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.     

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.     

Otilonium bromide inhibits muscle contractions via L-type calcium channels in the rat colon

The aim of this study is to evaluate in vitro the effect of otilonium bromide (OB) on the mechanical and electrical activities of the rat colonic smooth muscle using muscle bath, microelectrodes and patch-clamp techniques. Otilonium bromide dose dependently inhibited the spontaneous activity (logIC(50) +/- SE: -5.31 +/- 0.05). This effect was not modified by TTX (10(-6) mol L(-1)). Cyclic depolarizations were abolished by OB (10(-4) mol L(-1)). Electrical field stimulation induced inhibitory junction potentials (IJPs) followed by a depolarization with superimposed spikes causing a contraction. In the presence of OB (10(-4) mol L(-1)) IJPs were recorded, but spikes and contractions were abolished. Otilonium bromide (3 x 10(-6) mol L(-1)) inhibited inward current obtained in isolated cells (amphotericin perforated patch technique). The otilonium-sensitive current amplitude was maximal (75pA) around 0 mV. The effect of different doses of OB was tested by depolarizing cells from -70 mV to 0 mV. OB dose dependently inhibited the inward current with an EC(50) of 885 nmol L(-1). Abolishment of the otilonium-sensitive current by 3 x 10(-6) mol L(-1) nifedipine confirmed that it was an L-type Ca(2+) current. Our results show that OB inhibits the spontaneous and triggered muscular contractions. This effect is produced by the inhibition of muscular action potentials carried by L-type calcium current, confirming the spasmolytic properties of OB.     

Ca2+ involvement in the action potential generation of myenteric neurones in the rat oesophagus

Intracellular recordings were used to study the physiological behaviour of rat oesophageal myenteric neurones, which are embedded in striated muscle. Injection of depolarizing pulses evoked action potentials with a clear 'shoulder' in all neurones. This shoulder disappeared under low Ca2+/high Mg2+ conditions. Tetrodotoxin (TTX; 1 micromol L-1) did not impede spike firing, whereas under combined TTX and low Ca2+/high Mg2+ conditions the action potentials were completely abolished, indicating that TTX- resistant action potentials are mediated by a Ca2+ current. Further experiments with omega-conotoxin GVIA (100 nmol L-1) revealed that these Ca2+ currents enter the cell via N-type voltage-activated Ca2+ channels (see also accompanying paper). Tetraethylammonium (10 mmol L-1) caused broadening of the action potentials, which probably resulted from prolonged Ca2+ influx due to blockade of the delayed rectifier K+ channel. Although Ca2+ appears to be involved in the spike generation of all rat oesophageal myenteric neurones, only a minority (14%) shows a slow afterhyperpolarization. Thus, no strict correlation exists between the presence of a shoulder and a slow afterhyperpolarization. Furthermore, morphological identification of 25 of the impaled neurones revealed that there was no strict correlation between morphology and electrophysiological behaviour. Consequently, rat oesophageal myenteric neurones appear to differ in several aspects from myenteric neurones in smooth muscle regions of the gastrointestinal tract.     

mGluR1 agonists elicit a Ca 2+ signal and membrane hyperpolarization mediated by apamin-sensitive potassium channels in immature rat purkinje neurons

The type 1 metabotropic glutamate receptor (mGluR1) plays an import role in the synaptic physiology and development of cerebellar Purkinje neurons. mGluR1 expression occurs early in the developmental program of Purkinje neurons, at an age that precedes expression of the dendritic structure. Few studies have investigated the physiological response produced by mGluR1 activation in early-developing Purkinje neurons. To address this question, simultaneous recording of membrane potential and intracellular Ca(2+) was performed in immature cultured Purkinje neurons coupled with exogenous application of mGluR1 agonists. Membrane potential was measured using the perforated patch method of whole-cell recording, and intracellular Ca(2+) was measured using fura-2-based Ca(2+) imaging. Brief, 1-sec micropressure application of the group I mGluR-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked a prominent Ca(2+) signal and coincident fast hyperpolarization in the immature neurons. The mGluR1-selective antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester blocked the Ca(2+) signal and fast hyperpolarization, confirming the involvement of mGluR1s. Amplitude of the fast hyperpolarization varied as a function of membrane potential and intracellular Ca(2+) and was blocked by apamin, an antagonist of the small-conductance Ca(2+)-activated K(+) channel (SK), identifying this K(+) channel as an underlying mechanism. In similar experiments with mature cultured Purkinje neurons, DHPG elicited a Ca(2+) signal, but fast membrane hyperpolarization was not evident. These results suggest that mGluR1 activation and the resulting release of Ca(2+) from intracellular stores and activation of SK channels may be a mechanism through which mGluR1 can modulate neuronal excitability of Purkinje neurons during early development.     

