Purinergic inhibitory regulation of esophageal smooth muscle is mediated by P2Y receptors and ATP-dependent potassium channels in rats.

Purines such as ATP are regulatory transmitters in motility of the gastrointestinal tract. The aims of this study were to propose functional roles of purinergic regulation of esophageal motility. An isolated segment of the rat esophagus was placed in an organ bath, and mechanical responses were recorded using a force transducer. Exogenous application of ATP (10-100 µM) evoked relaxation of the esophageal smooth muscle in a longitudinal direction under the condition of carbachol (1 µM) -induced precontraction. Pretreatment with a non-selective P2 receptor antagonist, suramin (500 µM), and a P2Y receptor antagonist, cibacron blue F3GA (200 µM), inhibited the ATP (100 µM) -induced relaxation, but a P2X receptor antagonist, pyridoxal phosphate-6-azophenyl-2,4-disulfonic acid (50 µM), did not affect it. A blocker of ATP-dependent potassium channels (KATP channels), glibenclamide (200 µM), inhibited the ATP-induced relaxation and application of an opener of KATP channels, nicorandil (50 µM), produced relaxation. The findings suggest that ATP is involved in inhibitory regulation of the longitudinal smooth muscle in the muscularis mucosae of the rat esophagus via activation of P2Y receptors and then opening of KATP channels.

ATP-Induced Contractile Response of Esophageal Smooth Muscle in Mice.

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Alterations in descending brain-spinal pathways regulating colorectal motility in a rat model of Parkinson's disease.

Patients with Parkinson's disease (PD) often have constipation. It is assumed that a disorder of the regulatory mechanism of colorectal motility by the central nervous system is involved in the constipation, but this remains unclear. The aim of this study was to investigate whether central neural pathways can modulate colorectal motility in a rat model of PD. PD model rats were generated by injection of 6-hydroxydopamine into a unilateral medial forebrain bundle and destruction of dopaminergic neurons in the substantia nigra. Colorectal motility was measured in vivo in anesthetized rats. Intraluminal administration of capsaicin, as a noxious stimulus, induced colorectal motility in sham-operated rats but not in PD rats. Intrathecally administered dopamine (DA) and serotonin (5-HT), which mediate the prokinetic effect of capsaicin, at the L6-S1 levels enhanced colorectal motility in PD rats similarly to that in sham-operated rats. In PD rats, capsaicin enhanced colorectal motility only when a GABAA receptor antagonist was preadministered into the lumbosacral spinal cord. Capsaicin-induced colorectal motility was abolished by intrathecal administration of a D2-like receptor antagonist but not by administration of 5-HT2 and 5-HT3 receptor antagonists. These findings demonstrate that the inhibitory GABAergic component becomes operative and the stimulatory serotonergic component is suppressed in PD rats. The alteration of the central regulatory mechanism of colorectal motility is thought to be related to the occurrence of constipation in PD patients. Our findings provide a new insight into the pathogenesis of defecation disorders observed in PD.NEW & NOTEWORTHY In a rat model of Parkinson's disease, the component of descending brain-spinal pathways that regulate colorectal motility through a mediation of the lumbosacral defecation center was altered from stimulatory serotonergic neurons to inhibitory GABAergic neurons. Our findings suggest that chronic constipation in Parkinson's disease patients may be associated with alterations in central regulatory mechanisms of colorectal motility. The plasticity in the descending pathway regulating colorectal motility may contribute to other disease-related defecation abnormalities.

Essential roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of colorectal motility in rats.

The supraspinal brain regions controlling defecation reflex remain to be elucidated. The purpose of this study was to determine the roles of the hypothalamic A11 region and the medullary raphe nuclei in regulation of defecation. For chemogenetic manipulation of specific neurons, we used the double virus vector infection method in rats. hM3Dq or hM4Di was expressed in neurons of the A11 region and/or the raphe nuclei that send output to the lumbosacral defecation center. Immunohistological and functional experiments revealed that both the A11 region and the raphe nuclei directly connected with the lumbosacral spinal cord through descending pathways composed of stimulatory monoaminergic neurons. Stimulation of the hM3Dq-expressing neurons in the A11 region or the raphe nuclei enhanced colorectal motility only when GABAergic transmission in the lumbosacral spinal cord was blocked by bicuculline. Experiments using inhibitory hM4Di-expressing rats revealed that enhancement of colorectal motility caused by noxious stimuli in the colon is mediated by both the A11 region and the raphe nuclei. Furthermore, suppression of the A11 region and/or the raphe nuclei significantly inhibited water avoidance stress-induced defecation. These findings demonstrate that the A11 region and the raphe nuclei play an essential role in the regulation of colorectal motility. This is important because brain regions that mediate both intracolonic noxious stimuli-induced defecation and stress-induced defecation have been clarified for the first time.NEW & NOTEWORTHY The A11 region and the raphe nuclei, constituting descending pain inhibitory pathways, are related to both intracolonic noxious stimuli-induced colorectal motility and stress-induced defecation. Our findings may provide an explanation for the concurrent appearance of abdominal pain and defecation disorders in patients with irritable bowel syndrome. Furthermore, overlap of the pathway controlling colorectal motility with the pathway mediating stress responses may explain why stress exacerbates bowel symptoms.

