lithium-chloride and inositol-4-5-bisphosphate

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (InsP(x)) accumulation and Ca(2+) mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [(3)H]InsP(x) accumulation and Ca(2+) mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y(1) receptors, N(6)-cyclopentyladenosine and AMP, alpha,beta-methylene-ATP and beta, gamma-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [(3)H]InsP(x) accumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [(3)H]InsP(x) accumulation and Ca(2+) mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [(3)H]InsP(x) accumulation and Ca(2+) mobilization in canine TECs. P2Y(2) receptors may be predominantly mediating [(3)H]InsP(x) accumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca(2+) mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Calcium; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Female; Hydrolysis; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Lithium Chloride; Male; Phosphatidylinositols; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Respiratory Mucosa; Thionucleotides; Trachea; Uridine Triphosphate

Agonist-stimulated phosphoinositide hydrolysis is the principal mechanism underlying pharmacomechanical coupling in airways smooth muscle. In bovine tracheal smooth muscle, activation of muscarinic cholinoceptors results in sustained phospholipase C-mediated PtdIns(4,5)P2 hydrolysis but transient Ins(1,4,5)P3 accumulation, which implies agonist-stimulated metabolism of Ins(1,4,5)P3. To investigate the metabolic fate of Ins(1,4,5)P3 in bovine tracheal smooth muscle, we developed a [3H]inositol-labeling protocol wherein more than 98% of the [3H]inositol polyphosphates that accumulated over a 0 to 30-min incubation with 100 microM carbachol in the presence of 5 mM LiCl were derived from [3H]Ins(1,4,5)P3 and wherein the Ins(1,4,5)P3 3-kinase (EC 2.7.1.127) and 5-phosphatase (EC 3.1.3.56) pathways generated a set of mutually exclusive [3H]-inositol polyphosphate isomers. Under these conditions, the 5-phosphatase pathway was shown to be the dominant route for [3H]Ins(1,4,5)P3 metabolism at all time intervals measured, especially at early times (0-300 sec), where it accounted for more than 85% of [H]Ins(1,4,5)P3 metabolism. We also observed accumulation of a novel agonist and LiCl-sensitive [3H]InsP2 isomer identified as [3H]Ins(4,5)P2. The presence of a LiCI-sensitive inositol polyphosphate 1-phosphatase (EC 3.1.3.57) was demonstrated, and high LiCl concentrations (30 mM) caused a significant enhancement of [3H]Ins(1,4)P2 accumulation and a corresponding decline in [3H]Ins4P levels. Because nearly identical bell-shaped LiCl concentration-response curves were obtained for [H]Ins4P and [3H]Ins(4,5)P2 accumulation, and [3H]Ins(4,5)P2 was not generated under conditions expected to stimulate phospholipase D, these data suggest that the most likely precurser of [3H]Ins(4,5)P2 is [3H]Ins(1,4,5)P3. This is the first demonstration of Ins(4,5)P2 accumulation in a non-neuronal cell type, and the foregoing data suggest a novel route of formation via an Ins(1,4,5)P3 1-phosphatase, which would represent an additional pathway for [H]Ins(1,4,5)P3 removal.

Topics: Animals; Carbachol; Cattle; Cell-Free System; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Lithium Chloride; Muscarinic Agonists; Muscle, Smooth; Phosphoric Monoester Hydrolases; Receptors, Muscarinic; Trachea; Tritium