Biomedical subjects

K M Sanders

Publications and source records attributed to K M Sanders.

At least 19 recordsLinked to original sources

PubMed2000

Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons.

Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal muscles. The role of intramuscular ICC (IC-IM) in mediating enteric excitatory neural inputs was studied using gastric fundus muscles of wild-type animals and W/W(v) mutant mice, which lack IC-IM. Excitatory motor neurons, labeled with antibodies to vesicular acetylcholine transporter or substance-P, were closely associated with IC-IM. Immunocytochemistry showed close contacts between enteric neurons and IC-IM. IC-IM also formed gap junctions with smooth muscle cells. Electrical field stimulation yielded fast excitatory junction potentials in the smooth muscle that were blocked by atropine. Neural responses were greatly reduced in muscles of W/W(v) animals. Loss of cholinergic responses in W/W(v) muscles seemed to be caused by the loss of close synaptic contacts between motor neurons and IC-IM, because these muscles were not less responsive to exogenous acetylcholine than were wild-type muscles. W/W(v) muscles also responded to excitatory nerve stimulation when the breakdown of acetylcholine was blocked by neostigmine. The density of cholinergic nerve bundles within the muscles was not significantly different in wild-type and W/W(v) muscles, and similar amounts of (14)[C]choline were released from preloaded wild-type and W/W(v) muscles in response to nerve stimulation. The impact of losing IC-IM on gastric compliance was also evaluated in intact stomachs. Pressure increased as a function of fluid volume and infusion rate in wild-type animals, but W/W(v) animals showed little basal tone and minimal increases in pressure with fluid infusions. These data suggest that IC-IM play a major role in receiving cholinergic excitatory inputs from the enteric nervous system in the murine fundus.

Acetylcholine↗

PubMed2000

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes.

Spontaneous transient outward currents (STOCs) were recorded from smooth muscle cells of the guinea pig taenia coli using the whole cell patch-clamp technique. STOCs were resolved at potentials positive to -50 mV. Treating cells with caffeine (1 mM) caused a burst of outward currents followed by inhibition of STOCs. Replacing extracellular Ca(2+) with equimolar Mn(2+) caused STOCs to "run down. " Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM) inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs" remained in the presence of these drugs. Mini-STOCs were reduced by apamin (500 nM), an inhibitor of small-conductance Ca(2+)-activated K(+) channels (SK channels). Application of ATP or 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATP persisted in the presence of charybdotoxin but were blocked by combination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did not increase STOCs in the presence of pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, a P(2) receptor blocker. Similarly, pretreatment of cells with U-73122 (1 microM), an inhibitor of phospholipase C (PLC), abolished the effects of 2-MeS-ATP. Xestospongin C, an inositol 1,4,5-trisphosphate (IP(3)) receptor blocker, attenuated STOCs, but these events were not affected by ryanodine. The data suggest that purinergic activation through P(2Y) receptors results in localized Ca(2+) release via PLC- and IP(3)-dependent mechanisms. Release of Ca(2+) is coupled to STOCs, which are composed of currents mediated by large-conductance Ca(2+)-activated K(+) channels and SK channels. The latter are thought to mediate hyperpolarization and relaxation responses of gastrointestinal muscles to inhibitory purinergic stimulation.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1999

Rebound excitation and alternating slow wave patterns depend upon eicosanoid production in canine proximal colon.

