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      Prejunctional and postjunctional actions of heptanol and 18β-glycyrretinic acid in the rodent vas deferens

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          Abstract

          Heptanol and 18β-glycyrrhetinic acid (18βGA) block gap junctions, but have other actions on transmitter release that have not been characterised. This study investigates the prejunctional and postjunctional effects of these compounds in guinea pig and mouse vas deferens using intracellular electrophysiological recording and confocal Ca 2+ imaging of sympathetic nerve terminals. In mice, heptanol (2 mM) reversibly decreased the amplitude of purinergic excitatory junction potentials (EJPs; 52 ± 5%, P < 0.05) while having little effect on spontaneous excitatory junction potentials (sEJPs). Heptanol (2 mM) reversibly abolished the nerve terminal Ca 2+ transient in 52% of terminals. 18βGA (10 μM) decreased the mean EJP amplitude, and increased input resistance in both mouse (137 ± 17%, P < 0.05) and guinea pig (354 ± 50%, P < 0.001) vas deferens indicating gap junction blockade. Further, 18βGA increased the sEJP frequency significantly in guinea pigs (by 71 ± 25%, P < 0.05) and in 5 out of 6 tissues in mice (19 ± 3%, P < 0.05). Moreover, 18βGA depolarised cells from both mice (11 ± 1%, P < 0.01) and guinea pigs (8 ± 1%, P < 0.005). Therefore, we conclude that heptanol (2 mM) decreases neurotransmitter release (given the decrease in EJP amplitude) by abolishing the nerve terminal action potential in a proportion of nerve terminals. 18βGA (10 μM) effectively blocks the gap junctions, but the increase in sEJP frequency suggests an additional prejunctional effect, which might involve the induction of spontaneous nerve terminal action potentials.

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          Most cited references30

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          Expression and functions of neuronal gap junctions.

          Gap junctions are channel-forming structures in contacting plasma membranes that allow direct metabolic and electrical communication between almost all cell types in the mammalian brain. At least 20 connexin genes and 3 pannexin genes probably code for gap junction proteins in mice and humans. Gap junctions between murine neurons (also known as electrical synapses) can be composed of connexin 36, connexin 45 or connexin 57 proteins, depending on the type of neuron. Furthermore, pannexin 1 and 2 are likely to form electrical synapses. Here, we discuss the roles of connexin and pannexin genes in the formation of neuronal gap junctions, and evaluate recent functional analyses of electrical synapses that became possible through the characterization of mouse mutants that show targeted defects in connexin genes.
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            Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries.

            1. Phenylephrine (10 microM) evoked rises in tension in isolated rings of endothelium-denuded rabbit superior mesenteric artery. These increases consisted of a tonic component with superimposed rhythmic activity, the frequency of which generally remained constant over time but whose amplitude exhibited cycle-to-cycle variability. 2. The amplitude, but not the frequency, of the rhythmic activity was affected by a series of short peptides possessing sequence homology with extracellular loops 1 and 2 of connexin 43 (Cx43). Oscillatory behaviour was abolished at concentrations of 100-300 microM (IC50 of 20-30 microM), without change in average tone. No synergy was evident between peptides corresponding to the extracellular loops, and cytoplasmic loop peptides were biologically inactive. 3. The putative gap junction inhibitor heptanol mimicked the action of the extracellular loop peptides and abolished rhythmic activity at concentrations of 100-300 microM without effects on frequency. However, in marked contrast to the peptides, heptanol completely inhibited the contraction evoked by phenylephrine (IC50, 283 +/- 28 microM). 4. The presence of mRNA encoding Cx32, Cx40 and Cx43 was detected in the rabbit superior mesenteric artery by reverse transcriptase-polymerase chain reaction. Western blot analysis showed that Cx43 was the major connexin in the endothelium-denuded vessel wall. 5. We conclude that intercellular communication between vascular smooth muscle cells via gap junctions is essential for synchronized rhythmic activity in isolated arterial tissue, whereas tonic force development appears to be independent of cell-cell coupling. The molecular specificity of the peptide probes employed in the study suggests that the smooth muscle relaxant effects of heptanol may be non-specific and unrelated to inhibition of gap junctional communication.
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              Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries.

              In resistance arteries, spread of hyperpolarization from the endothelium to the adjacent smooth muscle is suggested to be a crucial component of dilation resulting from endothelium-derived hyperpolarizing factor (EDHF). To probe the role of endothelial gap junctions in EDHF-mediated dilation, we developed a method, which was originally used to load membrane impermeant molecules into cells in culture, to load connexin (Cx)-specific inhibitory molecules rapidly (approximately 15 minutes) into endothelial cells within isolated, pressurized mesenteric arteries of the rat. Validation was achieved by luminally loading cell-impermeant fluorescent dyes selectively into virtually all the arterial endothelial cells, without affecting either tissue morphology or function. The endothelial monolayer served as an effective barrier, preventing macromolecules from entering the underlying smooth muscle cells. Using this technique, endothelial cell loading either with antibodies to the intracellular carboxyl-terminal region of Cx40 (residues 340 to 358) or mimetic peptide for the cytoplasmic loop (Cx40; residues 130 to 140) each markedly depressed EDHF-mediated dilation. In contrast, multiple antibodies directed against different intracellular regions of Cx37 and Cx43, and mimetic peptide for the intracellular loop region of Cx37, were each without effect. Furthermore, simultaneous intra- and extraluminal incubation of pressurized arteries with inhibitory peptides targeted against extracellular regions of endothelial cell Cxs (43Gap 26, 40Gap 27, and (37,43)Gap 27; 300 micromol/L each) for 2 hours also failed to modify the EDHF response. High-resolution immunohistochemistry localized Cx40 to the end of endothelial cell projections at myoendothelial gap junctions. These data directly demonstrate a critical role for Cx40 in EDHF-mediated dilation of rat mesenteric arteries.
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                Author and article information

                Journal
                Auton Neurosci
                Auton Neurosci
                Autonomic Neuroscience
                Elsevier
                1566-0702
                1872-7484
                15 June 2009
                15 June 2009
                : 148
                : 1-2
                : 69-75
                Affiliations
                [a ]Department of Pharmacology, University of Oxford, Mansfield Rd., Oxford, OX3 0RP, UK
                [b ]School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India
                Author notes
                [* ]Corresponding author. keith.brain@ 123456pharm.ox.ac.uk
                Article
                AUTNEU1042
                10.1016/j.autneu.2009.03.006
                2758546
                19375392
                92fe1221-d10c-4dc4-8a33-519dc7201e9e
                © 2009 Elsevier B.V.

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 18 December 2008
                : 10 March 2009
                : 18 March 2009
                Categories
                Article

                Neurosciences
                sympathetic neurotransmission,18β-glycyrretinic acid,heptanol,gap junctions,smooth muscle

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