Elizabeth A. H. Beckett

Loss of Enteric Motor Neurotransmission in the Gastric Fundus of Sl/Sld Mice

Studies of W/WV mice, which lack intramuscular interstitial cells of Cajal (IC‐IM), have suggested that IC‐IM act as mediators of enteric motor neurotransmission in the gastrointestinal tract. We have studied Sl/Sld mice, which lack the ability to make membrane‐bound stem cell factor, to determine the consequences of inappropriate stem cell factor expression on IC‐IM populations and on enteric motor neurotransmission. IC‐IM were found within the circular and longitudinal muscles of the gastric fundus of wild‐type mice. IC‐IM were intimately associated with motor nerve terminals and nerve varicosities formed synaptic structures with these cells. IC‐IM were also connected with neighbouring smooth muscle cells via gap junctions. Immunohistochemistry and electron microscopy showed that IC‐IM were absent from fundus muscles of Sl/Sld mice, but the density of excitatory and inhibitory nerves was not significantly different than in wild‐type muscles. Loss of IC‐IM was associated with decreased membrane noise (unitary potentials) and significant reductions in post‐junctional excitatory and inhibitory enteric nerve responses. Reductions in neural responses were not due to defects in smooth muscle cells as responses to exogenous ACh and K+‐induced depolarization were normal in Sl/Sld mice. Responses to neurally released ACh were revealed in Sl/Sld mice by inhibiting ACh breakdown with the acetylcholinesterase inhibitor neostigmine. Inhibitory nerve stimulation elicited inhibitory junction potentials (IJPs) and relaxations in wild‐type mice. IJPs were reduced in amplitude and relaxation responses were absent in Sl/Sld mice. These observations suggest that membrane‐bound stem cell factor is essential for development of IC‐IM and that the close, synaptic‐like relationship between nerve terminals and IC‐IM may be the primary site of innervation by enteric motor neurons in gastric muscles.

Inhibition of Aquaporin-1 and Aquaporin-4 water permeability by a derivative of the loop diuretic bumetanide acting at an internal pore-occluding binding site

Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na+-K+-2Cl- cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (≥100 μM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC50 ∼20 μM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC50 ∼8 μM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 μM had no effect, and 20 μM caused 20% block of human Na+-K+-2Cl- cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl- currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Regional variation in contribution of myenteric and intramuscular interstitial cells of Cajal to generation of slow waves in mouse gastric antrum

When intracellular recordings were made from the antral region of murine stomach, cells with three different patterns of electrical activity were detected. One group of cells generated follower potentials, the second group generated pacemaker potentials and the third group generated slow waves that consisted of primary and secondary components. Slow waves recorded in different regions of the gastric antrum had similar amplitudes but different characteristic shapes. At the greater curvature, slow waves had large initial components. Midway between the greater and lesser curvature, the amplitude of the initial component was reduced and at the lesser curvature an initial component was difficult to detect. When the distributions of myenteric (ICC‐MY) and intramuscular interstitial cells of Cajal (ICC‐IM) were determined, using an antibody to Kit, ICC‐MY were found to be present at the greater curvature but were greatly reduced in density at the lesser curvature. In contrast, ICC‐IM were found in the circular layer of each region. When recordings were made from the antrum of W/WV mice, which lack ICC‐IM, incomplete slow waves were detected and their amplitudes fell from the greater to the lesser curvature. Again, a corresponding fall in the density of ICC‐MY was detected. The observations indicate that the contribution of ICC‐MY and ICC‐IM to the generation of slow waves varies in different regions of the mouse gastric antrum.