1. We tested the hypothesis that eicosanoid production could be related to the long-duration slow waves that occur after brief periods of inhibitory neurotransmission (rebound excitation) and the alternating patterns of long- and short-duration slow waves observed in the canine proximal colon. 2. Electrical field stimulation of colonic muscles inhibited slow waves during the stimulus and a long-duration slow wave occurred after the stimulus. Indomethacin reduced the post-stimulus response without affecting the inhibitory response. 3. ATP or 2-methylthio-ATP produced post-stimulus rebound responses similar to the response to field stimulation. Indomethacin inhibited the rebound response caused by ATP or 2-methylthio-ATP. 4. Alternating patterns consisting of long- and short-duration slow waves occurred spontaneously in some colonic muscles. These patterns could also be induced with acetylcholine. 5. Indomethacin, acetylsalicylic acid and ibuprofen abolished the alternating pattern and shifted the bimodal distribution of slow wave durations toward an intermediate duration. 6. Patch clamp experiments on isolated colonic myocytes showed that indomethacin blocked L-type Ca2+ currents. The effects of indomethacin on rebound excitation and alternating slow waves were accomplished at concentrations that blocked cyclooxygenase activity without significantly inhibiting L-type Ca2+ currents. 7. The results demonstrate that rebound excitation and alternating slow wave patterns in the canine colon have similar dependence on endogenous eicosanoid production. Rebound excitation may result from reduced production of an inhibitory eicosanoid during inhibitory nerve stimulation, and the alternating pattern may result from oscillations in eicosanoid production as a function of changes in cytoplasmic Ca2+ during long and short slow waves.

Animals↗

PubMed1999

Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle.

1. The membrane potential of gastrointestinal smooth muscles determines the open probability of ion channels involved in rhythmic electrical activity. The role of Ba2+-sensitive K+ conductances in the maintenance of membrane potential was examined in canine proximal colon circular muscle. 2. Application of Ba2+ (1-100 microM) to strips of tunica muscularis produced depolarization of cells along the submucosal surface of the circular muscle layer. Significantly higher concentrations of Ba2+ were needed to depolarize preparations from which the submucosal and myenteric pacemaker regions were removed. 3. Elevation of extracellular [K+]o (from 5.9 to 12 mM) brought membrane potentials closer to EK (the Nernst potential for K+ ions), suggesting activation of a K+ conductance. This occurred at potentials much more negative than the activation range for delayed rectifier channels (Kv). 4. Forskolin (1 microM) caused hyperpolarization and a leftward shift in the dose-response relationship for Ba2+, suggesting that forskolin may activate a Ba2+-sensitive conductance. 5. Patch-clamp recordings from interstitial cells of Cajal (ICC) revealed the presence of a Ba2+-sensitive inward rectifier potassium conductance. Far less of this conductance was present in smooth muscle cells. 6. Kir2.1 was expressed in the circular muscle layer of the canine proximal colon, duodenum, jejunum and ileum. Kir2.1 mRNA was expressed in greater abundance along the submucosal surface of the circular muscle layer in the colon. 7. These results demonstrate that ICC express a Ba2+-sensitive conductance (possibly encoded by Kir2.1). This conductance contributes to the generation and maintenance of negative membrane potentials between slow waves.

Animals↗

PubMed1999

Interstitial cells of cajal generate electrical slow waves in the murine stomach.

1. The gastric corpus and antrum contain interstitial cells of Cajal (ICC) within the tunica muscularis. We tested the hypothesis that ICC are involved in the generation and regeneration of electrical slow waves. 2. Normal, postnatal development of slow wave activity was characterized in tissues freshly removed from animals between birth and day 50 (D50). Slow wave amplitude and frequency increased during this period. Networks of myenteric ICC (IC-MY) were present in gastric muscles at birth and did not change significantly in appearance during the period of study as imaged by confocal immunofluorescence microscopy. 3. IC-MY networks were maintained and electrical rhythmicity developed in organ culture in a manner similar to normal postnatal development. Electrical activity was maintained for at least 48 days in culture. 4. Addition of a neutralizing antibody (ACK2) for the receptor tyrosine kinase, Kit, to the culture media caused progressive loss of Kit-immunoreactive cells. Loss of Kit-immunoreactive cells was associated with loss of slow wave activity. Most muscles became electrically quiescent after 3-4 weeks of exposure to ACK2. 5. In some muscles small clusters of Kit-immunoreactive IC-MY remained after culturing with ACK2. These muscles displayed slow wave activity but only in the immediate regions in which Kit-positive IC-MY remained. These data suggest that regions without Kit-immunoreactive cells cannot generate or regenerate slow waves. 6. After loss of Kit-immunoreactive cells, the muscles could not be paced by direct electrical stimulation. Stimulation with acetylcholine also failed to elicit slow waves. The data suggest that the generation of slow waves is an exclusive property of IC-MY; smooth muscle cells may not express the ionic apparatus necessary for generation of these events. 7. We conclude that IC-MY are an essential element in the spontaneous rhythmic electrical and contractile activity of gastric muscles. This class of ICC appears to generate slow wave activity and may provide a means for regeneration of slow waves.

