cyclic-gmp and 2-aminoethoxydiphenyl-borate

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

Topics: Boron Compounds; Bradykinin; Bradykinin B2 Receptor Antagonists; Carbazoles; Chloride Channels; Cyclic GMP; Estrenes; Flow Cytometry; HEK293 Cells; Humans; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Natriuretic Peptides; Nitrobenzoates; Patch-Clamp Techniques; Potassium Channel Blockers; Protein Kinase Inhibitors; Pyrrolidinones; RGS Proteins; Signal Transduction; Thionucleotides; Type C Phospholipases

Dermal photoreceptors in the pond snail Lymnaea stagnalis mediate the whole-body withdrawal response, including pneumostome closure, elicited by a shadow passing over the pneumostome area. The pneumostome closure response is part of the defense reaction in Lymnaea. The shadow or 'light-off' stimulus elicits activity in a higher order interneuron, RPeD11, which has a major role in mediating defensive withdrawal behavior elicited by noxious or threatening stimuli. Here, we tested our hypothesis that cyclic nucleotide-gated (CNG) channels are involved in the dermal photoreceptor-mediated transduction of the shadow stimulus. The response to the shadow stimulus recorded in RPeD11 was abolished by 500 μM cis-diltiazem, which blocks cGMP-activated conductance of CNG channels. On the other hand, the shadow response elicited in RPeD11 was not blocked by 2-amino ethyldiphenyl borate (2-APB), a transient receptor potential (TRP) channel blocker. Consistent with the electrophysiologic data, cis-diltiazem blocked the shadow-evoked withdrawal response, whereas 2-APB did not block the withdrawal response evoked by the shadow stimulus in intact freely behaving Lymnaea. Together, these findings support the hypothesis that the second messenger in dermal photoreceptors involves CNG and not TRP channels.

Topics: Animals; Boron Compounds; Cyclic GMP; Cyclic Nucleotide-Gated Cation Channels; Diltiazem; Interneurons; Light Signal Transduction; Lymnaea; Photic Stimulation; Photoreceptor Cells; Skin Physiological Phenomena

1. To investigate the cellular mechanisms underlying spontaneous excitation of smooth muscle of the guinea-pig urinary bladder, isometric tension was measured in muscle bundles while recording the membrane potential from a cell in the bundle with a microeletrode. Changes in the intracellular calcium concentration ([Ca(2+)](i); calcium transients) were recorded in strips loaded with the fluorescent dye, fura-PE3. 2. In 40% of preparations, individual action potentials and contractions, which were abolished by nifedipine (1 microm), were generated. In the remaining preparations, bursting action potentials and contractions were generated. Contractions were again abolished by nifedipine (1 microm), while higher concentrations of nifedipine (10-30 microm) were required to prevent the electrical activity. 3. Carbachol (0.1 microm) increased the frequency of action potentials and corresponding contractions. Apamin (0.1 microm) potentiated bursting activity and enhanced phasic contraction. Charybdotoxin (CTX, 50 nm) induced prolonged action potentials that generated enlarged contractions. In contrast, levcromakalim (0.1 microm) reduced the frequency of action potentials, action potential bursts and the size of the contractions. 4. Forskolin (0.1 microm), 8-bromoguanosin 3', 5' cyclic monophosphate (8Br-cGMP, 0.1 mm) and Y-26763 (10 microm) suppressed contractions without reducing the amplitude of either action potentials or Ca transients. 5. This paper confirms that action potentials and associated calcium transients are fundamental mechanisms in generating spontaneous contractions in smooth muscles of the guinea-pig bladder. However, in parallel with the excitation-contraction coupling, the sensitivity of the contractile proteins for Ca(2+) may play an important role in regulating spontaneous excitation and can be modulated by cyclic nucleotides and Rho kinase.

Topics: Action Potentials; Amides; Animals; Apamin; Boron Compounds; Calcium; Calcium Signaling; Carbachol; Charybdotoxin; Colforsin; Cromakalim; Cyclic GMP; Dose-Response Relationship, Drug; Drug Synergism; Female; Fura-2; Guinea Pigs; Isotonic Contraction; Male; Mechanotransduction, Cellular; Microelectrodes; Muscle Contraction; Muscle, Smooth; Nifedipine; Pyridines; Transducers; Urinary Bladder