Unpacking the homeostasis core concept in physiology: an Australian perspective.

Australia-wide consensus was reached on seven core concepts of physiology, which included homeostasis, a fundamental concept for students to understand as they develop their basic knowledge of physiological regulatory mechanisms. The term homeostasis is most commonly used to describe how the internal environment of mammalian systems maintains relative constancy. The descriptor "the internal environment of the organism is actively regulated by the responses of cells, tissues, and organs through feedback systems" was unpacked by a team of three Australian Physiology educators into 5 themes and 18 subthemes arranged in a hierarchy. Using a five-point Likert scale, the unpacked concept was rated by 24 physiology educators from 24 Australian Universities for level of importance and level of difficulty for students. Survey data were analyzed using a one-way ANOVA to compare between and within concept themes and subthemes. There were no differences in main themes for level of importance, with all ratings between essential or important. Theme 1: the organism has regulatory mechanisms to maintain a relatively stable internal environment, a process known as homeostasis was almost unanimously rated as essential. Difficulty ratings for unpacked concept themes averaged between slightly difficult and moderately difficult. The Australian team concurred with published literature that there are inconsistencies in the way the critical components of homeostatic systems are represented and interpreted. We aimed to simplify the components of the concept so that undergraduates would be able to easily identify the language used and build on their knowledge.NEW & NOTEWORTHY The homeostasis core concept of physiology was defined and unpacked by an Australian team with the goal of constructing a resource that will improve learning and teaching of this core physiology concept in an Australian Higher Education context.

Identification of a novel distension-evoked motility pattern in the mouse uterus.

The dynamic changes in uterine contractility in response to distension are incompletely understood. Rhythmic, propagating contractions of nonpregnant uterine smooth muscle occur in the absence of nerve activity (i.e., myogenic), events that decline during pregnancy and reemerge at parturition. We therefore sought to determine how myogenic contractions of the nonpregnant uterus are affected by distension, which might provide mechanistic clues underlying distension-associated uterine conditions such as preterm birth. Uteri isolated from nulliparous adult female mice in proestrus were video imaged to generate spatiotemporal maps, and myoelectrical activity simultaneously recorded using extracellular suction electrodes. Motility patterns were examined under basal conditions and following ramped intraluminal distension with fluid to 5 and 10 cmH2O. Intraluminal distension caused pressure-dependent changes in the frequency, amplitude, propagation speed, and directionality of uterine contractions, which reversed upon pressure release. Altered burst durations of underlying smooth muscle myoelectric events were concurrently observed, although action potential spike intervals were unchanged. Voltage-gated sodium channel blockade [tetrodotoxin (TTX); 0.6 µM] attenuated both the amplitude of contractions and burst duration of action potentials, whereas all activity was abolished by L-type calcium channel blockade (nifedipine; 1 µM). These data suggest that myogenic motility patterns of the nonpregnant mouse uterus are sensitive to changes in intraluminal pressure and, at high pressures, may be modulated by voltage-gated sodium channel activity. Future studies may investigate whether similar distension-evoked changes occur in the pregnant uterus and the possible pathophysiological role of such activity in the development of preterm birth.

Lesion development is modulated by the natural estrous cycle and mouse strain in a minimally invasive model of endometriosis.

Many rodent models of endometriosis are invasive, involving surgery to implant donor endometrial tissue into recipient animals. Moreover, few studies have compared and contrasted lesions between rodent strains and estrous stages without exogenous hormone manipulation. This is despite extensive data demonstrating that genetic and hormonal factors can influence endometriosis progression. Here, we have refined a minimally invasive model of endometriosis using naturally cycling mice (donor and recipient matched for cycle phase) to investigate lesion development in two different strains (C57BL/6 and BALB/c), induced in estrous stages of high and low estrogen (proestrus or estrus, respectively), and with varying amounts of donor endometrial tissue (7.5-40 mg), injected intraperitoneally. The overall probability of developing endometriosis-like lesions was higher in proestrus than estrus, and increased with greater masses of donor tissue. Similarly, the total number of lesions (0-3) increased from 7.5 to 40 mg, and was significantly greater in proestrus C57BL/6 mice but not BALB/cs. The dominant lesion type also differed between mouse strains; C57BL/6 mice were more likely to develop dense-type lesions, whereas BALB/c mice developed a greater proportion of cystic type. These data further support a role for estrogen in the development of endometriosis, and that genetic variance can influence the degree and characteristics of lesions. Our minimally invasive model would be beneficial for studies with outcome measurements particularly sensitive to incisional injury, such as pain, or alterations to sex hormones, including fertility.