Animals↗

PubMed1999

Health burden of hip and other fractures in Australia beyond 2000. Projections based on the Geelong Osteoporosis Study.

OBJECTIVE: To calculate the expected increase in the number of fractures in adults attributable to the predicted increase in the number of elderly Australians. DATA SOURCES: All fractures in adult residents (> or = 35 years) of the Barwon Statistical Division (total population, 218,000) were identified from radiological reports from February 1994 to February 1996. The Australian Bureau of Statistics supplied predictions of Australia's population (1996 to 2051). MAIN OUTCOME MEASURE: The projected annual number of fractures in Australian adults up to 2051 (based on stable rates of fracture in each age group). RESULTS: The number of fractures per year is projected to increase 25% from 1996 to 2006 (from 83,000 fractures to 104,000). Hip fractures are projected to increase 36% (from 15,000 to 21,000) because of a substantial rise in the number of elderly aged 85 years and over. Hip fractures are expected to double by 2026 and increase fourfold by 2051. CONCLUSIONS: In contrast to Europe and North America, where numbers of hip fractures are expected to double by 2026 and then stabilise, in Australia hip fractures will continue to place a growing demand on healthcare resources for many decades. These projections can be used for setting goals and evaluating the costs and benefits of interventions in Australia.

Adult↗

PubMed1999

Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II.

1. The kinetics of inactivation of delayed rectifier K+ current in murine colonic myocytes differed in amphotericin-permeabilized patch and conventional patch clamp. The difference was accounted for by Ca2+ buffering. 2. Calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitors increased the rate of inactivation and slowed recovery from inactivation of the outward current. This was seen in single steps and in the envelope of the current tails. The effect was largely on the TEA-insensitive component of current. 3. Dialysis of myocytes with autothiophosphorylated CaMKII slowed inactivation. This effect was reversed by addition of CaMKII inhibitor. 4. Antibodies revealed CaMKII-like immunoreactivity in murine colonic myocytes and other cells. Immunoblots identified a small protein with CaMKII-like immunoreactivity in homogenates of colonic muscle. 5. We conclude that CaMKII regulates delayed rectifier K+ currents in murine colonic myocytes. The changes in the delayed rectifier current may participate in the Ca2+-dependent regulation of gastrointestinal motility.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1999

Role of PI3-kinase in the development of interstitial cells and pacemaking in murine gastrointestinal smooth muscle.

1. Development of the pacemaker system in the small intestine depends upon signalling via tyrosine kinase (Kit) receptors. The downstream pathways initiated by Kit in interstitial cells of Cajal (ICC) have not been investigated. Wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibitors of phosphatidylinositol 3'-kinase (PI3-kinase), were used to test the involvement of this pathway in the development and maintenance of ICC and electrical rhythmicity in the murine small intestine. 2. ICC and electrical slow waves were present in the murine jejunum at birth. ICC and electrical rhythmicity continued to develop in neonates such that adult activity was recorded after 1 week. Development of ICC and rhythmicity were maintained in organ culture. 3. Wortmannin or LY 294002 inhibited the development of slow waves and blocked rhythmicity within 2-4 days. Loss of slow waves was preceded by disappearance of Kit-positive cells from the myenteric (IC-MY) and deep muscular plexus (IC-DMP) regions. Wortmannin or LY 294002 had no acute effect on slow waves. 4. Muscles from older animals (day 10-day 30) developed resistance to wortmannin treatment, but when the exposure to wortmannin was increased to 35 days, damage to ICC networks and electrical dysrhythmias were observed. 5. PI3-kinase appears to be a critical downstream signalling element linking Kit receptors to ICC development and maintenance of phenotype. ICC are more sensitive to Kit or PI3-kinase blockade at birth, but the importance of the PI3-kinase signalling in the maintenance of ICC persists into adulthood. Interference with PI3-kinase signalling in immature or adult animals could result in disruption of ICC and gastrointestinal dysrhythmias.

Aging↗

PubMed1999

Contribution of delayed rectifier potassium currents to the electrical activity of murine colonic smooth muscle.

1. We used intracellular microelectrodes to record the membrane potential (Vm) of intact murine colonic smooth muscle. Electrical activity consisted of spike complexes separated by quiescent periods (Vm approximately -60 mV). The spike complexes consisted of about a dozen action potentials of approximately 30 mV amplitude. Tetraethylammonium (TEA, 1-10 mM) had little effect on the quiescent periods but increased the amplitude of the action potential spikes. 4-Aminopyridine (4-AP, >= 5 mM) caused continuous spiking. 2. Voltage clamp of isolated myocytes identified delayed rectifier K+ currents that activated rapidly (time to half-maximum current, 11.5 ms at 0 mV) and inactivated in two phases (tauf = 96 ms, taus = 1.5 s at 0 mV). The half-activation voltage of the permeability was -27 mV, with significant activation at -50 mV. 3. TEA (10 mM) reduced the outward current at potentials positive to 0 mV. 4-AP (5 mM) reduced the early current but increased outward current at later times (100-500 ms) consistent with block of resting channels relieved by depolarization. 4-AP inhibited outward current at potentials negative to -20 mV, potentials where TEA had no effect. 4. Qualitative PCR amplification of mRNA identified transcripts encoding delayed rectifier K+ channel subunits Kv1.6, Kv4.1, Kv4.2, Kv4.3 and the Kvbeta1.1 subunit in murine colon myocytes. mRNA encoding Kv 1.4 was not detected. 5. We find that TEA-sensitive delayed rectifier currents are important determinants of action potential amplitude but not rhythmicity. Delayed rectifier currents sensitive to 4-AP are important determinants of rhythmicity but not action potential amplitude.

4-Aminopyridine↗

PubMed1999

Age- and gender-specific rate of fractures in Australia: a population-based study.

There is little population-based data concerning fracture rates in Australia. We ascertained all fractures occurring during 2 years in adults aged 35 years and over residing within a defined region (population 218 000), representative of the Australian population. The major strength of this study is the comprehensive ascertainment of fractures, which was ensured by regular searches of the only two radiologic providers in the Geelong Osteoporosis Study region. Nevertheless, vertebral fractures are likely to be underestimated since our ascertainment relied on a clinical indication for a medical imaging procedure. Among those aged 35-55 years, the fracture rate (persons per 10,000/year) in men was about double the rate in women (65 vs 35). The fracture rate was almost 7 times higher in women over 60 years versus women less than 55 years of age. In contrast, the fracture rate in men over 60 years was only 50% higher than in men less than 55 years of age (72 vs 104). Fracture rates in women and men were highest at the hip (28 and 10 respectively), spine (21 and 7), distal forearm (Colles') (18 and 4) and humerus (11 and 3), and were 3-4 times higher in women than men. These fractures accounted for 63% of all fractures in women and 32% in men. By contrast, the rate of lower leg and ankle fractures was less than 10 per 10,000 in both women and men and did not increase to the same extent with age. Hip fracture rates appear high, particularly among the older age strata, compared with retrospective ascertainment in other populations. In Australia, as in many other countries, there is an increasing longevity of the population. The number of women aged 90 years and over increased by 32% and the number of men of this age increased by 48% in the 5 years between the Australian national census of 1991 and 1996. Given stable fracture rates, the substantial health burden imposed by age-related fractures, particularly hip fractures, will continue to escalate in both women and men.

Adult↗

PubMed1999

Intimate relationship between interstitial cells of cajal and enteric nerves in the guinea-pig small intestine.

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 micrometer. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.

Animals↗

PubMed1999

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.

Animals↗

PubMed1999

Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS: Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS: When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS: These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.

Animals↗

PubMed1999

Parallel pathways mediate inhibitory effects of vasoactive intestinal polypeptide and nitric oxide in canine fundus.