Brain-derived neurotrophic factor induces long-lasting Ca2+-activated K+ currents in rat visual cortex neurons

Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+-activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+-activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of -50 mV, BDNF application (20 ng/mL) for 1-2 min induced an outward current (IBDNF-OUT; 80.0 +/- 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 microm), a specific phospholipase C (PLC)-gamma inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+-activated K+ channels. BAPTA-AM (150 microm) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+-activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+-activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current.     

Fluoxetine dilates isolated small cerebral arteries of rats and attenuates constrictions to serotonin, norepinephrine, and a voltage-dependent Ca(2+) channel opener.

BACKGROUND AND PURPOSE: Recent clinical observations question that the antidepressant effect of fluoxetine (Prozac) can be explained solely with serotonin reuptake inhibition in the central nervous system. We hypothesized that fluoxetine affects the tone of vessels and thereby modulates cerebral blood flow. METHODS: A small branch of rat anterior cerebral artery (195+/-15 microm in diameter at 80 mm Hg perfusion pressure) was isolated, cannulated, and pressurized (at 80 mm Hg), and changes in diameter were measured by videomicroscopy. RESULTS: Fluoxetine dilated small cerebral arteries with an EC(50) of 7.7+/-1.0x10(-6) mol/L, a response that was not affected by removal of the endothelium or application of 4-aminopyridine (an inhibitor of aminopyridine-sensitive K(+) channels), glibenclamide (an inhibitor of ATP-sensitive K(+) channels), or tetraethylammonium (a nonspecific inhibitor of K(+) channels). The presence of fluoxetine (10(-6) to 3x10(-5) mol/L) significantly attenuated constrictions to serotonin (10(-9) to 10(-5) mol/L) and norepinephrine (10(-9) to 10(-5) mol/L). Increasing concentrations of Bay K 8644 (a voltage-dependent Ca(2+) channel opener, 10(-10) to 10(-6) mol/L) elicited constrictions, which were markedly reduced by 2x10(-6) and 10(-5) mol/L fluoxetine, whereas 3x10(-5) mol/L fluoxetine practically abolished the responses. CONCLUSIONS: Fluoxetine elicits substantial dilation of isolated small cerebral arteries, a response that is not mediated by endothelium-derived dilator factors or activation of K(+) channels. The finding that fluoxetine inhibits constrictor responses to Ca(2+) channel opener, as well as serotonin and norepinephrine, suggests that fluoxetine interferes with the Ca(2+) signaling mechanisms in the vascular smooth muscle. We speculate that fluoxetine increases cerebral blood flow in vivo, which contributes to its previously described beneficial actions in the treatment of mental disorders.     

Calcium signalling and removal mechanisms in myenteric neurones.

To characterize further the Ca2+ signalling mechanisms of myenteric neurones, we studied the effect of thapsigargin, a blocker of the Ca2+-store ATPase, and the mechanisms involved in restoring the intracellular Ca2+ concentration ([Ca2+]i) after activation. Thapsigargin (5 x 10(-6) mol L(-1)) induced an oscillatory [Ca2+]i response in 86.6% of the neurones (n=276), which was blocked by the removal of extracellular Ca2+ and by omega-conotoxin MVIIA (5 x 10(-7) mol L(-1)). The IP3-blocker, 2-aminoethyl-diphenyl-borate (75 x 10(-6) mol L(-1)), blocked or reduced the responses in 74.5% of the neurones. The oscillatory responses induced by the depletion of Ca2+ stores suggest that myenteric neurones might recruite N-type Ca2+ channels as a refill mechanism. Thapsigargin pretreatment increased the amplitude, the upstroke and duration of the K+-induced [Ca2+]i responses. Mitochondrial blockers (rotenone and antimycin/oligomycin) also prolonged the responses, but without affecting the amplitude. Furthermore, it was found that for high [Ca2+]i, the thapsigargin-sensitive Ca2+ uptake was crucial, while mitochondrial blockade affected the Ca2+ uptake over a wide range of concentrations. The Ca2+-sequestering components might also have been compensating for each other, as most drugs only delayed and not inhibited Ca2+ removal.     

The development and pharmacological characterization of calcium channel currents in cultured embryonic rat septal cells

We characterized the development and pharmacology of Ca(2+) channel currents in NGF-treated embryonic day 21 cultured rat septal cells. Using standard whole-cell voltage clamp techniques, cells were held at -80 mV and depolarized to construct current-voltage relations in conditions that eliminated Na(+) or K(+) currents. Barium (10 mM) was used as the charge carrier. Maximum current was produced when cells were depolarized to 0 or +10 mV. Recordings from 77 cells revealed that Ca(2+) channel current density increases over time in culture from nearly 0 pA/pF on day 2 in vitro (0.65+/-0.65 pA/pF) to (6.95+/-1.59 pA/pF) on days 6-8. This was followed by a period where currents became nearly 3 times more dense (21.05+/-7.16 pA/pF) at days 9-17. There was little or no evidence for low voltage activated currents. Bath application of 50-100 microM CdCl(2) abolished approximately 95% of the current. Application of 10 microM nimodipine produced a 50.5+/-3.22% reduction in current, 2 microM omega-CTx-GVIA produced a 32.4+/-7.3% reduction, and application of 4 microM omega-Aga-IVA produced a 29.5+/-5.73% reduction in current. When all three inhibitors (10 microM nimodipine, 2 microM omega-CTx-GVIA, and 4 microM omega-Aga-IVA) were applied simultaneously, a residual current remained that was 18.0+/-4.9% of the total current and was completely abolished by application of CdCl(2). This is the first report to characterize Ca(2+) channel currents in cultured embryonic septal cells. These data indicate that there is a steady increase in Ca(2+) channel expression over time in vitro, and show that like other cultured neuronal cells, septal cells express multiple Ca(2+) channel types including L, N, P/Q and R-type channels.     

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.     

α2-Adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.     

Voltage- and Ca2+-dependent burst generation in neuroendocrine Dahlgren cells in the teleost Platichthys flesus

The neuroendocrine Type 1 Dahlgren cells of the caudal neurosecretory system of the flounder display characteristic bursting activity, which may increase secretion efficiency. The firing activity pattern in these cells was voltage-dependent; when progressively depolarized, cells moved from silent (approximately -70 mV), through bursting and phasic to tonic firing (< -65 mV). Brief (10 s) evoked bursts of spikes were followed by a slow after-depolarization (ADP; amplitude up to 10 mV, duration 10-200 s), which was also voltage-dependent and could trigger a prolonged burst. The ADP was significantly reduced in the absence of external Ca(2+) ions or the presence of the L-type Ca(2+) channel blocker, nifedipine. BayK 8644 (which increases L-type channel open times) significantly increased ADP duration, whereas the Ca(2+)-activated nonselective cation channel blocker, flufenamic acid, had no effect. Pharmacological blockade of Ca(2+)-activated K(+) channels, using apamin and charybdotoxin, increased the duration of both ADP and evoked bursts. However, action potential waveform was unaffected by either apamin/charybdotoxin, nifedipine, BayK 8644 or removal of external Ca(2+). The short duration (approximately 100 ms), hyperpolarization-activated, postspike depolarizing afterpotentials (DAP), were significantly reduced by nifedipine. We propose that long duration ADPs underlie bursts and that short duration DAPs play a role in modulation of spike frequency.     

