Prejunctional modulation of non-adrenergic non-cholinergic (NANC) inhibitory responses in the isolated guinea-pig gastric fundus

The inhibitory neurotransmission of the stomach was investigated in isolated guinea-pig gastric fundus. In preparations treated with guanethidine (1 micro mol L-1) and p-fluoro-hexahydro-sila-difenidol (1 micro mol L-1), electrical stimulation evoked neurogenic inhibitory responses not modified by hexamethonium (100 micro mol L-1), suggesting that inhibitory postganglionic non-adrenergic non-cholinergic (NANC) nerve fibres are involved. The nitric oxide (NO)-synthase inhibitor Nomega-nitro-l-argininine-methyl-ester hydrochloride (1-100 micro mol L-1) and the soluble guanylyl cyclase inhibitor ODQ (0.1-3 micro mol L-1) also abolished such relaxant response, suggesting the involvement of NO/Cyclic Guanosine 3',5' monophosphate (cGMP) system as the final mechanism of muscle relaxation. The alpha2-adrenoceptor agonist, UK 14 304 (10 nmol L-1-10 micro mol L-1) did not influence the electrical field stimulation (EFS)-evoked NANC responses. These latter responses were also refractory to a variety of receptor agonists and antagonists, acting at Gamma Aminobutyric Acid (GABA), serotonin 5HT1a, opioid micro , delta and kappa, muscarinic M1 and M2, histamine H2 and H3 and cannabinoid receptors. The NANC response was insensitive to the P/Q-type Ca2+-channel blocker omega-agatoxin TK (1 nmol L-1-0.1 micro mol L-1), but partially inhibited by the N-type Ca2+-channel blocker omega-conotoxin GVIA (0.1 nmol L-1-0.1 micro mol L-1), and by the L-type Ca2+-channel blockers nifedipine and calcicludine (0.1 nmol L-1-0.1 micro mol L-1). These data suggest that the NANC relaxation of the isolated guinea-pig gastric fundus is mediated by NO as the final inhibitory (neuro)transmitter at the longitudinal smooth muscle cells. The mechanism(s) promoting NO production is/are Ca2+-dependent, but apparently insensitive to presynaptic modulation. Both N- and L-type channels seem to occur in nitrergic nerve endings, where they contribute to trigger NO diffusion at the synaptic cleft.     

Impaired propulsive motility in the distal but not proximal colon of BK channel β1‐subunit knockout mice

Background Large‐conductance Ca²⁺‐activated K⁺ (BK) channels regulate smooth muscle tone. The BK channel β1‐subunit increases Ca²⁺ sensitivity of the α‐subunit in smooth muscle. We studied β1‐subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. Methods Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. Key Results Bethanechol‐induced contractions were larger in the distal colon of β1‐subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsβ1‐subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of β1‐subunit KO compared to WT mice. Gastrointestinal transit was unaffected by β1‐subunit KO. Fecal output was decreased and glass bead expulsion times were increased in β1‐subunit KO mice. Membrane potential of distal colon smooth muscle cells from β1‐subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. Conclusions & Inferences BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.     

Effect of the stable thromboxane derivative, carbocyclic thromboxane A2, on membrane potential of rat myenteric neurones in culture