Sex differences in the central regulation of colorectal motility in response to noxious stimuli.

Distinct sex differences in the prevalence and symptoms of abnormal bowel habits in patients with irritable bowel syndrome (IBS) have been reported. We have elucidated the sex differences in the regulation of colorectal motility via the central nervous system. Noxious stimuli in the colorectum of anesthetized male rats enhance colorectal motility by activating monoaminergic neurons in descending pain inhibitory pathways from the brainstem to the lumbosacral spinal cord. These monoaminergic neurons release serotonin and dopamine into the lumbosacral spinal cord, resulting in the increment of colorectal motility. In female rats, in contrast, noxious stimuli in the colorectum have no effect on colorectal motility. We clarified that GABAergic inhibition in the lumbosacral spinal cord masks the enhancement of colorectal motility induced by monoamines in female animals. Considering that IBS patients often show visceral hypersensitivity and hyperalgesia, our studies suggest that differences in the descending neurons that respond to painful stimuli are involved in various sex differences in abnormal bowel habits.

Contribution of sex hormones to the sexually dimorphic response of colorectal motility to noxious stimuli in rats.

Our recent studies have shown that noxious stimuli in the colorectum enhance colorectal motility via the brain and spinal defecation centers in male rats. In female rats, however, noxious stimuli have no effect on colorectal motility. The purpose of this study was to determine whether sex hormones are major contributing factors for sex-dependent differences in neural components of the spinal defecation center. Colorectal motility was measured using an in vivo method under ketamine and α-chloralose anesthesia in rats. Capsaicin was administered into the colorectal lumen as noxious stimuli. Orchiectomy in male rats had no effect on the capsaicin-induced response of colorectal motility. However, in ovariectomized female rats, capsaicin administration enhanced colorectal motility, though intact female animals did not show enhanced motility. When estradiol was administered by using a sustained-release preparation in ovariectomized female rats, capsaicin administration did not enhance colorectal motility unless a GABAA receptor antagonist was intrathecally administered to the lumbosacral spinal cord. These findings suggest that estradiol allowed the GABAergic neurons to operate in response to intracolonic administration of capsaicin. The operation of GABAergic inhibition by the action of estradiol could be manifested in male rats only when the effects of male sex hormones were removed by orchiectomy. Taken together, our results indicate that sex hormones contribute to the sexually dimorphic response in colorectal motility enhancement in response to noxious stimuli through modulating GABAergic pathways.NEW & NOTEWORTHY This study demonstrated that estradiol permits inhibitory regulation in the spinal defecation center not only in female rats but also in orchiectomized male rats. GABAergic pathways are likely involved in the effect of estradiol. This is the first report showing that sex hormones affect colorectal motility through the alteration of neural components of the regulatory pathways. Our findings provide a novel insight into pathophysiological mechanisms of defecation disorders related to changes in sex hormones.

Intrathecally administered substance P activated the spinal defecation center and enhanced colorectal motility in anesthetized rats.

