U50,488 modulation of sodium currents in colon sensory neurons

1049 Diversity of K÷ Channels in Circular Smooth Muscle of OpossumLower Esophageal Sphincter Yong Zhang, David V. Miller, William G. Paterson, Queen's Univ, Kingston Canada Background: We previously demonstrated that the balanceof K+ and Ca2+-activatedCI- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES) (AJP 279(6): G1226-G1234, Dec. 2000). In current studies, the contribution of major K÷ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. Method: Mechanical and electrical activity was recorded in strips perfused with Kreb's solution pre-gased with 95% 02 + 5% C02 at 35°C using standard isometric tension and intracellular microelectrode recordings. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Results: The K* channel blockers, tetraethylammonium (TEA, 2 mM), charybdotoxin (CTX, 100 nM) and 4aminopyridine (4-AP, 2 raM), enhanced resting tone, whereas apamin (300 nM) and glibenclamide (10 /~M) were without effects (n=6). Intracellular recordings showed on-going, spike-like action potentials lAPs)with resting membrane potential (MP)of -41.3 _+ 0.9 mV, amplitude of 19.9 _+ 1.0 mV and frequency of 43.0 -+ 2.7 min" (n=24). TEA and 4-AP depolarized the MPs to -37.6 _+ 1.9 mV and -38.6 -+ 2.1 mV, and increased the frequency of APs to 66.5 -+ 9.9 min-~and 53.9 _+ 4.0 min~ (n = 5), respectively. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting for 400 ms (-120 mY - + 100 mV) with the increment of 20 mV from holding potential of -80 mY ([K+]I = 150 mM, [K+]o= 2.5 raM). However, no inward rectification was observed. The outward current reached the peak within 50 ms and showed little or no deactivation. It was significantly decreasedby bath application of nifedipine (1 p,M), TEA, 4-AP and iberiotoxin 0BTN, 100 nM)(n = 6, ANOVAtest, p
1050 Chronic Inflammation Disturbs the Caz* Handling of Ileal SmoMk Muscle. Tom C. Seerden, Joris G. De Man, 8enedicte Y. De Winter, Tom G. Moreels, Univ of Antwerp, Div Gastroenterology, Antwerp Belgium; Gunther G. Vrolix, Univ of Antwerp, Div Pathology, Antwerp Belgium; Arnold G. Herman, Univ of Antwerp, Div Pharmacology, Antwerp Belgium; Erik A. Van Marck, Univ of Antwerp, Div Pathology, Antwerp Belgium; Paul A. Pelckmans, Univ of Antwerp, Div Gastroenterology, Antwerp Belgium Increase in cytosolic Ca2" initiates smooth muscle contraction. Chronic inflammation disturbs intestinal motility by incompletely understood mechanisms. We therefore studied the effect of granulomatous inflammation on the Ca2÷ pathways in the ileum. Mice were infected with the parasite Schistosoma mansoni which induces granulomatous intestinal inflammation. 16 weeks later, ileal longitudinal muscle strips from control and infected mice were mounted in organ baths for pharmacological study. Ileum from infected mice showed granulomas, increased wall thickness and blunted villi and an enhanced activity of the neutrophyl marker MPO (2_+lU/g vs 10_+2U/g, control vs inflamed ileum). All experiments were performed in the presence of the neuronal blocker TTX (lp, M). Carbachol (CCh, 0.01-1pM)induced dosedependent contractions that were higher in inflamed ileum. After normalisation to the weight and lenght of muscle strips, the amplitude of these contractions was similar in control and inflamed ileum (1vM CCh: 1.4_+0.2g/mm2 vs 1.5_+0.1g/ram2, controls vs inflamed ileum). On CCh-precontracted muscle strips, miconazole (blocks CRAC channels), genistein (blocks tyrosine kinase) and dantrolene (blocks ryanodine receptors) induced dose-dependent relaxations of similar amplitude in control and inflamed ileum. However, relaxations to HALO77 (blocks protein kinase, inhibits Ca2÷ sensitisation) and nifedipine (blocks L-type Ca2+ channels) were less pronounced in inflamed ileum (for 10p,M HA1077: 83+-6% vs 63_+6%; for 30nM nifedipine: 94_+4% vs 52+3%, control vs inflamed ileum). Dose-responsecurves to carhachol were inhibited to the same extent in control and inflamed ileum by miconazole, genistein and dantrolene (all 10/~M) whereas HA1077 (10/~M) and nifedipine (3OHM) inhibited the contractions to CCh to a lesser extent in inflamed ileum (% inhibition of a 0.3pM CCh contraction: 80+_3% vs 46-+9% by HA1077 and 58_+13% vs 37_+8% by nitedipine, control vs inflamed ileum). The contraction to thapsigargin (0.1/~M, blocks sarcoplasmic reticulum Ca2+/ATPase) and to ryanodine (3/~M, activates sarcoplasmic reticulum ryanndine receptors) was less pronounced in inflamed ileum (thapsigargin: 0.65_+0.16g/mm2 vs 0.19_0.08g/mm z and ryanodine: 1.99_+0.55g/mm2 vs 0.79-+0.15g/mm2, control vs inflamed ileum). These results indicate that chronic inflammation disturbs the Ca2* handling of ileal smooth muscle. This involves L-type Ca2÷ channels, intracellular Ca2+ stores and protein kinases that are involved in cellular Ca2+ sensitisation.

