Pharmacological characterisation of voltage-dependent Ca2+ channels in isolated ganglia from the myenteric plexus

Voltage-dependent Ca2+ channels of fura-2-loaded ganglionic cells from the myenteric plexus of newborn rats were pharmacologically characterised. In contrast to completely dissociated myenteric cells, intact ganglia showed a stronger loading with the Ca2+-sensitive dye and a reproducible stimulation of the fura-2 signal by the cholinergic agonist, carbachol. A depolarisation-induced increase in the intracellular Ca2+ concentration ([Ca2+]i) was induced by superfusion with 35 mmol l(-1) KCl. This increase in [Ca2+]i was sensitive to Ni2+ and Co2+ as well as omega-conotoxin MVIIA, omega-agatoxin IVA, and SNX-482. The strongest inhibition was achieved by nifedipine (5 x 10(-7) mol l(-1)) and omega-conotoxin GVIA (4.3 x 10(-7) mol l(-1)). These two blockers also inhibited the [Ca2+]i increase evoked by nicotinic receptor stimulation. Consequently, isolated myenteric ganglia in culture express different types of voltage-dependent Ca2+ channels, from which the L- and the N-type seem to be the most important. When exposed to mediators of inflammation such as tumor necrosis factor-alpha (TNF-alpha) or different prostaglandins, no pronounced alterations in the fura-2 ratio were observed suggesting that changes in the Ca2+-signalling are not centrally involved in the response of enteric ganglionic cells to these paracrine substances.     

Ca(2+) entry activated by S-nitrosylation. Relationship to store-operated ca(2+) entry

The coupling between Ca(2+) pools and store-operated Ca(2+) entry channels (SOCs) remains an unresolved question. Recently, we revealed that Ca(2+) entry could be activated in response to S-nitrosylation and that this process was stimulated by Ca(2+) pool emptying (Favre, C. J., Ufret-Vincenty, C. A., Stone, M. R., Ma, H-T. , and Gill, D. L. (1998) J. Biol. Chem. 273, 30855-30858). In DDT(1)MF-2 smooth muscle cells and DC-3F fibroblasts, Ca(2+) entry activated by the lipophilic NO donor, GEA3162 (5-amino-3-(3, 4-dichlorophenyl)1,2,3,4-oxatriazolium), or the alkylator, N-ethylmaleimide, was observed to be strongly activated by transient external Ca(2+) removal, closely resembling activation of SOC activity in the same cells. The nonadditivity of SOC and NO donor-activated Ca(2+) entry suggested a single entry mechanism. Calyculin A-induced reorganization of the actin cytoskeleton prevented SOC but had no effect on GEA3162-induced Ca(2+) entry. However, a single entry mechanism could account for both SOC and NO donor-activated entry if the latter reflected direct modification of the entry channel by S-nitrosylation, bypassing the normal coupling process between channels and pools. Small differences between SOC and GEA3162-activated Ba(2+) entry and sensitivity to blockade by La(3+) were observed, and in HEK293 cells SOC activity was observed without a response to thiol modification. It is concluded that in some cells, S-nitrosylation modifies an entry mechanism closely related to SOC and/or part of the regulatory machinery for SOC-mediated Ca(2+) entry.     

Nitric oxide inhibition of cAMP synthesis in parotid acini: regulation of type 5/6 adenylyl cyclase

The nitric oxide (NO) donor, GEA 3162, inhibited isoproterenol-induced cyclic AMP (cAMP) accumulation in a concentration- and time-dependent manner in mouse parotid acini; SIN-1 mimicked these effects. Inhibition of stimulated cAMP accumulation was independent of phosphodiesterase activity. GEA 3162 also inhibited forskolin-induced cAMP accumulation. Removal of extracellular Ca(2+), addition of La(3+), or the calmodulin (CaM) inhibitor, calmidazolium, did not prevent the NO-mediated response, and addition of the soluble guanylyl inhibitor, ODQ, did not reverse GEA 3162-induced inhibition of cAMP accumulation. GEA 3162 also inhibited adenylyl cyclase in vitro independently of Ca(2+)/CaM. Further studies revealed that the NO synthase (NOS) inhibitor, 7-nitroindazole (7-NI), reduced significantly thapsigargin-induced Ca(2+) release and capacitative Ca(2+) entry and reversed thapsigargin inhibition of the AC Type 5/6 isoform (AC5/6). Data suggest that NO produced endogenously has dual effects on cAMP accumulation in mouse parotid acini, an inhibitory effect on AC activity and a modulatory effect on capacitative Ca(2+) entry resulting in AC5/6 inhibition.     

