Colonic smooth muscle and Cav 1.2 channel mutations that alter contractile activity
Helman, Justin M.
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Ca 2+ entry through Cav1.2 L-type voltage-gated Ca 2+ channels is a Ca 2+ source for intestinal smooth muscle contraction. We examined Cav1.2 in a Timothy Syndrome (TS) mouse with a TS1 G406R Cav 1.2 "gain of function" mutation and in a mouse with a TS2 (Neo) G406R mutation and a neo cassette suppressor of Cav 1.2 expression. Isometric force was measured in rings of proximal colon from TS1 and TS2 (Neo) positive and negative littermate (LM) mice. Spontaneous contractile activity occurred in tissues from all animals. Treatment with Nw-nitro-L-arginine (10 -3 M, L-NA), a nitric oxide synthase antagonist, caused rhythmic contractions. Treatment with atropine (10 -5 M) did not alter spontaneous activity. Treatment with TTX (10 -6 ) generated spontaneous contractile activity in 50% of tissues suggesting the release of inhibitory neurotransmitter acting on enteric pacemaker cells. Suggesting that spontaneous activity is generated by enteric pacemaker cells and modulated by the release of NO and inhibitory neurotransmitters. In TS1 positive animals increased tone was seen after L-NA treatment, which was significantly larger compared to LM animals. Tissue rings were placed in a high-K + zero-Ca 2+ bath causing all spontaneous activity to stop and basal tone to decrease. Ca 2+ was then returned to the bath in logarithmic doses causing the tissues to contract. Tissues from TS1 positive animals had a significantly stronger contraction to doses of Ca 2+ compared to LM tissues while TS2 (Neo) tissues had a significantly weaker contraction compared to LM tissues. This data shows that mutations associated the TS1 mice result in increased contractile activity in response to Ca 2+ while the TS2 (Neo) mutation results in decreased contractile activity in response to Ca 2+ . Electric field stimulation (EFS, 3-20 Hz) produced frequency-dependent tension in all tissues, suggesting the release of multiple neurotransmitters. A contraction followed by relaxation was seen during EFS, however only a contraction was present if pre-exposed to L-NA, and tension was significantly greater in TS1 and smaller in TS2 Neo mice. EFS induced tension was blocked by the muscarinic antagonist, atropine (10 -5 M), suggesting nitronergic and cholinergic control of muscle activity. An increase in tension was seen when stimulation was stopped, which was significantly larger in TS1 and less in TS2 (Neo). This data shows that mutations associated the TS1 mice result in increased contractile activity while the TS2 (Neo) mutation results in decreased contractile activity.