The effects of carbocyclic thromboxane A(2) (cTXA(2); 10(-6) mol L(-1)) on membrane potential and cytosolic Ca(2+) concentration were measured with the whole-cell patch-clamp or the fura-2 method, respectively, at rat myenteric ganglia. cTXA(2) caused a hyperpolarization of myenteric neurones from -19.3 +/- 2.5 to -29.3 +/- 2.3 mV. In addition, the eicosanoid potentiated the carbachol-induced depolarization from 4.2 +/- 1.0 mV under control conditions to 11.1 +/- 1.1 mV in the presence of the cTXA(2) (n = 9). The hyperpolarization was abolished by internal application of CsCl (140 mmol L(-1)), a non-selective blocker of K(+) channels, or EGTA (11 mmol L(-1)in the pipette solution), a chelator of intracellular Ca(2+). A similar inhibition was observed in the presence of charybdotoxin (10(-7) mol L(-1)). Fura-2 imaging experiments revealed a cTXA(2)-evoked increase in the intracellular Ca(2+) concentration as indicated by a rise in the fura-2 ratio signal. This response was mediated by a release of Ca(2+) from intracellular stores as sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase blockade with cyclopiazonic acid (5 x 10(-5) mol L(-1)) completely abolished the response to cTXA(2). A similar inhibition was observed after blockade of phospholipase C with U-73122 (10(-5) mol L(-1)). These results suggest an activation of Ca(2+)-activated K(+) channels by cTXA(2) after stimulation of phospholipase C.     

CaM kinase II in colonic smooth muscle contributes to dysmotility in murine DSS‐colitis

Background Altered calcium mobilization has been implicated in the development of colonic dysmotility in inflammatory bowel disease. The aim of this study was to investigate the mechanisms by which disrupted intracellular Ca²⁺ signalling contributes to the impaired contractility of colon circular smooth muscles. Methods Acute colitis was induced in C57Bl/6 mice with dextran sulphate sodium (DSS) in the drinking water for 5 days. Key Results Spontaneous and acetylcholine‐evoked contractions, caffeine‐evoked hyperpolarization, and SERCA2 and phospholamban expression were reduced compared with controls. Tetrodotoxin did not restore control levels of contractile activity. The amplitudes, but not the frequency, of intracellular Ca²⁺ waves were increased compared with controls. Caffeine abolished intracellular Ca²⁺ waves in control smooth muscle cells, but not in smooth muscle cells from DSS‐treated mice. CaM kinase II activity and cytosolic levels of HDAC4 were increased, and IκBα levels were decreased in distal colon smooth muscles from DSS‐treated mice. Conclusions & Inferences These results suggest that disruptions in intracellular Ca²⁺ mobilization due to down‐regulation of SERCA2 and phospholamban expression lead to increased CaM kinase II activity and cytosolic HDAC4 that may contribute to the dysmotility of colonic smooth muscles in colitis by enhancing NF‐κB activity.     

Effect of butyrate on membrane potential, ionic currents and intracellular Ca2+ concentration in cultured rat myenteric neurones

Myenteric neurones from 1-10-day-old rats were isolated from the small and large intestine by enzymatic digestion with collagenase. Single cells were collected and kept in culture for up to 1 week. After 1-5 days in culture, membrane potential and ionic currents were measured with the whole-cell patch-clamp technique. The intracellular Ca2+ concentration was measured with the fura-2 method. The short-chain fatty acid butyrate (50 mmol L-1) induced a reversible hyperpolarization of the myenteric neurones by about 10 mV. This hyperpolarization was concomitant with an inhibition of a TTX-sensitive Na+ current. The hyperpolarization could be suppressed by intracellular application of Cs+, a nonselective K+ channel blocker. Fura-2 experiments revealed that butyrate induced an increase of the intracellular Ca2+ concentration. The butyrate response was suppressed by thapsigargin, indicating that butyrate stimulates the release of intracellular Ca2+. This release is responsible for the voltage response, because intracellular chelation of Ca2+ inhibited the butyrate induced hyperpolarization. Consequently, butyrate acts on enteric neurones by releasing Ca2+ from intracellular stores with the consequence of the activation of K+ channels, followed by a hyperpolarization.     

