ryanodine and 1-oleoyl-2-acetylglycerol

Spontaneous local Ca(2+) release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca(2+) release events, we sought to investigate the signaling pathway for spontaneous local Ca(2+) release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca(2+) release events, indicating that these events are RyR-mediated Ca(2+) sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP(3)Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca(2+) sparks. Under patch clamp conditions, dialysis of IP(3) to activate IP(3)Rs increased the activity of local Ca(2+) events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca(2+) sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca(2+) sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca(2+) sparks, whereas the PKC inhibitor chelerythrine, PKCvarepsilon inhibitory peptide, or PKCvarepsilon gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca(2+) sparks in airway SMCs through two distinct signaling pathways: a positive IP(3)-IP(3)R pathway and a negative diacylglycerol-PKCvarepsilon pathway.

Topics: Alkaloids; Animals; Benzophenanthridines; Boron Compounds; Caffeine; Calcium; Cells, Cultured; Diglycerides; Estrenes; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Mice; Myocytes, Smooth Muscle; Oxazoles; Pyrrolidinones; Respiratory System; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Signal Transduction; Type C Phospholipases

1. In visceral smooth muscles, both M(2) and M(3) muscarinic receptor subtypes are found, and produce two major metabolic effects: adenylyl cyclase inhibition and PLCbeta activation. Thus, we studied their relevance for muscarinic cationic current (mI(CAT)) generation, which underlies cholinergic excitation. Experiments were performed on single guinea-pig ileal cells using patch-clamp recording techniques under conditions of weakly buffered [Ca(2+)](i) (either using 50 microm EGTA or 50-100 microm fluo-3 for confocal fluorescence imaging) or with [Ca(2+)](i) 'clamped' at 100 nm using 10 mm BAPTA/CaCl(2) mixture. 2. Using a cAMP-elevating agent (1 microm isoproterenol) or a membrane-permeable cAMP analog (10 microm 8-Br-cAMP), we found no evidence for mI(CAT) modulation through a cAMP/PKA pathway. 3. With low [Ca(2+)](i) buffering, the PLC blocker U-73122 at 2.5 microm almost abolished mI(CAT), in some cases without any significant effect on [Ca(2+)](i). When [Ca(2+)](i) was buffered at 100 nm, U-73122 reduced both carbachol- and GTPgammaS-induced mI(CAT) maximal conductances (IC(50)=0.5-0.6 microm) and shifted their activation curves positively. 4. U-73343, a weak PLC blocker, had no effect on GTPgammaS-induced mI(CAT), but weakly inhibited carbachol-induced current, possibly by competitively inhibiting muscarinic receptors, since the inhibition could be prevented by increasing the carbachol concentration to 1 mm. Aristolochic acid and D-609, which inhibit PLA(2) and phosphatidylcholine-specific PLC, respectively, had no or very small effects on mI(CAT), suggesting that these enzymes were not involved. 5. InsP(3) (1 microm) in the pipette or OAG (20 microm) applied externally had no effect on mI(CAT) or its inhibition by U-73122. Ca(2+) store depletion (evoked by InsP(3), or by combined cyclopiazonic acid, ryanodine and caffeine treatment) did not induce any significant current, and had no effect on mI(CAT) in response to carbachol when [Ca(2+)](i) was strongly buffered to 100 nm. 6. It is concluded that phosphatidylinositol-specific PLC modulates mI(CAT) via Ca(2+) release, but also does so independently of InsP(3), DAG, Ca(2+) store depletion or a rise of [Ca(2+)](i). Our present results explain the previously established 'permissive' role of the M(3) receptor subtype in mI(CAT) generation, and provide a new insight into the molecular mechanisms underlying the shifts of the cationic conductance activation curve.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclase Inhibitors; Animals; Aristolochic Acids; Bridged-Ring Compounds; Caffeine; Calcium; Carbachol; Diglycerides; Estrenes; Guanosine Triphosphate; Guinea Pigs; Ileum; Indoles; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Isoenzymes; Isoproterenol; Male; Membrane Potentials; Muscle, Smooth; Norbornanes; Patch-Clamp Techniques; Phospholipase C beta; Phospholipases A; Pyrrolidinones; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Ryanodine; Thiocarbamates; Thiones; Type C Phospholipases

1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta-adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7. Incubation with thapsigargin (1 microM) and ryanodine (10 microM) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8. The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate leve

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Caffeine; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Cyclic AMP; Diglycerides; Gallopamil; Guinea Pigs; Hydrolysis; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Intestine, Small; Isoproterenol; Muscle, Smooth; Protein Kinase C; Receptors, Adrenergic, beta; Receptors, Muscarinic; Ryanodine; Stereoisomerism; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin