h-89 and inositol-1-4-6-trisphosphate

Ca(2+) and cAMP signalling pathways interact in a complex manner at multiple sites. This crosstalk fine-tunes the spatiotemporal patterns of Ca(2+) and cAMP signals. In salivary glands of the blowfly Calliphora vicina fluid secretion is stimulated by serotonin (5-hydroxytryptamine, 5-HT) via activation of two different 5-HT receptors coupled to the InsP(3)/Ca(2+) (Cv5-HT(2α)) or the cAMP pathway (Cv5-HT(7)), respectively. We have shown recently in permeabilized gland cells that cAMP sensitizes InsP(3)-induced Ca(2+) release to InsP(3). Here we study the effects of the cAMP signalling pathway on 5-HT-induced oscillations in transepithelial potential (TEP) and in intracellular [Ca(2+)]. We show: (1) Blocking the activation of the cAMP pathway by cinanserin suppresses the generation of TEP and Ca(2+) oscillations, (2) application of 8-CPT-cAMP in the presence of cinanserin restores 5-HT-induced TEP and Ca(2+) oscillations, (3) 8-CPT-cAMP sensitizes the InsP(3)/Ca(2+) signalling pathway to 5-HT and the Cv5-HT(2α) receptor agonist 5-MeOT, (4) 8-CPT-cAMP induces Ca(2+) oscillations in cells loaded with subthreshold concentrations of InsP(3), (5) inhibition of protein kinase A by H-89 abolishes 5-HT-induced TEP and Ca(2+) spiking and mimics the effect of cinanserin. These results suggest that activation of the cyclic AMP pathway promotes the generation of 5-HT-induced Ca(2+) oscillations in blowfly salivary glands.

Topics: Animals; Calcium; Cinanserin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diptera; Epithelium; Inositol 1,4,5-Trisphosphate; Isoquinolines; Membrane Potentials; Receptor, Serotonin, 5-HT2A; Salivary Glands; Serotonin; Signal Transduction; Sulfonamides; Thionucleotides

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

Topics: Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dideoxyadenosine; Diglycerides; Enzyme Inhibitors; Estrenes; Grasshoppers; Inositol 1,4,5-Trisphosphate; Isoquinolines; Muscarine; Muscarinic Agonists; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Receptors, Muscarinic; Second Messenger Systems; Sphingosine; Sulfonamides; Thapsigargin; Thionucleotides; Type C Phospholipases