M‐type channels selectively control bursting in rat dopaminergic neurons

Midbrain dopaminergic neurons in the substantia nigra, pars compacta and ventral tegmental area are critically important in many physiological functions. These neurons exhibit firing patterns that include tonic slow pacemaking, irregular firing and bursting, and the amount of dopamine that is present in the synaptic cleft is much increased during bursting. The mechanisms responsible for the switch between these spiking patterns remain unclear. Using both in‐vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in‐vitro experiments, we have found that M‐type channels, which are present in midbrain dopaminergic cells, modulate the firing during bursting without affecting the background low‐frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10‐bis(4‐pyridinylmethyl)‐9(10H)‐anthracenone dihydrochloride, specifically potentiated burst firing. Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M‐type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine‐related diseases, including Parkinson’s disease and drug addiction. Moreover, our results demonstrate that the influence of M‐type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern.     

Mechanism of butyrate-induced hyperpolarization of cultured rat myenteric neurones

Short-chain fatty acids produced by the bacterial fermentation of carbohydrates are present in high concentrations within the colonic lumen and have been shown to alter the excitability of enteric neurones. The present study was designed to investigate the mechanisms of butyrate-induced changes in membrane potential of myenteric neurones. Myenteric neurones from 4-10-day-old rats were isolated from the small and large intestine by an enzymatic digestion with collagenase and kept in culture. Membrane potential was measured with the whole-cell patch-clamp technique and the intracellular Ca2+ concentration was measured with the fura-2 method. The short-chain fatty acid butyrate (10-100 mmol L(-1)) induced a reversible and concentration-dependent hyperpolarization of the membrane with a half-maximal effect at 30 mmol L(-1). The hyperpolarization evoked by butyrate (50 mmol L(-1)) was strongly inhibited by charybdotoxin (10(-7) mol L(-1)), a specific blocker of Ca2+ -dependent K+ channels. The butyrate-induced hyperpolarization was resistant against blockade of phospholipase C by U-73122 (10(-5) mol L(-1)), and resistant against inclusion of heparin (6 x 10(-6) mol L(-1)), an inositol-1,4,5-trisphosphate receptor antagonist, in the patch-pipette. In contrast, ruthenium red (3 x 10(-5) mol L(-1)), an inhibitor of ryanodine receptors, significantly reduced both the hyperpolarization of the membrane as well as the increase in the intracellular Ca2+ concentration evoked by butyrate. Even in neurones permeabilized with saponin (10 mg L(-1)), butyrate was able to stimulate a release of stored intracellular Ca2+ suggesting a direct action of the short-chain fatty acid at the stores without mediation of a soluble intracellular second messenger.     

Increased colonic motility in a rat model of irritable bowel syndrome is associated with up‐regulation of L‐type calcium channels in colonic smooth muscle cells

Objective This paper aimed to investigate the relationship between up‐regulation of L‐type calcium channels and altered motility disorder in a rat model of irritable bowel syndrome (IBS). Methods Male Sprague–Dawley rats were subjected to neonatal maternal separation (NMS) from postnatal day 2–14 or normal handling (NH), and used when weighted 250–300 g. Colonic smooth muscle contractions was studied in an organ bath system. L‐type Ca²⁺ channel α_1c subunit expression in smooth muscles from rat colon were studied by immunofluorescence and Western blotting analysis. The intracellular calcium concentration ([Ca²⁺]_i) of enzymatically isolated single colonic smooth muscle cell was studied with laser confocal fluorescent microscopy. Results The fecal pellets during 1 h water avoidance stress (WAS) were significantly increased; the amplitude of spontaneous contractions and contractions induced by Bay K 8644 (10 nm –1 μm ), KCl (10–60 mm ) and ACh (100 nm –10 μm ) were significantly increased in NMS rats, when comparing with that of NH rats. [Ca²⁺]i induced by Bay K 8644 (1 μm ), KCl (40 mm ), and ACh (10 μm ) significantly increased in muscle cells of NMS rats than NH rats. Further, α_1c protein expression was significantly up‐regulated in colonic smooth muscle of NMS rats than NH rats. Conclusion These results suggest that NMS lead to up‐regulation of L‐type Ca²⁺ channels expression in the colon, which contributes to the colonic motility disorder. Our findings provide direct evidence to help understanding the underlying mechanism of chronic stress‐induced colonic motility disorder in IBS.     