Noxious stimuli on the colorectum cause colorectal contractions through activation of descending monoaminergic pathways projecting from the supraspinal defecation center to the spinal defecation center. Since it is known that substance P is involved in the response to peripheral noxious stimuli in the spinal cord, we investigated the effects of intrathecally administered substance P at L6-S1 levels on colorectal motility in rats that were anesthetized with α-chloralose and ketamine. Intrathecally administered substance P enhanced colorectal motility, even after transection of the thoracic spinal cord at the T4 level. Severing the pelvic nerves, but not the colonic nerves, abolished substance P enhanced colorectal motility. In the spinal cord at L6-S1 levels, expression of mRNA coding neurokinin (NK) 1-3 receptors was detected by RT-PCR. Immunohistological experiments revealed that preganglionic neurons of the pelvic nerves express NK1 receptors, whereas expression of NK2 receptors was not found. In addition, substance P-containing fibers densely innervated around the preganglionic neurons expressing NK1 receptors. An intrathecally administered NK1 receptor antagonist (spantide) attenuated capsaicin-induced colorectal contractions. These results suggest that the colokinetic action of substance P is mediated by the NK1 receptor in the spinal defecation center. Our findings indicate that substance P may function as a neurotransmitter in the spinal defecation center.NEW & NOTEWORTHY We found that intrathecally administered substance P enhanced colorectal motility in anesthetized rats. Neurokinin (NK) 1 receptors, but not NK2 receptors, were detected in preganglionic neurons of the pelvic nerves. Blockade of NK1 receptors in the spinal cord attenuated the enhanced colorectal motility in response to intracolonic noxious stimuli. The findings indicate that substance P may function as a neurotransmitter in the spinal reflex pathway controlling defecation.

Successful induction of deep hypothermia by isoflurane anesthesia and cooling in a non-hibernator, the rat.

The aim of the present study was to establish a novel method for inducing deep hypothermia in rats. Cooling rats anesthetized with isoflurane caused a time-dependent decrease in rectal temperature, but cardiac arrest occurred before their body temperature reached 20 °C when isoflurane inhalation was continued during the cooling process. Stopping inhalation of isoflurane when the rectal temperature reached 22.5 °C successfully induced deep hypothermia, although stopping the inhalation at 27.5 °C resulted in spontaneous recovery of rectal temperature. The hypothermic condition was able to be maintained for up to 6 h. A large number of c-Fos-positive cells were detected in the hypothalamus during hypothermia. Both the maintenance of and recovery from hypothermia caused organ injury, but the damage was transient and recovered within 1 week. These findings indicate that the established procedure is appropriate for inducing deep hypothermia without accompanying serious organ injury in rats.

Sexually dimorphic response of colorectal motility to noxious stimuli in the colorectum in rats.

KEY POINTS: This study showed a remarkable sex difference in responses of colorectal motility to noxious stimuli in the colorectum in rats: colorectal motility was enhanced in response to intracolonic administration of a noxious stimulant, capsaicin, in male rats but not in female rats. The difference in descending neurons from the brain to spinal cord operating after noxious stimulation could be responsible for the sex difference. In male rats, serotoninergic and dopaminergic neurons are dominantly activated, both of which activate the spinal defaecation centre. In female rats, GABAergic neurons in addition to serotoninergic neurons are activated. GABA may compete for facilitative action of 5-HT in the spinal defaecation centre, and thereby colorectal motility is not enhanced in response to intracolonic administration of capsaicin. The findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome. ABSTRACT: We previously demonstrated that noxious stimuli in the colorectum enhance colorectal motility through activation of descending pain inhibitory pathways in male rats. It can be expected that the regulatory mechanisms of colorectal motility differ in males and females owing to remarkable sex differences in descending pain inhibitory pathways. Thus, we aimed to clarify sex differences in responses of colorectal motility to noxious stimuli in rats. Colorectal motility was measured in vivo in anaesthetized rats. Administration of a noxious stimulant, capsaicin, into the colorectal lumen enhanced colorectal motility in male rats but not in female rats. Quantitative PCR and immunohistochemistry showed that TRPV1 expression levels in the dorsal root ganglia and in the colorectal mucosa were comparable in male and female rats. When a GABAA receptor inhibitor was intrathecally administered to the L6-S1 level of the spinal cord, colorectal motility was facilitated in response to intracolonic capsaicin even in female rats. The capsaicin-induced response in the presence of the GABA blocker in female rats was inhibited by intrathecal administration of 5-HT2 and -3 receptor antagonists but not by a D2-like dopamine receptor antagonist. Our findings demonstrate that intracolonic noxious stimulation activates GABAergic and serotoninergic descending neurons in female rats, whereas serotoninergic and dopaminergic neurons are dominantly activated in male rats. Thus, the difference in the descending neurons operating after noxious stimulation would be responsible for the sexually dimorphic responses of colorectal motility. Our findings provide a novel insight into pathophysiological mechanisms of sex differences in functional defaecation disorders such as irritable bowel syndrome.

ATP-dependent potassium channels contribute to motor regulation of esophageal striated muscle in rats.