The K+-channel opener NS1619 increases endothelial NO-synthesis involving p42/p44 MAP-kinase

Ca(2+)-activated K(+) channels with large conductance (BK(Ca)) have been shown to play an important role in the regulation of vascular tone. We examined the role of the p42/p44 MAP-kinase (p42/p44(MAPK)) on nitric oxide (NO) production in human endothelial cells induced by the BK(Ca)-opener NS1619. Using DiBAC-fluorescence imaging a concentration-dependent (2.5-12.5 microM) hyperpolarization induced by NS1619 was observed. A significant increase of intracellular Ca(2+)-concentration by NS1619 was seen using Fura-2-fluorescence-imaging, which was blocked by 2-APB, or reduction of extracellular Ca(2+) (n=30; p<0.05). A cGMP-radioimmunoassay was used to examine NO synthesis. NS1619 significantly increased cGMP levels which was inhibited by LNMMA, iberiotoxin, BAPTA, 2-APB, reduction of extracellular Ca(2+), PD 98059, or U0126 (cGMP (pmol/mg protein): NS1619 3.25 +/- 0.85; NS1619 + L-NMMA 0.86 +/- 0.02; NS1619 + iberiotoxin 0.99 +/- 0.09; NS1619 + BAPTA 0.93 +/- 0.29; NS1619 + 2-APB 0.99 +/- 0.31; NS1619 + Ca(2+)-reduction 1.17 +/- 0.06; NS1619 + PD98059 1.06 +/- 0.49; NS1619 + U0126 1.10 +/- 0.24; n=10; p<0.05). The phosphorylation of eNOS and p42/p44(MAPK) was examined by immunocytochemistry. Phosphorylation of p42/p44(MAPK) was significantly increased after 10 minutes of NS1619 stimulation, whereas eNOS phosphorylation was not changed over a period of 1 to 30 minutes. NS1619-induced hyperpolarization was not affected by treatment with PD 98059 or U0126. Additionally, NS1619 inhibited endothelial proliferation involving a NO-dependent mechanism. Our data demonstrate that NS1619 causes a transmembrane Ca(2+)-influx leading to an increased NO production involving p42/p44(MAPK). This rise of NO formation is responsible for the NS1619 induced reduction of endothelial cell growth.     

Contractile effects and intracellular Ca2+ signalling induced by motilin and erythromycin in the circular smooth muscle of human colon

Motilin has excitatory effects on the colon of the rabbit and the dog, but little is known of its effect on the human colon. The aim of this study was to investigate the effects induced by motilin and erythromycin A (EMA) on muscle strips and on single cells from primary cultures from human colon. Isotonic contraction was recorded in circular muscle strips from macroscopically normal resection specimens of patients operated on for colonic neoplasm. Agonist-induced intracellular Ca2+ ([Ca2+]i) signalling was studied in primary cultures of colonic smooth-muscle cells using the ratiometric Ca2+ indicator Indo 1, on a laser-scanning confocal epifluorescence microscope. In circular muscle strips, norleucine13-porcine motilin ([Nle13]-pm)and EMA induced tonic contractions with an EC50 of 92 +/- 21 nmol L(-1) and 31 +/- 16 micromol L(-1), respectively. The maximal contraction was 21 +/- 4% (motilin) and 33 +/- 12% (EMA) of the response to 10(-4) mol L(-1) acetylcholine (ACh). The motilin antagonist OHM-11526 (10(-5.5) mol L(-1)) abolished the effects of both [Nle13]-pm and EMA. Neither tetrodotoxin (10(-5.5) mol L(-1)), L-nitro-D-arginine methyl ester (L-NAME) (10(-3.5) mol L(-1)) nor guanethidine (10(-5) mol L(-1)) interfered with the effects of [Nle13]-pm or EMA. [Nle13]-pm (10(-11)-10(-6) mol L(-1)) induced rises of [Ca2+]i in cultured colonic myocytes. At 10(-6) mol L-1, 94% of the cells responded, and half of the cells responded at 1.4 nmol L(-1) [Nle13]-pm. 81% (35/43) and 95% (75/79) responded to EMA (10(-6) mol L(-1)) and acetylcholine (ACh, 10(-4) mol L(-1)), respectively. The motilin antagonist GM-109 inhibited motilin- and EMA-induced [Ca2+]i rises. In the absence of extracellular Ca2+, only 13% (7/52) of the cells responded to [Nle13]-pm (10(-6) mol L(-1)) vs. 90% (47/52) to ACh (10(-4) mol L(-1)). Motilin and EMA have direct excitatory effects on circular smooth muscle from the human colon and these effects are mediated via a smooth-muscle motilin receptor. These findings suggest that motilin may regulate colonic motility and that motilides may have therapeutic potential for the treatment of colonic hypomotility.     