=nflammation that is paralleled by significant increases in MPO expression. At 7-10 days post infect=on, homogenates of inflamed jejunum were immunoprecipitated with anti-nitrotyrosine and anti-Ca2- -channel ~1C antibodies. The precipitates then were cross treated with antiCa2--channel oL1Cand nitrotyrosine antibodies using Western blotting techniques. Results from both experiments indicated that the L-type Ca2--channel protein was nitrated during inflammation. In another experiment, a segment of jejunum was taken from a control animal and strips of longitudinal muscle were removed and placed in tissue baths such that isometric contractions could be recorded. Treatment of a muscle strip with 50 nM MPO for 60 mins resulted in a marked delay in the onset and a decrease in the velocity of the phasic phase of a carbachol-inducedcontraction. However,the amplitude of the tonic phaseof the contraction was not affected. Becausethe phasic phase of smooth muscle contraction has been shown to be due mainly to the influx of Ca2- via voltage-gated Ca2+-channels, the response to potassium was determined. The potassium-induced contraction also was markedly inhibited. These preliminary results suggest that changes in muscle contractility perhaps due to nitration of voltage-dependent Ca2" channels occur in the presence of elevated MPO activity.

1052 U50,405 Modulation of Sodium Currents in Colon Sensory Neurons. Xin Su, Sherw L Kardos, Klaus Bielafeldt, Ruth E. Wachtel, G F. Gebhart, Univ of Iowa, Iowa C~, IA Background: We previously reported that the kappa-opioid receptor agonist 1S,2S-U50,488 attenuated afferent fiber responses to colorectal distension (CRD) in the rat. Subsequently, enantiomers 1S, 2R-U50,488 and 1R, 2R-U50,488 were found to dose-dependently inhibit responses of fibers to CRD. These effects could not be blocked by a high dose of naloxone, arguing against an opioid receptor-mediated mechanism. We hypothesized that these drugs function as ion channel blockers, thereby decreasing neuron excitability. Methods: Colon sensory neurons were acutely dissociated from $1 dorsal root ganglia to record voltage activated Na" currents, using the whole cell patch clamp technique. The retrograde tracer DiI was injected into the descending colon 1-2 weeks prior to experiments, allowing identification of colon sensory neurons. Na- currents were elicited by depolarization from -80 to + 10 inV. Results: Perfusion of the analgesic enantiomer 1S, 2S-U50,488 or either of the other two enantiomers (1S, 2R-U50,488 and 1R, 2R-U50,488; all 10/~M) decreased peak Na+ current to 51.4 _+0.4% (n =3), 66.4 _+7.9% (n = 7) and 49.1 _+7.8% (n =4) of control, respectively. The calculated half-inhibitory concentrations (IC5o)were 1.30 x 10-sM, 1.41 x 10-7Mand 4.08 x IO~M, respectively. The inhibition of Na+ current by 1S, 2S-U50,488 was use-depondent as demonstrated by a progressive decrease in current during repetitive stimulation at 0.5 Hz. After lOpM 1S, 2S-U50,488, the ratio of current amplitude at the 15th pulse to the 1st pulse was 0.67 _+0.08(n =3). In control experiments, a second stimulus pulse was given after 30 s without repetitive depolarization. None of the 3 cells tested showed a time-dependent decreasein peak Na" current under those conditions. Naloxone (10"~M), a nonselective opioid receptor antagonist, did not reverse the inhibitory effect of the 1S, 2S-U50,488 (39.8 _+8.1 % of control; n = 4). The peak Na" current also decreasedafter the addition of 1S, 2S-U50,488 (33.0 _+7.5 % of control; n =5) when the cells were dialyzed with GDP-~S, ruling out a Gprotein-mediated affect. Conclusions: These results suggest that U50,488 and its enantiomers inhibit voltage actk,ated Na" currents in colon sensory neurons by direct and use-dependent interaction with ion channels. This effect may contribute to their potential analgesic properties in vivo.

