to examine these transduction mechanisms in taste cells of the mudpuppy Necturus maculosus by calcium-imaging and whole-cell recording. Denatonium benzoate increased intracellular calcium levels and induced an outward current independently of extracellular calcium. The denatonium-induced increase in intracellular calcium was inhibited by U73122, an inhibitor of phospholipase C, and by thapsigargin, an inhibitor of calcium transport into intracellular stores. The denatonium-induced outward current was blocked by GDP-beta-S, a blocker of G-protein activation. Neither resting nor denatonium-induced intracellular calcium levels were affected by inhibition of phosphodiesterase (with IBMX) or adenylate cyclase (with SQ22536) or by raising intracellular cyclic nucleotides directly (with cell permeant analogs). Our results support the hypothesis that denatonium is transduced via a G-protein cascade involving phospholipase C, inositol 1,4,5-trisphosphate, and raised intracellular calcium levels. Our results do not support the hypothesis that denatonium is transduced via phosphodiesterase and cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylate Cyclase Toxin; Animals; Calcium; Calcium Channels; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Necturus; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pyrrolidinones; Quaternary Ammonium Compounds; Receptors, Cytoplasmic and Nuclear; Ryanodine; Signal Transduction; Taste; Taste Buds; Thapsigargin; Virulence Factors, Bordetella