3H-D-aspartate release from cerebellar granule neurons is differentially regulated by glutamate- and K(+)-stimulation.

Neurotransmitter release in response to either 55 mM K+ or 25 microM glutamate as well as its dependency on Ca2+ from different sources was compared in cultured glutamatergic cerebellar granule cells from rat brain. The intracellular Ca2+ concentration was monitored at the single cell level in neurites as well as cell bodies employing the fluorescent Ca2+ indicator fura-2. Transmitter release was assayed using 3H-D-aspartate to label the exogenously accessible glutamate pools, which in these neurons is believed to also include the transmitter pool. In an attempt to distinguish whether transmitter release was dependent on an intact cytoskeleton or not, the colchicine-like drug Nocodazole, which also blocks transport of vesicles, was used. K(+)-stimulated transmitter release consisted for the major part (around 70%) of a Ca(2+)-dependent, Nocodazole sensitive release component and this K(+)-induced release appeared to be almost exclusively dependent on N-type Ca2+ channels. In contrast, 50% of the glutamate-induced Ca(2+)-dependent release was triggered by Ca2+ from a Dantrolene sensitive intracellular Ca2+ pool. Since these neurons undergo a pronounced maturational change in which neurotransmitter vesicles become increasingly prominent, the Ca2+ responses and transmitter release evoked by the two different stimuli were investigated as a function of the culture period. K+ and glutamate were found to increase intracellular [Ca2+] differentially. In 1-day-old cultures K+ elicited a small albeit significant increase in [Ca2+]i while glutamate was completely without effect. In 7-day-old neurons both agents induced a large increase in [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel calcium-sensitive potassium conductance is coupled to P2X3 subunit containing receptors in myenteric neurons of guinea pig ileum

This study characterized P2X receptors in guinea pig ileum myenteric S neurons (n = 124) in vitro using electrophysiological methods. ATP or alpha,beta-methylene ATP (alpha,beta-mATP), an agonist at P2X(1) and P2X(3) subunit containing receptors, depolarized 103 neurons (85%). Pyridoxal-phosphate-6-azophenyl-2',4' disulfonic acid (10 micromol L(-1)) blocked ATP- and alpha,beta-mATP-induced depolarizations. ATP-induced depolarizations and fast excitatory postsynaptic potentials (fEPSPs) were reduced by trinitrophenyl-ATP (10 micromol L(-1)), an antagonist that can block P2X(3) receptors. Ivermectin (10 micromol L(-1)), a modulator of P2X(4) and P2X(4/6) receptors, had no effect on alpha,beta-mATP-induced depolarizations. In 58% of neurons, the alpha,beta-mATP induced-depolarization was followed by an afterhyperpolarization (AHP) (P2X-AHP). Under voltage clamp, alpha,beta-mATP induced an inward current followed by an outward current which reversed polarity at 0 and -80 mV respectively. The P2X-AHP was reduced in low extracellular Ca(2+) solutions. Blockers of large, intermediate and small conductance Ca(2+)-activated K(+) channels or voltage-gated K(+) channels did not inhibit the P2X-AHP. Half of the neurons exhibiting the P2X-AHP contained nitric oxide synthase (NOS)-immunoreactivity (ir). In summary, NOS-ir S neurons express P2X(3) subunit containing P2X receptors. P2X receptors couple to activation of a Ca(2+)-activated K(+) conductance that mediates an AHP. As P2X receptors contribute to fEPSPs, the P2X-AHP may modulate S neuron excitability during purinergic synaptic transmission.     

P2Y1 receptors mediate inhibitory neuromuscular transmission and enteric neuronal activation in small intestine.