Synaptic specializations exist between enteric motor nerves and interstitial cells of Cajal in the murine stomach

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC‐IM) in the stomach and colon and ICC in the deep muscular plexus (ICC‐DMP) of the small intestine. In the absence of ICC‐IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC‐IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse‐associated proteins between nerve terminals and ICC‐IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP‐25 were detected. Synaptotagmin and SNAP‐25‐immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC‐IM but not smooth muscle. W/WV mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC‐IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP‐25 were detected in W/WV tissues. In the absence of ICC‐IM, synaptotagmin and SNAP‐25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT‐PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD‐93 and PSD‐95 in the stomach and expression levels of PSD‐93 and PSD‐95 were reduced in W/WV mutants. These data support the existence of synaptic specializations between enteric nerves and ICC‐IM in gastric tissues. In the absence of ICC‐IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals. J. Comp. Neurol. 493:193–206, 2005.

Kit signaling is essential for development and maintenance of interstitial cells of Cajal and electrical rhythmicity in the embryonic gastrointestinal tract

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC‐MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC‐MY networks and pacemaker activity. ICC‐MY and pacemaker activity developed normally in W/+ and WV/+ heterozygotes, but failed to develop between E17 to P0 in W/WV embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17‐P0 for 3 days caused loss of functionally developed ICC‐MY networks, but ICC‐MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC‐MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit‐positive cell population is either lost or amplified. Developmental Dynamics, 2006.

Pacing of interstitial cells of Cajal in the murine gastric antrum: neurally mediated and direct stimulation

Phase advancement of electrical slow waves and regulation of pacemaker frequency was investigated in the circular muscle layer of the gastric antra of wild‐type and W/WV mice. Slow waves in the murine antrum of wild‐type animals had an intrinsic frequency of 4.4 cycles min−1 and were phase advanced and entrained to a maximum of 6.3 cycles min−1 using 0.1 ms pulses of electrical field stimulation (EFS) (three pulses delivered at 3–30 Hz). Pacing of slow waves was blocked by tetrodotoxin (TTX) and atropine, suggesting phase advancement was mediated via intrinsic cholinergic nerves. Phase advancement and entrainment of slow waves via this mechanism was absent in W/WV mutants which lack intramuscular interstitial cells of Cajal (ICC‐IM). These data suggest that neural regulation of slow wave frequency and regulation of smooth muscle responses to slow waves are mediated via nerve‐ICC‐IM interactions. With longer stimulation parameters (1.0–2.0 ms), EFS phase advanced and entrained slow waves in wild‐type and W/WV animals. Pacing with 1–2 ms pulses was not inhibited by TTX or atropine. These data suggest that stimulation with longer pulse duration is capable of directly activating the pacemaker mechanism in ICC‐MY networks. In summary, intrinsic excitatory neurons can phase advance and increase the frequency of antral slow waves. This form of regulation is mediated via ICC‐IM. Longer pulse stimulation can directly activate ICC‐MY in the absence of ICC‐IM.

The first intestinal motility patterns in fetal mice are not mediated by neurons or interstitial cells of Cajal

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC‐MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC‐MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W/Wv embryos, which lack KIT+ ICC‐MP and slow waves. Third, KIT‐immunoreactive cells with the morphology of ICC‐MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC‐MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L‐type Ca2+ channel antagonist nicardipine (2.5 μm) abolished ripples in the duodenum and reduced their frequency and size in the colon. Our findings demonstrate that prominent propagating contractions (ripples) are present in the duodenum and colon of fetal mice. Ripples are not mediated by neurons or ICC‐MP, but entry of extracellular Ca2+ through L‐type Ca2+ channels is essential. Thus, during development of the intestine, the first motor patterns to develop are myogenic.

Properties of unitary potentials generated by intramuscular interstitial cells of Cajal in the murine and guinea-pig gastric fundus