The aim of the present study was to clarify roles of ATP-dependent potassium channels (KATP channels) in motility of the striated muscle portion in the esophagus. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response of striated muscle in the esophageal segment. Application of glibenclamide, an antagonist of KATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of KATP channels, decreased amplitude of twitch contractions. RT-PCR revealed the expression of subunits of KATP channels in esophageal tissue. In addition, immunopositivity for subunits of KATP channels was observed in the striated muscle cells of the esophageal muscle layer. These findings indicate that KATP channels contribute to motor regulation of striated muscle in the rat esophagus.

Mild hypothermia causes a shift in the alternative splicing of cold-inducible RNA-binding protein transcripts in Syrian hamsters.

Cold-shock proteins are thought to participate in the cold-tolerant nature of hibernating animals. We previously demonstrated that an alternative splicing may allow rapid induction of functional cold-inducible RNA-binding protein (CIRBP) in the hamster heart. The purpose of the present study was to determine the major cause of the alternative splicing in Syrian hamsters. RT-PCR analysis revealed that CIRBP mRNA is constitutively expressed in the heart, brain, lung, liver, and kidney of nonhibernating euthermic hamsters with several alternative splicing variants. In contrast, the short variant containing an open-reading frame for functional CIRBP was dominantly found in the hibernating animals. Keeping the animals in a cold and dark environment did not cause a shift in the alternative splicing. Induction of hypothermia by central administration of an adenosine A1-receptor agonist reproduced the shift in the splicing pattern. However, the agonist failed to shift the pattern when body temperature was kept at 37°C, suggesting that central adenosine A1 receptors are not directly linked to the shift of the alternative splicing. Rapid reduction of body temperature to 10°C by isoflurane anesthesia combined with cooling did not alter the splicing pattern, but maintenance of mild hypothermia (~28°C) for 2 h elicited the shift in the pattern. The results suggest that animals need to be maintained at mild hypothermia for an adequate duration to induce the shift in the alternative splicing. This is applicable to natural hibernation because hamsters entering hibernation show a gradual decrease in body temperature, being maintained at mild hypothermia for several hours.

Induction of hibernation-like hypothermia by central activation of the A1 adenosine receptor in a non-hibernator, the rat.

Central adenosine A1-receptor (A1AR)-mediated signals play a role in the induction of hibernation. We determined whether activation of the central A1AR enables rats to maintain normal sinus rhythm even after their body temperature has decreased to less than 20 °C. Intracerebroventricular injection of an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), followed by cooling decreased the body temperature of rats to less than 20 °C. Normal sinus rhythm was fundamentally maintained during the extreme hypothermia. In contrast, forced induction of hypothermia by cooling anesthetized rats caused cardiac arrest. Additional administration of pentobarbital to rats in which hypothermia was induced by CHA also caused cardiac arrest, suggesting that the operation of some beneficial mechanisms that are not activated under anesthesia may be essential to keep heart beat under the hypothermia. These results suggest that central A1AR-mediated signals in the absence of anesthetics would provide an appropriate condition for maintaining normal sinus rhythm during extreme hypothermia.

Neonatal lethality of neural crest cell-specific Rest knockout mice is associated with gastrointestinal distension caused by aberrations of myenteric plexus.

RE1-silencing transcription factor (REST), also known as NRSF (neuron-restrictive silencer factor), is a well-known transcriptional repressor of neural genes. Rest null mice have embryonic lethality which prevents further investigations of the functions of the Rest gene in vivo. We studied neonatal but not embryonic lethality that was characterized by gastrointestinal tract dilation in the neural crest cell (NCC)-specific Rest conditional knockout (CKO) mice. While no histological abnormalities except the thinning of the digestive tract as a consequence of the gas accumulation were found in the digestive tract of the mutant mice, they do not have proper gastric retention after oral dye administration and the reduction of acetylcholinesterase (AChE) activity in NCC-derived myenteric plexus in the stomach was detected. High CO2 concentration in the dilated digestive tract of the Rest CKO mice indicates a failure of gut function by underdeveloped cholinergic transmission in the enteric nervous system. The observed gastrointestinal distension phenotype provides a model for understanding the genetic and molecular basis of NCC defects in humans.

Postnatal development of excitatory innervations in longitudinal smooth muscle of the chicken anterior mesenteric artery.