1. The gastric adaptation reflex is activated by the release of non-adrenergic, non-cholinergic (NANC) inhibitory transmitters, including nitric oxide (NO) and vasoactive intestinal polypeptide (VIP). The role of NO in this reflex is not disputed, but some investigators suggest that NO synthesis is stimulated by VIP in post-junctional cells or in nerve terminals. We investigated whether the effects of these transmitters are mediated by independent pathways in the canine gastric fundus. 2. VIP and NO produced concentration-dependent relaxation of the canine fundus. Nomega-nitro-L-arginine (L-NNA) reduced relaxation induced by electrical field stimulation (EFS; 0.5-8 Hz), but had no effect on responses to exogenous VIP and sodium nitroprusside (SNP, 10 microM). 3. Oxyhaemoglobin reduced relaxations produced by EFS and SNP. Oxyhaemoglobin also reduced relaxation responses to low concentrations of VIP (<10 nM), but these effects were non-specific and mimicked by methaemoglobin which had no effect on nitrergic responses. 4. A blocker of guanylyl cyclase, 1H-[1,2,4]oxidiazolo [4,3,-a]quinoxalin-1-one, (ODQ) inhibited responses to EFS, SNP and DETA/NONOate (an NO.donor), but had no effect on responses to VIP. cis-N-(2-phenylcyclopentil)-azacyclotridec-1en-2-amine monohydrochloride (MDL 12,330A), a blocker of adenylyl cyclase, reduced responses to EFS, VIP and forskolin, but did not affect responses to SNP. 5. Levels of cyclic GMP were enhanced by the NO donor S-nitroso-n-acetylpenicillamine (SNAP) but were unaffected by VIP (1 microM). The increase in cyclic GMP in response to SNAP was blocked by ODQ. 6. The results suggest that at least two transmitters, possibly NO and VIP, mediate relaxation responses in the canine fundus. NO and VIP mediate responses via cyclic GMP- and cyclic AMP-dependent mechanisms, respectively. No evidence was found for a serial cascade in which VIP is coupled to NO-dependent responses.

Adenylyl Cyclase Inhibitors↗

PubMed1999

Evidence that nitric oxide acts as an inhibitory neurotransmitter supplying taenia from the guinea-pig caecum.

Nitric oxide synthase-containing nerve fibres are abundant within taenia of the guinea-pig caecum, but there is little previous evidence supporting a direct role for nitric oxide (NO) in responses to enteric inhibitory nerve stimulation. In this study we have attempted to identify an NO-dependent component of inhibitory transmission in isolated taenia coli. Isometric tension was recorded in the presence of atropine and guanethidine (both 1 microM). Tone was raised with histamine (1 microM), and intrinsic inhibitory neurons stimulated using either a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide; DMPP) or electrical field stimulation (EFS). DMPP (1-100 microM) produced concentration-dependent biphasic relaxations, comprising an initial peak relaxation followed by a sustained relaxation. Responses to DMPP were antagonized by tetrodotoxin (1 microM) or apamin (0.3 microM) and abolished by hexamethonium (300 microM). L-nitro-arginine (L-NOARG; 100 microM) and oxyhaemoglobin (2%) both significantly reduced sustained relaxations produced by DMPP. EFS (5 Hz, 30 s) also produced biphasic relaxations. Both L-NOARG and an inhibitor of soluble guanylate cyclase (ODQ, 1-10 microM) reduced the sustained component of EFS responses. Two NO donors, sodium nitroprusside (SNP) and diethylenetriamine-nitric oxide adduct (DENO), produced concentration-dependent relaxations. Responses to SNP and DENO were antagonized by ODQ (1 microM) and by apamin (0.3 mM). These results suggest that NO contributes directly to a component of inhibitory transmission in guinea-pig taenia coli. The actions of NO appear to be mediated via cyclic GMP synthesis, and may involve activation of small conductance calcium activated K+ channels. A role for NO is most evident during sustained relaxations evoked by longer stimulus trains or chemical stimulation of intrinsic neurons.

Animals↗

PubMed1999

Effects of anion channel antagonists in canine colonic myocytes: comparative pharmacology of Cl-, Ca2+ and K+ currents.

