290

Bxpression of Intermediate-Conductance Calcium-Activated Potassium Channels in Enteric Neurons john B Furuess, Bilfie Hunne, Gareth A. Hicks, Stephen E. Moore, Craig B. Neylon

Background: Ca2 + -activated K + channels are crucially important in modulating neuronal excitability because they determine the shape of the repolarization phase of the action potential and contribute to after-hyperpolarizing potentials. Two major classes of Ca2 + activated potassium channels have been previously identified in nerve cells; large conductance (BK) channels and small conductance (SK) channels. However, there is evidence that these channels are not the channels responsible for after-hyperpolarizing potentials in myenteric AH neurons. Methods. Channel activity was recorded in myentenc AH neurons of the guinea pig small intestine with patch electrodes, mRNA and protein were extracted from isolated ganglia, and channels were localized by immunohistochemistry. Results: We detected inter- mediate conductance K channels (about 40 nS) in patch clamp, which were opened during the delayed afier-hyperpolarization following action potentials in AH type neurons. RT-PCR cloning from enteric ganglion extracts revealed a sequence with close sequence homology In the IK channel expressed other cell types. Using antisera raised against a peptide sequence from rat :K channels, we located IK channeI immunoreaetivity to myenteric neurons in the rat small intestine, but not gha or other cell types. We found a single immunoreactive band o[ approx. 55kD in Westem blots of ganglion protein. Conclusions: These results provide biophysical, gene sequence, Western blot and immunohistochemical data that 1K channels are expressed in enteric neurons. This is the first evidence for IK channels in enteric neurons, and indicates that these channels may be important in determining the excitability states of the enteric nervous system.

291

Enteric Sensory Neurons in the Mouse: Identification, Electrophysiology and Morphology W01fgang Kunze, Roland Khffscbenkel, Jutta Hahn, Mario Mueller, Wen Jiang, David Grundy

The mouse is a popular model species for studying gut disease, yet there have been few recordings from mouse enteric neurons. We have made whole cell patch clamp recordings from neurons in the longitudinal muscle myenteric plexus preparation using c578U6 mouse ileum Pipettes were rifled with a potassium rich saline containing 0.2O6 Neurubiotin. 2 classes of neurons were revealed: H-neurons had broad action potentials (1/2width = 2.3ms) with a prominent hump on the descending limb and a Vrest-53mV. S-neurons had narrower action potentials (1/2width~ 1.7ms) with no hump and a Vrest-49mV. Only H neurons had a Cs-sensinve hyperpolarization-activated cationic current (lh). This current had a V1/ 2-82mV, a Bofizmann sensitivity factor k ~ 12mV and a reversal potential of -29inV. They als0 expressed a persistent inward current with a threshold of -60inV. This current (INa,p) was unafkcted by extracellular 2M TTX or 0.5 mM CdC12, but substitution of NMDG+ for Na + abolished it. Unlike for guinea pig AH-neurons( 1 ), single spike, slow afterhyperpo- larlzation potentials (IAHP) were generally absent. H neurons (5/5) responded to ganglion dlstonion(2) with graded generator potentials and antidromic action potentials this response was sensory because it was not abolished by synaptic blockade with 10mM extracelhilar Mg2+. In contrast, 2/6 S neurons responded to distortion with fEPSPs; this response was dimivated by blocking synaptic transmission. Neurubiotin-staimng revealed that H neurons had Dogiel type 11 (DID morphology; S neurons were unipolar. We conclude that the mouse ikam contains sensory neurons that are DII morphologically; lh and 1Na,p currents and the hump but ROt 1AHP are conserved between guinea pig(I) and mouse. (I) Rugiero et aI 2002 J Physiol. 538:447-463 (2) Kunze et ai 2000 J Physiol. 526:375-85

292

Role Of Calcium Channels In Extrinsic Afferent Sensitivity In The Mouse Jejunum Mann Mueller, Martin Kreis, Joerg Glatzle, Wolfgang Kunze, David Grundy