There is increasing evidence that adenosine 5'-triphosphate or a related purine plays a crucial role in smooth muscle relaxation and enteric synaptic neurotransmission. Accordingly, the aim of the present work is to investigate the role P2Y(1) receptors in purinergic inhibitory neurotransmission (pig ileum) and enteric neuronal activation in the small intestine (guinea-pig ileum). Using contractility measurements, micro-electrode recordings and Ca(2+) imaging we found that (i) adenosine 5'-Omicron-2-thiodiphosphate (ADPbetaS) (10 micromol L(-1)) caused smooth muscle relaxation and hyperpolarization that was antagonized by MRS2179 (10 micromol L(-1)) a P2Y(1) receptor antagonist and apamin (1 micromol L(-1)); (ii) electrical field stimulation (EFS) caused a non-nitrergic inhibitory junction potential (IJP) and relaxation that was antagonized by MRS2179 (10 micromol L(-1)); (iii) P2Y(1) receptors were immunolocalized in smooth muscle cells and enteric neurons; (i.v.) superfusion of ADPbetaS (1 micromol L(-1)) induced Ca(2+) transients in myenteric neurons that were inhibited by MRS2179 (1 micromol L(-1)), but not by tetrodotoxin (1 micromol L(-1)); and (v) EFS induced calcium transients were partially inhibited by MRS2179 (1 micromol L(-1)). We conclude that in the small intestine purinergic neuromuscular transmission responsible for the IJP and non-nitrergic relaxation is mediated by P2Y(1) receptors located in smooth muscle cells. Functional P2Y(1) receptors are also present in guinea-pig myenteric neurons. Therefore, P2Y(1) receptors might be an important pharmacological target to modulate gastrointestinal functions.     

Functional importance of Ca2+-activated K+ channels for lysophosphatidic acid-induced microglial migration

Abstract Migration of microglial cells towards damaged tissue plays a key role in central nervous system regeneration under pathological conditions. Using time lapse video microscopy we show that lysophosphatidic acid (LPA) enhances chemokinetic migration of murine microglial cells. In the presence of 1 micro m LPA, the mean migration rate of microglial cells was increased 3.8-fold. In patch-clamp studies we demonstrate that LPA induces activation of a Ca(2+)-activated K(+) current. Microglial Ca(2+)-activated K(+) currents were abolished by either 50 nm charybdotoxin or 10 micro m clotrimazole. In contrast, 5 micro m paxilline did not have any significant effects on Ca(2+)-activated K(+) currents. The LPA-stimulated migration of microglial cells was inhibited by blockers of IKCa1 Ca(2+)-activated K(+) channels. The mean migration rate of LPA-stimulated cells was decreased by 61% in the presence of 50 nm charybdotoxin or by 51% during exposure to 10 micro m clotrimazole. Microglial migration was not inhibited by 5 micro m paxilline. It is concluded that IKCa1 Ca(2+)-activated K(+) channels are required for LPA-stimulated migration of microglial cells.     

Characterization of cholera toxin B subunit-induced Ca(2+) influx in neuroblastoma cells: evidence for a voltage-independent GM1 ganglioside-associated Ca(2+) channel

The role of endogenous GM1 ganglioside in neurite outgrowth has been studied in N18 and NG108-15 neuroblastoma cells with the GM1-specific ligand cholera toxin B subunit (Ctx B), which stimulates Ca(2+) influx together with neuritogenesis. Our primary goal has been to identify the nature of the calcium channel that is modulated by GM1. An L-type voltage-operated Ca(2+) channel (VOCC) was previously proposed as the mediator of this phenomenon. This investigation, employing fura-2 fluorescent measurements and specific channel blockers and other agents, revealed that GM1 modulates a hitherto unidentified Ca(2+) channel not of the L type. It was opened by Ctx B; was permeable to Ca(2+) and Ba(2+) but not Mn(2+); and was blocked by Ni(2+), Cd(2+), and La(3+). Although most dihydropyridines inhibited Ctx B-induced Ca(2+) influx as well as neurite outgrowth at higher concentrations, they and other VOCC blockers at normally employed concentrations failed to do so, suggesting uninvolvement of VOCC. In addition, Ca(2+) influx induced by Ctx B was not mediated by cGMP-dependent or G-protein-coupled nonselective cation channels, as demonstrated by the cGMP antagonist Rp-cGMPS or the G-protein/receptor uncoupling agent suramin, respectively. Finally, Ca(2+) influx was unlikely to be due to inhibition or reversal of Na(+)-Ca(2+) exchanger via Ctx B induction of Na(+) uptake, insofar as no effect was seen on blocking Na(+) channels, inhibiting Na(+)-K(+)-ATPase, or eliminating extracellular Na(+). The results suggest that this novel channel is gated by interaction with GM1, which, when associated with the channel and bound by appropriate ligand, promotes Ca(2+) influx. This in turn induces signaling for the onset of neuritogenesis.