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.     

Arachidonic acid regulates two Ca2+ entry pathways via nitric oxide

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.     

Adenylyl cyclase co-distribution with the CaBPs, calbindin-D28 and calretinin, varies with cell type: assessment with the fluorescent dye, BODIPY forskolin, in enteric ganglia

The aims of the present study were: (1) to evaluate BODIPY forskolin as a suitable fluorescent marker for membrane adenylyl cyclase (AC) in living enteric neurons of the guinea-pig ileum; (2) to test the hypothesis that AC is distributed in several subpopulations of enteric neurons; (3) to test the hypothesis that the distribution of AC in the myenteric plexus is not unique to AH/Type 2 neurons. BODIPY forskolin was used to assess the co-distribution of AC in ganglion cells expressing the specific calcium-binding proteins (CaBPs), calretinin, calbindin-D₂₈, and s-100. Cultured cells or tissues were incubated with 10?μM BODIPY forskolin for 30?min and fluorescent labeling was monitored by using laser scanning confocal microscopy. BODIPY forskolin stained the cell soma, neurites, and nerve varicosities of Dogiel Type I or II neurons. About 99% of myenteric and 27% of submucous ganglia contained labeled neurons. About 14% of myenteric and 3% of submucous glia with immunoreactivity for s-100 protein displayed BODIPY forskolin fluorescence. BODIPY forskolin differentially labeled myenteric neurons immunoreactive for calbindin-D₂₈ (80%) and calretinin (17%). The majority (63%) of BODIPY forskolin-labeled myenteric neurons displayed no immunoreactivity for either CaBP. In submucous ganglia, the dye labeled 44.6% of calretinin-immunoreactive neurons, representing 21% of all labeled neurons; it also labeled varicose nerve fibers running along blood vessels. AC thus exists in myenteric Dogiel type II/AH neurons, enteric cholinergic S/Type 1 neurons, and other unidentified non-cholinergic S/Type 1 neurons. Our data also support the hypothesis that AC is expressed in distinct functional subpopulations of AH and S neurons in enteric ganglia, and show that BODIPY forskolin is a suitable marker for AC in immunofluorescence co-distribution studies involving living cells or tissues.     

Upregulation of cyclooxygenase-2 and thromboxane A2 production mediate the action of tumor necrosis factor-alpha in isolated rat myenteric ganglia

Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-alpha (100 ng/ml) hyperpolarized the membrane (from -31.0 +/- 2.7 mV under control conditions to -61.2 +/- 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 +/- 0.7 mV under control conditions to 27.5 +/- 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (10(-6) mol/l), a stable thromboxane A2 agonist. The TNF-alpha action was inhibited by 1-benzylimidazole (2 x 10(-4) mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 10(-4) mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-alpha. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-alpha.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

CRF-induced calcium signaling in guinea pig small intestine myenteric neurons involves CRF-1 receptors and activation of voltage-sensitive calcium channels