AIMS: The anterior mesenteric artery of chickens contains a well-developed outer longitudinal smooth muscle layer in addition to an inner circular layer. Cholinergic and purinergic neurons play crucial roles in excitatory transmission at the longitudinal smooth muscle. The aim of this study was to clarify postnatal development of excitatory neurotransmission of the longitudinal smooth muscle. MAIN METHODS: Membrane potentials of smooth muscle were recorded with a microelectrode technique. Perivascular nerves were stimulated by applying electrical field stimulation (EFS). KEY FINDINGS: Histological examination showed that longitudinal smooth muscles exist in the artery at birth. EFS failed to evoke membrane response in 1-day-old chickens, though it caused depolarization (excitatory junction potential; EJP) in 12-week-old chickens. However, exogenous application of acetylcholine (ACh) or ATP produced depolarization in longitudinal smooth muscle of 1-day-old chickens, suggesting that responsiveness of smooth muscle to excitatory neurotransmitters is already established at birth. In preparations isolated from 10-day-old chickens, EFS caused EJP, which was totally blocked by atropine but not by a non-specific purinoceptor antagonist, suramin. Several purinoceptor subtypes including P2Y1, which may be related to depolarizing response in smooth muscle of adult chickens, were expressed in the anterior mesenteric artery of 10-day-old chickens. SIGNIFICANCE: Excitatory innervation in longitudinal smooth muscle of the chicken anterior mesenteric artery is not established at birth but develops during the early postnatal period. Moreover, development of cholinergic excitatory innervation precedes that of purinergic excitatory innervation, although receptors that mediate purinergic control are already expressed in smooth muscle.

Extract from Calotropis procera latex activates murine macrophages.

Calotropis procera latex has long been used in traditional medicines. Extracts from C. procera latex have been reported to have various pharmacological actions, including protection from myocardial infarction, hepatoprotective action, antitumor activity, antinociceptive, and pro- and anti-inflammatory actions. To evaluate the immunomodulatory functions of the water-soluble C. procera extract (CPE), we investigated its ability to activate macrophages-effector cells in inflammatory and immune responses. Intraperitoneal injection of CPE in mice (2 mg/mouse) induced migration of macrophages to the intraperitoneal cavity, confirming the proinflammatory effects of water-soluble CPE. The direct effects of CPE on macrophages were then assessed by measuring the production of nitric oxide (NO) as an indicator for macrophage activation. Addition of CPE (1-10 microg/ml) to the culture medium of the murine monocyte/macrophage cell line RAW264.7 caused an increase in NO production in a time- and dose-dependent manner. CPE-elicited NO production was blocked by application of an inhibitor of inducible nitric oxide synthase (iNOS). Expression of iNOS mRNA was induced by treatment of cultured macrophages with CPE. Injection of CPE in mice also resulted in an increase in plasma NO level. The results suggest that CPE activates macrophages and facilitates NO production via up-regulation of iNOS gene expression.

Central A1-receptor activation associated with onset of torpor protects the heart against low temperature in the Syrian hamster.

Body temperature drops dramatically during hibernation, but the heart retains the ability to contract and is resistant to induction of arrhythmia. Although adaptive changes in the heart prior to hibernation may be involved in the cold-resistant property, it remains unclear whether these changes are sufficient for maintaining cardiac pulsatility under an extreme hypothermic condition. We forcibly induced hypothermia in Syrian hamsters by pentobarbital anesthesia combined with cooling of the animals. This allows reproduction of a hypothermic condition in the absence of possible hibernation-specific reactions. Unlike hypothermia in natural hibernation, the forced induction of hypothermia caused atrioventricular block. Furthermore, J-waves, which are typically observed during hypothermia in nonhibernators, were recorded on an ECG. The origin of the J-wave seemed to be related to irreversible injury of the myocardium, because J-waves remained after recovery of body temperature. An abnormal ECG was also found when hypothermia was induced in hamsters that were well adapted to a cold and darkened environment or hamsters that had already experienced hibernation. These results suggest that acclimatization prior to hibernation does not have a crucial effect at least on acquisition of cardiac resistance to low temperature. In contrast, an abnormal ECG was not observed in the case of hypothermia induced by central administration of an adenosine A1-receptor agonist and subsequent cooling, confirming the importance of the adenosine system for inducing hibernation. Our results suggest that some specific mechanisms, which may be driven by a central adenosine system, operate for maintaining the proper cardiac pulsatility under extreme hypothermia.

Evidence that stimulation of ghrelin receptors in the spinal cord initiates propulsive activity in the colon of the rat.