Introduction: Enteric sensory neurons respond to a variety of mucosally applied chemicals in addition to mechanical stimulation. Extrinsic afferents respond to the same stimuli. We have previously postulated that there may be "crosstalk" between these two sensory systems but in vitro studies under Ca2 + -free conchtions, used in an attempt to synapticafly uncouple enteric and extrinsic afferents, proved inconclusive because of the altered neuronal excitability Ihat accompanies Ca2 removal. (Muefler MH etal. Gastroenterol 2002, G364). The aim of tht~ study therefore was to examine extrinsic afferent sensitivity following manipulation of synapfic transmission using the N-type Ca2 + blocker w-conotoxin GVIA and cadmium. Methods: Jejunal segments with mesenteric arcade attached were removed from pentoharhi- tone sodium (60rng/kg) anaesthetized mice and perfused in vitro with Kreb's buffer (340 C) Extracelluiar muhninit recordings were made from a paravascular nerve bundle within the mesenteric fan in a separate oil-filled recording chamber. The lumen was perfused separately wnh pH neutral buffered normal saline except when this was switched for 2 toni periods to acidified buffer containing HCI (pH 2) or buffer containing 5-hydroxytryptamine (5-HT, 500 ~M). Afferent responses to luminal stimulation or distension with saline up to 60 mmHg are quoted as peak increases over baseline (mean _+ SEM) and were compared using ANOVA followed by Bonferroni correction for multiple comparisons. Results: Disten- sion elicited a marked increase in afferent discharge from a baseline rate of 15 _+ 3 irnp/s to a maximum at 60 mmHg of 85 -4" 8 imp/s, n = 7). Acid and 5-HT evoked a robust but smaller increase in firing (peak 17-+ 3 imp/s, n = 10 and 21 _4_-4 imp/s, n = 13, respectively). In the presence of w-conotoxin (500aM) the peak responses to distension (80-+ 9 imp/s, n = 6), 5-8T (27 • 11 imp/s, n = 6 ) and acid (peak 20_+4 imp/s, n ~ 6 ) were unchanged (all P>005) Cadmium ions (250p, M) also had no effect on the peak response to distension /76 + 5 imp/s, n = 7, P>0.05) but significantly reduced the responses to acid (peak 6 _+ 2 imp/s, n ~ 7, P<0.05) and 5-HT (peak 9 _+ 3 imp/s, n = 7, P<0.05). Conclusion: The response of extrinsic afferents to distension appears to be direct and does not depend on synaptic mechanisms. In contrast, the response to acid and 5-HT are attenuated by blocking Ca2 +

channels with cadmium but not w-conotoxin suggesting an intermediary step in the afferent response to lumi~aI chemicals Supported by the fortuene-program University of Tuebingen

293

Expression of Temperature-Sensitive Ion Channels in Nodose Ganglia and Gut Tissue, Detected by RT-PCR and Immunohistochemistry Lei Zhang, Kate Brudy, Marcello Costa, Simon J. Brookes

Our understanding of the molecular basis of neuronal thermoreception has advanced with reports that 5 ion channels of the transient receptor potential (TRP) class are temperature sensitive; TRPvl (VR-1, capsaicin receptor), TRPv2 (VRL-1), TRPv3, TRPv4 (VR-OAC) and TRPm8 (CMR-1) have all been detected in dorsal root ganglia as either mRNA or protein. We have investigated the presence of these channels in the nodose ganglion of the mouse using a combination of reverse-transcriptase polymerase chain reaction (RT-PCR), immuno- histochemistry and retrograde labelling. Transcripts of 4 channels were strongly detected in the whole mouse nodose ganglion, but TRPv3 appeared to be weakly expressed. Both the position of the band on agarose gels and results of sequencing PCR products confirmed the authemicity of the amplified transcripts. TRPvl,2 and 4 were present at lower levels in the superior cervical ganglion and TRPv3 and TRPm8 were not detected in this sympathetic ganglion. We also tested for transcripts in single nodose cell bodies, retrogradely labeled from the stomach with the carbocyanine dye, Dil TRPvl,2,4 and TRPm8 have all been detected in single, labeled netlrons, indicating that they are present m a population of vagal primary afferents projecting to the upper gut. In the mouse gut wall, moderate levels of TRPv2 and v3 transcripts were detected in mnscularis externa from the stomach and ileum but not in mueosal tissue, whereas TRPv4 and TRPm8 were detected in ileal mucosa but were sparse or absent in the muscularis extema. Immunohistochemistry using TRPvl antibodies revealed strong TRI~I immunofiuorescence in a population of mouse nodose ganglion cells. The expression of a range of temperature-sensitive TRP channels in nodose ganglion cells provides a molecular basis for thermoreception by vagal primary afferent neurons to the upper gut. In addition, the presence of transcripts in mucosal and muscular layers of the gut wall suggest that these channels may have functional roles apart from temperature sensitivity This work was supported by a grant-in-aid from AstraZeneca