Uterine Contractility in the Nonpregnant Mouse: Changes During the Estrous Cycle and Effects of Chloride Channel Blockade.

Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca(2+)-activated Cl(-) currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 µM), anthracene-9-carboxylic acid (0.1-1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 µM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl(-) channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca(2+)-activated Cl(-) channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl(-) channel.

AqF026 is a pharmacologic agonist of the water channel aquaporin-1.

Aquaporin-1 (AQP1) facilitates the osmotic transport of water across the capillary endothelium, among other cell types, and thereby has a substantial role in ultrafiltration during peritoneal dialysis. At present, pharmacologic agents that enhance AQP1-mediated water transport, which would be expected to increase the efficiency of peritoneal dialysis, are not available. Here, we describe AqF026, an aquaporin agonist that is a chemical derivative of the arylsulfonamide compound furosemide. In the Xenopus laevis oocyte system, extracellular AqF026 potentiated the channel activity of human AQP1 by >20% but had no effect on channel activity of AQP4. We found that the intracellular binding site for AQP1 involves loop D, a region associated with channel gating. In a mouse model of peritoneal dialysis, AqF026 enhanced the osmotic transport of water across the peritoneal membrane but did not affect the osmotic gradient, the transport of small solutes, or the localization and expression of AQP1 on the plasma membrane. Furthermore, AqF026 did not potentiate water transport in Aqp1-null mice, suggesting that indirect mechanisms involving other channels or transporters were unlikely. Last, in a mouse gastric antrum preparation, AqF026 did not affect the Na-K-Cl cotransporter NKCC1. In summary, AqF026 directly and specifically potentiates AQP1-mediated water transport, suggesting that it deserves additional investigation for applications such as peritoneal dialysis or clinical situations associated with defective water handling.

Morphological identification of thoracolumbar spinal afferent nerve endings in mouse uterus.

Major sensory innervation to the uterus is provided by spinal afferent nerves, whose cell bodies lie predominantly in thoracolumbar dorsal root ganglia (DRG). While the origin of the cell bodies of uterine spinal afferents is clear, the identity of their sensory endings has remained unknown. Hence, our major aim was to identify the location, morphology, and calcitonin gene-related peptide (CGRP)-immunoreactivity of uterine spinal afferent endings supplied by thoracolumbar DRG. We also sought to determine the degree of uterine afferent innervation provided by the vagus nerve. Using an anterograde tracing technique, nulliparous female C57BL/6 mice were injected unilaterally with biotinylated dextran into thoracolumbar DRG (T13-L3). After 7-9 days, uterine horns were stained to visualize traced nerve axons and endings immunoreactive to CGRP. Whole uteri from a separate cohort of animals were injected with retrograde neuronal tracer (DiI) and dye uptake in nodose ganglia was examined. Anterogradely labeled axons innervated each uterine horn, these projected rostrally or caudally from their site of entry, branching to form varicose endings in the myometrium and/or vascular plexus. Most spinal afferent endings were CGRP-immunoreactive and morphologically classified as "simple-type." Rarely, uterine nerve cell bodies were labeled in nodose ganglia. Here, we provide the first detailed description of spinal afferent nerve endings in the uterus of a vertebrate. Distinct morphological types of spinal afferent nerve endings were identified throughout multiple anatomical layers of the uterine wall. Compared to other visceral organs, uterine spinal afferent endings displayed noticeably less morphological diversity. Few neurons in nodose ganglia innervate the uterus.