Previous studies have failed to reveal an effect of the gastrointestinal peptide hormone ghrelin on colonic motility. In the present work, ghrelin was applied into the lumbo-sacral spinal cord in the region of defecation control centres, and a synthetic ghrelin receptor agonist, CP464709, which crosses the blood-brain barrier, was applied intravenously or into the lumbo-sacral cord. Both ghrelin and CP464709 elicited propulsive contractions and emptying of the colon in anaesthetized rats. In conscious rats, subcutaneous CP464709 caused fecal expulsion. The sites of action and nerve pathways involved in the stimulation of the colon by ghrelin receptor activation were investigated in anaesthetized rats. Intrathecal application of CP464709 at L6-S1, but not application at ponto-medullary levels or to the thoracic spinal cord, elicited propulsive contractions. The stimulation evoked by intravenous CP464709 was prevented if the pelvic nerve outflows were severed, but not if the spinal cord was cut rostral to the defecation centre at L6-S3. The response was also blocked by hexamethonium. When ghrelin, applied intrathecally, was used to desensitize its receptors, the effect of intravenous CP464709 was blocked. CP464709 did not affect small intestine motility or the amplitudes of visceromotor reflexes caused by colorectal distension. It is concluded that activation of ghrelin receptors in the lumbo-sacral spinal cord triggers co-ordinated propulsive contractions that empty the colo-rectum. The pathways through which these responses are generated pass out of the spinal cord via the pelvic nerves and cause propulsive contractions through activation of enteric neurons.

The distribution of intermediate-conductance, calcium-activated, potassium (IK) channels in epithelial cells.

Intermediate-conductance, calcium-activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid-secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid-exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell-protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current-dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown.

Neurally released ATP mediates endothelium-dependent hyperpolarization in the circular smooth muscle cells of chicken anterior mesenteric artery.

The object of the present study was to clarify the neurotransmitter(s) controlling membrane responses to electrical field stimulation (EFS) in the circular smooth muscle cells of first-order branches of chicken anterior mesenteric artery.EFS (five pulses at 20 Hz, 1 ms) evoked a hyperpolarization of amplitude--21.6+/-1.2 mV, total duration 21.8+/-1.2 s and latency 641.7+/-81.9 ms. The response was tetrodotoxin-sensitive and nonadrenergic noncholinergic (NANC) in nature. The NANC response was blocked by the nonspecific purinergic antagonist, suramin, indicating that the response is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). Either desensitization or blockade of P2Y receptor with its putative agonist 2-methylthioATP (1 microM for 30 min) or with its antagonist cibacron blue F3GA (10 microM), respectively, abolished the purinergic hyperpolarization. PPADS at concentrations up to 100 microM had no effect on the EFS-induced response, indicating that this response is mediated through P2Y, but not P2X, receptor. In addition, the response was completely abolished by two specific P2Y1 receptor antagonists, namely, MRS 2179 (300 nM) and A3P5PS (10 microM). Removal of the endothelium abolished the purinergic hyperpolarization, which was converted, in some preparations, to a small depolarization, indicating that the hyperpolarizing response is endothelium-dependent. The present study suggests that in first-order branches of chicken anterior mesenteric artery, ATP released from perivascular nerves may diffuse to the endothelium-activating P2Y1 receptor to induce release of an inhibitory substance that mediates hyperpolarization in the circular smooth muscle.

An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery.

1. The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2. EFS (5 pulses at 20 Hz) evoked a depolarization of amplitude 19.7+/-2.1 mV, total duration 29.6+/-3.1 s and latency 413.0+/-67.8 ms. This depolarization was tetrodotoxin (TTX)-sensitive and its amplitude was partially decreased by atropine (0.5 microM); however, its duration was shortened by further addition of prazosin (10 microM). 3. Atropine/prazosin-resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose-dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5'-triphosphate (ATP). 4. Neither desensitization nor blocking of P2X receptor with its putative receptor agonist alpha,beta-methylene ATP (alpha,beta-MeATP, 1 microM) and its antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS, up to 50 microM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2-methylthioATP (2-MeSATP, 1 microM) and its antagonist Cibacron blue F3GA (CBF3GA, 10 microM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5. The purinergic depolarization was inhibited by pertussis toxin (PTX, 600 ng ml(-1)). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U-73122 (10 microM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX-sensitive G-protein, which is involved in a PLC-mediated signaling pathway. 6. Data of the present study suggest that the EFS-induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.