Intracellular recordings were made from isolated bundles of the circular muscle layer of mouse and guinea‐pig gastric fundus. These preparations displayed an ongoing discharge of membrane noise (unitary potentials), similar to that recorded from similar preparations made from the circular layer of the antrum. Bundles of muscle from the fundus of W/WV mice, which lack intramuscular interstitial cells of Cajal (ICCIM) lacked the discharge of membrane noise observed in wild‐type tissues. When the membrane potential was changed by passing depolarizing or hyperpolarizing current pulses, the discharge of membrane noise was little changed. The membrane noise was unaffected by adding chloride channel blockers; however, agents which buffered the internal concentration of calcium ions reduced the discharge of membrane noise. Treatment of tissues with CCCP, which interferes with the uptake of calcium ions by mitochondria, also reduced the membrane noise and caused membrane hyperpolarization. Similar observations were made on bundles of tissue isolated from the circular layer of the guinea pig antrum. Together the observations indicate that membrane noise is generated by a pathway located in ICCIM. The properties of this pathway appear to vary dramatically within a given organ. The lack of voltage sensitivity of the discharge of membrane noise in the fundus provides a possible explanation for the lack of rhythmic electrical activity in this region of the stomach.

Regulation of pacemaker frequency in the murine gastric antrum

PGE2 has been linked to the production of gastric arrhythmias such as tachygastria. The interstitial cells of Cajal (ICC) generate electrical rhythmicity in gastrointestinal muscles, and may therefore be a target for PGE2 in gastric muscles. We cultured ICC from the murine gastric antrum, verified that cells were Kit immunoreactive, and measured spontaneous slow waves. These events were caused by spontaneous inward (pacemaker) currents that were not blocked by nifedipine. Forskolin and 8‐bromoadenosine 3′:5′‐cyclic monophosphate (8‐Br‐cAMP) reduced the frequency of pacemaker currents in ICC and of slow waves in intact antral muscles. The effects of forskolin and 8‐Br‐cAMP were not blocked by inhibitors of protein kinase A, suggesting that cAMP has direct effects on pacemaker activity. PGE2 mimicked the effects of forskolin and 8‐Br‐cAMP on ICC, but increased slow‐wave frequency in intact muscles. Therefore, the chronotropic effects of specific prostaglandin EP receptor agonists were examined. Butaprost and ONO‐AE1‐329, EP2 and EP4 receptor agonists, mimicked the effects of forskolin and 8‐Br‐cAMP on ICC and intact muscles. Sulprostone (EP3>EP1 agonist), GR63799, and ONO‐AE‐248 (EP3 agonists) enhanced the frequencies of pacemaker currents in ICC and slow waves in intact muscles. The effects of sulprostone were not blocked by SC‐19220, an EP1 receptor antagonist. These observations suggest that the positive chronotropic effects of PGE2 in intact muscles are mediated by EP3 receptor stimulation. The effects of PGE2 in intact muscles may be dependent upon the relative expression of EP receptors and/or proximity of receptors to sources of PGE2.

A pH-sensitive potassium conductance (TASK) and its function in the murine gastrointestinal tract

The excitability of smooth muscles is regulated, in part, by background K+ conductances that determine resting membrane potential. However, the K+ conductances so far described in gastrointestinal (GI) muscles are not sufficient to explain the negative resting potentials of these cells. Here we describe expression of two‐pore K+ channels of the TASK family in murine small and large intestinal muscles. TASK‐2, cloned from murine intestinal muscles, resulted in a pH‐sensitive, time‐dependent, non‐inactivating K+ conductance with slow activation kinetics. A similar conductance was found in native intestinal myocytes using whole‐cell patch‐clamp conditions. The pH‐sensitive current was blocked by local anaesthetics. Lidocaine, bupivacaine and acidic pH depolarized circular muscle cells in intact muscles and decreased amplitude and frequency of slow waves. The effects of lidocaine were not blocked by tetraethylammonium chloride, 4‐aminopyridine, glibenclamide, apamin or MK‐499. However, depolarization by acidic pH was abolished by pre‐treatment with lidocaine, suggesting that lidocaine‐sensitive K+ channels were responsible for pH‐sensitive changes in membrane potential. The kinetics of activation, sensitivity to pH, and pharmacology of the conductance in intestinal myocytes and the expression of TASK‐1 and TASK‐2 in these cells suggest that the pH‐sensitive background conductance is encoded by TASK genes. This conductance appears to contribute significantly to resting potential and may regulate excitability of GI muscles.