Spinal Glial Adaptations Occur in a Minimally Invasive Mouse Model of Endometriosis: Potential Implications for Lesion Etiology and Persistent Pelvic Pain.

Glial adaptations within the central nervous system are well known to modulate central sensitization and pain. Recently, it has been suggested that activity of glial-related proinflammatory cytokines may potentiate peripheral inflammation, via central neurogenic processes. However, a role for altered glial function has not yet been investigated in the context of endometriosis, a chronic inflammatory condition in women associated with peripheral lesions, often manifesting with persistent pelvic pain. Using a minimally invasive mouse model of endometriosis, we investigated associations between peripheral endometriosis-like lesions and adaptations in central glial reactivity. Spinal cords (T13-S1) from female C57BL/6 mice with endometriosis-like lesions (ENDO) were imaged via fluorescent immunohistochemistry for the expression of glial fibrillary acidic protein (GFAP; astrocytes) and CD11b (microglia) in the dorsal horn (n = 5). Heightened variability ( P = .02) as well as an overall increase ( P = .04) in the mean area of GFAP immunoreactivity was found in ENDO versus saline-injected control animals. Interestingly, spinal levels showing the greatest alterations in GFAP immunoreactivity appeared to correlate with the spatial location of lesions within the abdominopelvic cavity. A subtle but significant increase in the mean area of CD11b immunostaining was also observed in ENDO mice compared to controls ( P = .02). This is the first study to describe adaptations in nonneuronal, immune-like cells of the central nervous system attributed to the presence of endometriosis-like lesions.

Functional and molecular identification of pH-sensitive K+ channels in murine urinary bladder smooth muscle.

OBJECTIVE: To examine the role of pH-sensitive K(+) channels in setting the resting membrane potential in murine bladder smooth muscle, as bladder contractility is influenced by the resting membrane potential, which is mainly regulated by background K(+) conductances. MATERIALS AND METHODS: Using conventional microelectrode recordings, isometric tension measurements, patch-clamp recordings, reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry, we assessed bladder smooth muscle cells and tissues. RESULTS: Acidic pH (pH 6.5) depolarized the resting membrane potential of murine bladder smooth muscles and increased muscle tone and contractility. The pH-induced changes were not abolished by neuronal blockers or classical K(+)-channel antagonists. Lidocaine (1 mM) and bupivacaine (100 microm) mimicked the effects of acidifying the external solution, and in the presence of lidocaine no further increase in contractility was induced by reducing the pH to 6.5. Voltage-clamp experiments on freshly dispersed bladder myocytes showed that pH 6.5 decreased the outward current. Pre-treatment of bladder myocytes with the classical K(+) antagonists tetraethylammonium (10 mm), 4-aminopyridine (5 mM), glibenclamide (10 microm) or apamin (300 nM) did not inhibit the effects of low pH on outward current. However, treatment with lidocaine (1 mM) abolished the effects of acidic pH on outward current. RT-PCR showed the expression of the acid-sensitive K(+) channel (TASK)-1 and TASK-2 gene transcripts in murine bladder, and immunohistochemistry and Western blot analysis showed TASK-1 and TASK-2 channel expression and distribution in smooth muscle tissues and cells. CONCLUSION: TASK channels are expressed in bladder smooth muscle and contribute to the basal K(+) conductances responsible for resting membrane potential.

Differential effect of morphine on gastrointestinal transit, colonic contractions and nerve-evoked relaxations in Toll-Like Receptor deficient mice.