Dopamine D1 receptor‐mediated upregulation of BKCa currents modifies Müller cell gliosis in a rat chronic ocular hypertension model

Müller cell gliosis is a common response in many retinal pathological conditions. We previously demonstrated that downregulation of Kir channels contributes to Müller cell gliosis in a rat chronic ocular hypertension (COH) model. Here, the possible involvement of outward K⁺ currents in Müller cell gliosis was investigated. Outward K⁺ current densities in Müller cells isolated from COH rats, as compared with those in normal rats, showed a significant increase, which was mainly contributed by large‐conductance Ca²⁺‐activated K⁺ (BK_Ca) channels. The involvement of BK_Ca channels in Müller cell gliosis is suggested by the fact that glial fibrillary acidic protein (GFAP) levels were augmented in COH retinas when these channels were suppressed by intravitreal injections of iberiotoxin. In COH retinas an increase in dopamine (DA) D1 receptor (D1R) expression in Müller cells was revealed by both immunohistochemistry and Western blotting. Moreover, protein levels of tyrosine hydroxylase were also increased, and consistent to this, retinal DA contents were elevated. SKF81297, a selective D1R agonist, enhanced BK_Ca currents of normal Müller cells through intracellular cAMP‐PKA signaling pathway. Furthermore, GFAP levels were increased by the D1R antagonist SCH23390 injected intravitreally through eliminating the BK_Ca current upregulation in COH retinas, but partially reduced by SKF81297. All these results strongly suggest that the DA‐D1R system may be activated to a stronger extent in COH rat retinas, thus increasing BK_Ca currents of Müller cells. The upregulation of BK_Ca channels may antagonize the Kir channel inhibition‐induced depolarization of Müller cells, thereby attenuating the gliosis of these cells.     

Heterotrimeric guanosine triphosphate‐binding protein‐coupled modulatory actions of motilin on K+ channels and postsynaptic γ‐aminobutyric acid receptors in mouse medial vestibular nuclear neurons

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice‐patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti‐motilin receptor antibody. Bath application of motilin (0.1 μm ) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose‐dependent manner (K_d, 0.03 μm ). The motilin action on spontaneous action potentials was blocked by apamin (100 nm ), a blocker of small‐conductance Ca²⁺‐activated K⁺ channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate‐γ‐S (1 mm ) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild‐type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ‐aminobutyric acid transporter‐Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small‐conductance Ca²⁺‐activated K⁺ channels and postsynaptic γ‐aminobutyric acid receptors through heterotrimeric guanosine triphosphate‐binding protein‐coupled receptor in a group of glutamatergic MVNs.     

Ca2+‐dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20–70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca²⁺ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca²⁺ transients in circadian pacemakers accompany tetrodotoxin (TTX)‐blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX‐sensitive sustained Na⁺ channels. Instead, they are predominantly driven by mibefradil‐sensitive, low‐voltage‐activated Ca²⁺ channels and DK‐AH269‐sensitive hyperpolarisation‐activated, cyclic nucleotide‐gated cation channels. Rhythmic depolarisations activate voltage‐gated Na⁺ channels and nifedipine‐sensitive high‐voltage‐activated Ca²⁺ channels. Together with Ca²⁺ rises, the depolarisations open repolarising small‐conductance but not large‐conductance Ca²⁺‐dependent K⁺ channels. In contrast, we hypothesise that P/Q‐type Ca²⁺ channels coupled to large‐conductance Ca²⁺‐dependent K⁺ channels are involved in input‐dependent activity.     