1. Volume-Sensitive, Outwardly Rectifying (VSOR) Cl- currents were measured in canine colonic myocytes by whole-cell patch clamp. Decreasing extracellular osmolarity 50 milliosmoles l-1 activated current that was carried by Cl- and 5 - 7 times greater in the outward direction. 2. Niflumic acid, an inhibitor of Ca2+-activated Cl- channels, did not inhibit VSOR Cl- current. Glibenclamide, an antagonist of CFTR, and anthracene-9-carboxylate (9-AC) inhibited current less than 25% at 100 microM. 3. DIDS (4, 4-diisothiocyanato-stilbene-2,2'disulphonate) inhibited VSOR Cl- current more potently than SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonate). IC50s were 0.84 and 226 microM, respectively. 4. VSOR Cl- current was strongly inhibited by tamoxifen ([Z]-1-[p-dimethylaminoethoxy-phenyl]-1,2-diphenyl-1-butene), an anti-oestrogen compound (IC50=0.57 microM). 5. Gd3+ antagonized VSOR Cl- current more potently than La3+. The IC50 for Gd3+ was 23 microM. In contrast, 100 microM La3+ inhibited current only 35+/-7%. 6. Antagonists of VSOR Cl- current had non-specific effects. These compounds blocked voltage-dependent K+ and Ca2+ currents in colonic myocytes. Tamoxifen (10 microM) and DIDS (10 microM) inhibited L-type Ca2+ current 87+/-7 and 31+/-5%, respectively. Additionally, in the presence of 300 nM charybdotoxin, tamoxifen (1 microM) and DIDS (10 microM) inhibited delayed rectifier K+ current 38+/-8 and 10+/-2%, respectively. 7. The pharmacology of VSOR Cl- channels overlaps with voltage-dependent cation channels. DIDS and tamoxifen inhibited VSOR Cl- equally. However, because DIDS had much less effect on L-type Ca2+ and delayed rectifier K+ channels than did tamoxifen, it might be useful in experiments to investigate the physiological and pathophysiological role of this conductance in whole tissues.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Development and plasticity of interstitial cells of Cajal.

Interstitial cells of Cajal (ICC) are the pacemakers in gastrointestinal (GI) muscles, and these cells also mediate or transduce inputs from the enteric nervous system. Different classes of ICC are involved in pacemaking and neurotransmission. ICC express specific ionic conductances that make them unique in their ability to generate and propagate slow waves in GI muscles or transduce neural inputs. Much of what we know about the function of ICC comes from developmental studies that were made possible by the discoveries that ICC express c-kit and proper development of ICC depends upon signalling via the Kit receptor pathway. Manipulating Kit signalling with reagents to block the receptor or downstream signalling pathways or by using mutant mice in which Kit or its ligand, stem cell factor, are defective has allowed novel studies into the specific functions of the different classes of ICC in several regions of the GI tract. Kit is also a surface antigen that can be used to conveniently label ICC in GI muscles. Immunohistochemical studies using Kit antibodies have expanded our knowledge about the ICC phenotype, the structure of ICC networks, the interactions of ICC with other cells in the gut wall, and the loss of ICC in some clinical disorders. Preparations made devoid of ICC have also allowed analysis of the consequences of losing specific classes of ICC on GI motility. This review describes recent advances in our knowledge about the development and plasticity of ICC and how developmental studies have contributed to our understanding of the functions of ICC. We have reviewed the clinical literature and discussed how loss or defects in ICC affect GI motor function.

Animals↗

PubMed1999

Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles.

Regulation of gastrointestinal (GI) motility is intimately coordinated with the modulation of ionic conductance expressed in GI smooth muscle and nonmuscle cells. Interstitial cells of Cajal (ICC) act as pacemaker cells and possess unique ionic conductances that trigger slow wave activity in these cells. The slow wave mechanism is an exclusive feature of ICC: Smooth muscle cells may lack the basic ionic mechanisms necessary to generate or regenerate slow waves. The molecular identification of the components for these conductances provides the foundation for a complete understanding of the ionic basis for GI motility. In addition, this information will provide a basis for the identification or development of therapeutics that might act on these channels. It is much easier to study these conductances and develop blocking drugs in expression systems than in native GI muscle cells. This review focuses on the relationship between ionic currents in native GI smooth muscle cells and ICC and their molecular counterparts.

Animals↗