Toll-like receptors (TLRs) are expressed in enteric neurons, glia, gastrointestinal (GI) smooth muscle and mucosa, yet their functional roles in the GI tract are not fully understood. TLRs have been linked to many of the undesirable central effects of chronic opioid administration including hyperalgesia and dependence via activation of central microglia. Opioid-induced bowel dysfunction (OIBD) remains a primary reason for the reduction or withdrawal of opioid analgesics. Morphine-induced inhibition of colonic motility was assessed in vivo by GI transit studies and in vitro using isolated colons from wildtype (WT) and TLR deficient mice. Morphine slowed movement of ingested content in WT but this retardation effect was attenuated in TLR4 -/- and TLR2/4 -/- . In isolated colons, morphine reduced amplitude and frequency colonic migrating motor contractions in both WT and TLR2/4 -/- . Electrical field stimulation elicited distal colon relaxation that was potentiated by morphine in WT but not in TLR2/4 -/- . Inhibitory junction potentials were of similar amplitude and kinetics in WT and TLR2/4 -/- distal colon and not altered by morphine. Enteric nerve density and proportion of nitrergic nerves were similar in WT and TLR2/4 -/- distal colon. These data suggest an involvement of TLRs in opioid pharmacodynamics and thus a potential interventional target for OIBD.

Inhibitory responses mediated by vagal nerve stimulation are diminished in stomachs of mice with reduced intramuscular interstitial cells of Cajal.

Intramuscular interstitial cells of Cajal (ICC-IM) are closely associated with enteric motor nerve terminals and electrically coupled to smooth muscle cells within the gastric musculature. Previous studies investigating the role of ICC-IM in motor neurotransmission have used indiscriminate electric field stimulation of neural elements within the gastric wall. To determine the role of ICC-IM in transduction of vagally-mediated motor input to gastric muscles electrical and mechanical responses to selective electrical vagal stimulation (EVS) were recorded from gastric fundus and antral regions of wild type and W/WV mice, which lack most ICC-IM. EVS evoked inhibitory junction potentials (IJPs) in wild type muscles that were attenuated or abolished by L-NNA. IJPs were rarely evoked in W/WV muscles by EVS, and not affected by L-NNA. EVS evoked relaxation of wild type stomachs, but the predominant response of W/WV stomachs was contraction. EVS applied after pre-contraction with bethanechol caused relaxation of wild type gastric tissues and these were inhibited by the nitric oxide synthase inhibitor L-NNA. Relaxation responses were of smaller amplitude in W/WV muscles and L-NNA did not attenuate relaxation responses in W/WV fundus muscles. These data suggest an important role for ICC-IM in vagally-mediated nitrergic relaxation in the proximal and distal stomach.

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/W(V) 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/W(V) 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/W(V) 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.

Microarray comparison of normal and W/Wv mice in the gastric fundus indicates a supersensitive phenotype.

Interstitial cells of Cajal (ICC) have been identified in specific areas throughout the smooth musculature of the gastrointestinal (GI) tract. Located within the circular and longitudinal muscle layers of the gastric fundus lies a specific type of ICC, termed "intramuscular" ICC or IC-IM. The principal function of this cell type is to act as "mediators of excitatory and inhibitory enteric neurotransmission." The functional role of these cells has been investigated using W/W(v) mutant mice that specifically lack IC-IM, resulting in disrupted enteric neurotransmission. The aim of the present study was to investigate differential gene expression in W/W(v) mutant mice, from the tunica muscularis of the gastric fundus using a mouse cDNA microarray containing 1,081 known genes. Verification of the microarray data was attained using real-time "quantitative" PCR (qPCR). Of the 1,081 arrayed genes, 36 demonstrated differential expression by >2-fold in the W/W(v) mice. An agreement rate of 50% (7 of 14 tested) was obtained using qPCR. Of the seven confirmed changes in expression, several were indicative of a supersensitive phenotype, observed in denervation models. Expression of several putative neurotransmitter receptors including P2Y, the receptor for the inhibitory neurotransmitter ATP, was upregulated. The functional role of the P2Y receptor was also investigated using electrophysiological recordings. These results offer a new insight into the molecular changes that occur in W/W(v) fundic smooth muscle and may also provide novel information with regard to the importance of IC-IM in enteric neurotransmission.

Characterization of primary afferent spinal innervation of mouse uterus.