Relation of exocytosis and Ca-activated K current during Ca release from intracellular stores in individual rat chromaffin cells

Measurement of the change in cell membrane capacitance (C_m) along with the change in I_K(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca²⁺-free external solution, bradykinin (100 nM) caused a transient increase in C_m with a concurrent change in I_K(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP₃-mediated increase of submembrane-free Ca²⁺. The increases in C_m and I_K(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca²⁺-free solution, and this was irreversibly blocked by ryanodine (1 μM) in a use-dependent manner. Caffeine-sensitive intracellular Ca²⁺ stores were also depleted in several seconds and recovered by an influx of external Ca²⁺. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca²⁺ release from the same or distinct but rapidly equilibrating intracellular Ca²⁺ stores. The single cell assay of exocytosis and the increase in I_K(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca²⁺ release from intracellular stores potentially increases submembrane Ca²⁺ concentration and modulates simultaneously two submembrane Ca²⁺-dependent processes, exocytosis and I_K(Ca), in rat adrenal chromaffin cells.     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.     

Differential regulation of HCN channel isoform expression in thalamic neurons of epileptic and non-epileptic rat strains

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels represent the molecular substrate of the hyperpolarization-activated inward current (I_h). Although these channels act as pacemakers for the generation of rhythmic activity in the thalamocortical network during sleep and epilepsy, their developmental profile in the thalamus is not yet fully understood. Here we combined electrophysiological, immunohistochemical, and mathematical modeling techniques to examine HCN gene expression and I_h properties in thalamocortical relay (TC) neurons of the dorsal part of the lateral geniculate nucleus (dLGN) in an epileptic (WAG/Rij) compared to a non-epileptic (ACI) rat strain. Recordings of TC neurons between postnatal day (P) 7 and P90 in both rat strains revealed that I_h was characterized by higher current density, more hyperpolarized voltage dependence, faster activation kinetics, and reduced cAMP-sensitivity in epileptic animals. All four HCN channel isoforms (HCN1-4) were detected in dLGN, and quantitative analyses revealed a developmental increase of protein expression of HCN1, HCN2, and HCN4 but a decrease of HCN3. HCN1 was expressed at higher levels in WAG/Rij rats, a finding that was correlated with increased expression of the interacting proteins filamin A (FilA) and tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Analysis of a simplified computer model of the thalamic network revealed that the alterations of I_h found in WAG/Rij rats compensate each other in a way that leaves I_h availability constant, an effect that ensures unaltered cellular burst activity and thalamic oscillations. These data indicate that during postnatal developmental the hyperpolarizing shift in voltage dependency (resulting in less current availability) is compensated by an increase in current density in WAG/Rij thereby possibly limiting the impact of I_h on epileptogenesis. Because HCN3 is expressed higher in young versus older animals, HCN3 likely does not contribute to alterations in I_h in older animals.     

DNA microarray analysis of striatal gene expression in symptomatic transgenic Huntington's mice (R6/2) reveals neuroinflammation and insulin associations

Huntington's disease (HD) is an inherited, progressive neurodegenerative disorder caused by CAG repeat expansion in the gene that codes for the protein huntingtin. The underlying neuropathological events leading to the selectivity of striatal neuronal loss are unknown. However, the huntingtin mutation interferes at several levels of normal cell function. The complexity of this disease makes microarray analysis an appealing technique to begin the identification of common pathways that may contribute to the pathology. In this study, striatal tissue was extracted for gene expression profiling from wild-type and symptomatic transgenic Huntington mice (R6/2) expressing part of the human Huntington's disease gene. We interrogated a 15 K high-density mouse EST array not previously used for HD and identified 170 significantly differentially expressed ESTs in symptomatic R6/2 mice. Of the 80 genes with known function, 9 genes had previously been identified as altered in HD. 71 known genes were associated with HD for the first time. The data obtained from this study confirm and extend previous observations using DNA microarray techniques on genetic models for HD, revealing novel changes in expression in a number of genes not previously associated with HD. Further bioinformatic analysis, using software to construct biological association maps, focused attention on proteins such as insulin and TH1-mediated cytokines, suggesting that they may be important regulators of affected genes. These results may provide insight into the regulation and interaction of genes that contribute to adaptive and pathological processes involved in HD.