Mitochonddai Caz* Uptake Regulates After-Hyperpoladzations in Guinea-Pig Myeofedc Neurons Pieter Vanden Berghe, Terence K. Smith, Dept of Physiology and Cell Biology, Reno, NV Besides their fundamental role in energy metabolism, mitochondria also play a key role in Ca2~ homeostasis. The effer-hyperpolafization (AH), following action potential lAP) firing in AH neurons, is essential for organizing incoming sensory signals, thereby controlling gut motility. The AH is dependent on the changes in intracellular Ca2* concentration ([Ca~+]=) through activation of ryanodine receptors (Hillsley et al., 2000). The aim of this study was to investigate to what extent mitochondria influence the [Ca2+],and thereby regulate neuronal excitability. Methods: Standard longitudinal myenteric plexus preparations of guinea pig ileum were mounted in a coverglass-bottom recording dish, constantly pertused with oxygenated Krebs (35°C). Nicardipine (10~ M) and atropine (2.10~ M) prevented contractions in the longitudinal muscle layer. Micro-electrodes (-+ 120 M) were used to record membrane potential changes and to iontophoretically load the neurons with the Ca2+ indicator Fura-2 (0.75-1 raM). The [Ca2*], was reflected in the ratio of fluorescence at either Fura-2 excitation wavelengths (Ionoptix®). Results: The AH amplitude and the accompanying increase [Ca2+],depended on the number ofAP s (9 _+ 0.7 mY; 12.9 _+ 1% [Ca2+],increase and 10.8 -+ 1.4 mV; 20.2 _ 0.9% [Ca2*], increase for 2 and 3 AP s respectively). In the presence of the protonophore FCCP(10~ M), the AH turned into a prominent hyperpolarization (13.0--- 1.lmV, n = 8) lasting for several minutes. This was paralleled by an increase in [Ca2+],(-10.5%) reaching a plateau phase. In S-type neurons, FCCP also induced hyperpolarizations (21.6 _+ 0.2 mV, n =2) again accompanied by a rise in [Ca2+],(-6.5%). This hyperpolarization developed more slowly (- 4 min) and was not triggered or amplified by AP firing. Similarly, the mitochondrial complex I blocker rotenone (10.5 M) induced hyperpolarizations in both AH (20 mV) and S-type neurons (15.2 -+ 3.8 mV). Generally, the neurons became inexcitable, but when current-clamped to the resting membrane potential, no differences in AH characteristics could be observed. Conclusion: Mitochondrial inhibitors cause myenteric neurons to hyperpolarize. The prolongation of the AH and the rise in [Ca2+], suggest that, at least in AH neurons, insufficient Ca2+ buffering and the subsequent opening of Ca2~ activated K÷ channels is part of the underlying mechanism. Therefore, we conclude that mitochondrial Ca2÷ uptake is crucial for the control of the AH in myentefic neurons. Supported by NIH Grant # PO1 DK-41315.

1051 Ca2+-Channel Protein Nitration and Intestinal Smooth Muscle Contraetility Ka Bian, Norman Weisbrodt, Ferid Murad, Univ of Texas Medical Sch, Houston, TX Intestinal smooth muscle function is altered in many conditions that are characterized by inflammation and~or sepsis, cond/tions that are not uncommon. Little is known about the pathways and mediators accounting for the dysfunction, and there are no current effective interventions. Important markers of inflammationa are infiltration of leukocytes and elevated myeloperoxidase (MPO) activity. MPO is a heine enzyme of neutrophils that uses hydrogen peroxide and nitrite to generate nitrogen dioxide and hypochlorous acid which in turn catalyzes nitration of tyrosine. The following experiments were designed to test the HYPOTHESISthat nitration of a key protein involved in smooth muscle contraction occurs in intestinal muscle during an inflamed state. Mice were infected with Trichinella spiralis (TS) to induce a jejunal

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