The primary afferent innervation of the uterus is incompletely understood. The aim of this study was to identify the location and characteristics of primary afferent neurons that innervate the uterine horn of mice and correlate the different morphological types of putative primary afferent nerve endings, immunoreactive to the sensory marker, calcitonin gene related peptide (CGRP). Using retrograde tracing, injection of 5-10 µL of 1,1'-didodecyl-3,3,3,3'-tetramethylindocarbocyanine perchlorate (DiI) into discrete single sites in each uterine horn revealed a biomodal distribution of sensory neurons in dorsal root ganglia (DRG) with peak labeling occurring between T13-L3 and a second smaller peak between L6-S1. The mean cross sectional area of labeled cells was 463 µm(2) ± s.e.m. A significantly greater proportion of labeled neurons consisted of small cell bodies (<300 µm(2)) in the sacral spinal cord (S2) compared with peak labeling at the lumbar (L2) region. In both sections and whole mount preparations, immunohistochemical staining for CGRP revealed substantial innervation of the uterus by CGRP-positive nerve fibers located primarily at the border between the circular and longitudinal muscle layers (N = 4). The nerve endings were classified into three distinct types: "single," "branching," or "complex," that often aligned preferentially in either the circular or longitudinal axis of the smooth muscles. Complex endings were often associated with mesenteric vessels. We have identified that the cell bodies of primary afferent neurons innervating the mouse uterus lie primarily in DRG at L2 and S1 spinal levels. Also, the greatest density of CGRP immunoreactivity lies within the myometrium, with at least three different morphological types of nerve endings identified. These findings will facilitate further investigations into the mechanisms underlying sensory transduction in mouse uterus.

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 W(V)/+ heterozygotes, but failed to develop between E17 to P0 in W/W(V) 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.

Effects of female steroid hormones on A-type K+ currents in murine colon.

Idiopathic constipation is higher in women of reproductive age than postmenopausal women or men, suggesting that female steroid hormones influence gastrointestinal motility. How female hormones affect motility is unclear. Colonic motility is regulated by ion channels in colonic myocytes. Voltage-dependent K(+) channels serve to set the excitability of colonic muscles. We investigated regulation of Kv 4.3 channel expression in response to acute or chronic changes in female hormones. Patch clamp experiments and quantitative PCR were used to compare outward currents and transcript expression in colonic myocytes from male, non-pregnant, pregnant and ovariectomized mice. Groups of ovariectomized mice received injections of oestrogen or progesterone to investigate the effects of hormone replacement. The capacitance of colonic myocytes from non-pregnant females was larger than in males. Net outward current density in male and ovariectomized mice was higher than in non-pregnant females and oestrogen-treated ovariectomized mice. Current densities in late pregnancy were lower than in female controls. Progesterone had no effect on outward currents. A-type currents were decreased in non-pregnant females compared with ovariectomized mice, and were further decreased by pregnancy or oestrogen replacement. Kv 4.3 transcripts did not differ significantly between groups; however, expression of the potassium channel interacting protein KChIP1 was elevated in ovariectomized mice compared with female controls and oestrogen-treated ovariectomized mice. Delayed rectifier currents were not affected by oestrogen. In the mouse colon, oestrogen suppresses A-type currents, which are important for regulating excitability. These observations suggest a possible link between female hormones and altered colonic motility associated with menses, pregnancy and menopause.

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/W(V) 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/W(V) 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/W(V) 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.

Loss of enteric motor neurotransmission in the gastric fundus of Sl/Sl(d) mice.

Studies of W/W(V) 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/Sl(d) 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/Sl(d) 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/Sl(d) mice. Responses to neurally released ACh were revealed in Sl/Sl(d) 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/Sl(d) 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.

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues.

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.

Regulation of pacemaker frequency in the murine gastric antrum.

PGE(2) 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 PGE(2) 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. PGE(2) 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, EP(2) and EP(4) receptor agonists, mimicked the effects of forskolin and 8-Br-cAMP on ICC and intact muscles. Sulprostone (EP(3)>EP(1) agonist), GR63799, and ONO-AE-248 (EP(3) 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 EP(1) receptor antagonist. These observations suggest that the positive chronotropic effects of PGE(2) in intact muscles are mediated by EP(3) receptor stimulation. The effects of PGE(2) in intact muscles may be dependent upon the relative expression of EP receptors and/or proximity of receptors to sources of PGE(2).