294

AS1C Ion Channels Contribute to Colonic Mechanosensory Function in Mice Stuart M. Brierley, John A. Wemmie, Michael J. Welsh, L. A. Blackshaw

Background: Mechannsensitive ion channels are essential for noxious and non-noxious sensing of the mechanical environment. They include ASIC, BNC1 and DRASIC. Deletion of BNC1 and DRAS1C may increase or decrease mechanosensory function in skin afferents (1,2); the role of ASIC is not yet reported. None of these channels have so far been investigated in visceral sensory pathways. In rat and mouse colon, there are three types of afferents: mucosal and muscular (low threshold to luminal stimuli) and serosal/mesenteric (high threshold). Aims: Investigate the role of the ASIC ion channel in mechanotransduction of high threshold colonic primary afferents using ASIC wild-type (WT) and null mutant (KO) mice. Methods: Using a novel in vitro mouse colonic afferent preparation (3), distal colon and lumbar splanchnic nerve (LSN) were removed intact. In a specialized organ bath, the colon was opened and pinned fiat. The INN was led through to an nil-filled chamber and single units recorded. 44 Serosal & mesenteric afferents were classibed according to the location of their mechanoreceptive field and responses to mechanical stimuli: blunt probing and application of calibrated yon Frey hairs (70, I60, :000rag for 3 sec). Results: SerosaI & mesenteric afferents in both wild-type and null mutant mice were rapidly adapting & responded in a graded manner to mechanical stimuli (70-1000rag). Disrupting the ASIC gone increased significantly the sensitivity of serosal & mesenteric mechanoreceptors to a 1000mg yon Frey hair. ASIC null-mutants generated significantly more action potentials per probe (1000rag) than wild-type mice (P<0.05) and displayed significantly higher instan- taneous frequencies (P<0.001) and mean frequencies (P<0.01) than wild-type mice. More- over, compared to wild-type mice ASIC null mutants demonstrated slower initial adaptation (WT: 0.3 sec to 50~ of initial response vs KO: O. 7 sec to 50% of initial response) followed by more rapid adaptation (WT: 1.9 sec to 25% of initial response vs KO: 1.1 sec to 25% of initial response). Conclusions: This study identifies a prominent contribution of the AS1C ion channel to the mechanosensory function of colonic high threshold primary afferents in the mouse. These results provide an insight into the fundamental mechanisms responsible for viscera/mechanosensation. Supported by NHMRC Australia & University of Adelaide. (1) Price MP etal., (2000) Nature 407, 1007-11 (2) Price MP e t a l , (2001) Neuron 32, 1071-83 (3) 8rierley & Blackshaw (2003) Proc Anst Neurosci Soc. 14. In press

295

Inhibit/on of Inducible Nitric Oxide Production Reverses The Inhibition of Na- Glucose Co-Transport in The Chronically Inflamed Rabbit Intestine Steven Coon, James K Kim, Urea Sundaram

Background. Na-ghicose co-transport (SGLT-1) is inhibited during chronic intestinal inflam- mation secondary to a decrease in the number of co-transporters. Constitutive nitric oxide has been shown to regulate SGLT-1 in the normal rabbit intestine. However, whether and how inducible nitric oxide (iNn), known to be elevated in the inflamed intestinal mucosa, may regulate the SGLT-1 alteration is unknown. Aims. Determine whether i n n mediates the inhibition of SGLT-1 during chronic enteritis. Methods. The rabbit model of niflarnmatory bowel disease induced by Eimeria magna was utilized. Villas cells were isolated from the rabbit intestine by a Ca + + chelation technique. Brush border membrane vesicles (BBMV) were prepared from villus cefis by Ca + + precipitation and differentia] centrifugation. Results. Treatment of rabbits with chronic enteritis with N6-(1-iminoethyl)-L-lysine, dihy- druchlorine (L-NIL), an inhibitor of inducible NO synthase, resulted in the reversal of inhihition on SGLT-I in villus cells (Na-dependent 3-O-methyI glucose (3-OMG) uptake was 3.9_0.5nmols/mg protein 5 minutes in normal, 0.9-+0.2 in inflamed and 40-+0.5

A-39 AGA Abstracts