Corticotropin-releasing factor (CRF) is a 41-amino acid peptide with distinct effects on gastrointestinal motility involving both CRF-1 and CRF-2 receptor-mediated mechanisms that are generally claimed to be centrally mediated. Evidence for a direct peripheral effect is rather limited. Electrophysiological studies showed a cAMP-dependent prolonged depolarization of guinea pig myenteric neurons on application of CRF. The current study aimed to test the direct effect of CRF on myenteric neurons and to identify the receptor subtype and the possible mechanisms involved. Longitudinal muscle myenteric plexus preparations and myenteric neuron cultures of guinea pig small intestine were incubated with the calcium indicator Fluo-4. Confocal Ca(2+) imaging was used to visualize activation of neurons on application of CRF. All in situ experiments were performed in the presence of nicardipine 10(-6) M to reduce tissue movement. Images were analyzed using Scion image and a specifically developed macro to correct for residual minimal movements. A 75 mM K(+)-Krebs solution identified 1,076 neurons in 46 myenteric ganglia (16 animals). Administration of CRF 10(-6) M and CRF 10(-7) M during 30 s induced a Ca(2+) response in 22.4% of the myenteric neurons (n = 303). Responses were completely abolished in the presence of the nonselective CRF antagonist astressin (n = 55). The selective CRF-1 receptor antagonist CP 154,526 (n = 187) reduced the response significantly to 2.1%. Stresscopin, a CRF-2 receptor agonist, could not activate neurons at 10(-7) M, and its effect at 10(-6) M (15.3%, n = 59) was completely blocked by CP 154,526. TTX 10(-6) M (n = 70) could not block the CRF-induced Ca(2+) transients but reduced the amplitude of the signals significantly. Removal of extracellular Ca(2+) blocked all responses to CRF (n = 47). L-type channels did not contribute to the CRF-induced Ca(2+) transients. Blocking N- or P/Q-type Ca(2+) channels did not reduce the responses significantly. Combined L- and R-type Ca(2+) channel blocking (SNX-482 10(-8) M, n = 64) abolished nearly all responses in situ. Combined L-, N-, and P/Q-type channel blocking also significantly reduced the response to 8.6%. Immunohistochemical staining for CRF-1 receptors clearly labeled individual cell bodies in the ganglia, whereas the CRF-2 receptor staining was barely above background. CRF induces Ca(2+) transients in myenteric neurons via a CRF-1 receptor-dependent mechanism. These Ca(2+) transients highly depend on somatic calcium influx through voltage-operated Ca(2+) channels, in particular R-type channels. Action potential firing through voltage-sensitive sodium channels increases the amplitude of the Ca(2+) signals. Besides centrally mediated effects, CRF is likely to modulate gastrointestinal motility on the myenteric neuronal level.     

Cationic amino acid transport across the blood-brain barrier is mediated exclusively by system y+

Cationic amino acid (CAA) transport is brought about by two families of proteins that are found in various tissues: Cat (CAA transporter), referred to as system y+, and Bat [broad-scope amino acid (AA) transporter], which comprises systems b0,+, B0,+, and y+L. CAA traverse the blood-brain barrier (BBB), but experiments done in vivo have only been able to examine the BBB from the luminal (blood-facing) side. In the present study, plasma membranes isolated from bovine brain microvessels were used to identify and characterize the CAA transporter(s) on both sides of the BBB. From these studies, it was concluded that system y+ was the only transporter present, with a prevalence of activity on the abluminal membrane. System y+ was voltage dependent and had a Km of 470 +/- 106 microM (SE) for lysine, a Ki of 34 microM for arginine, and a Ki of 290 microM for ornithine. In the presence of Na+, system y+ was inhibited by several essential neutral AAs. The Ki values were 3-10 times the plasma concentrations, suggesting that system y+ was not as important a point of access for these AAs as system L1. Several small nonessential AAs (serine, glutamine, alanine,and glycine) inhibited system y+ with Ki values similar to their plasma concentrations, suggesting that system y+ may account for the permeability of the BBB to these AAs. System y+ may be important in the provision of arginine for NO synthesis. Real-time PCR and Western blotting techniques established the presence of the three known nitric oxide synthases in cerebral endothelial cells: NOS-1 (neuronal), NOS-2 (inducible), and NOS-3 (endothelial). These results confirm that system y+ is the only CAA transporter in the BBB and suggest that NO can be produced in brain endothelial cells.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Calbindin immunoreactivity of enteric neurons in the guinea-pig ileum

Previous studies have identified Dogiel type II neurons with cell bodies in the myenteric plexus of guinea-pig ileum to be intrinsic primary afferent neurons. These neurons also have distinctive electrophysiological characteristics (they are AH neurons) and 82-84% are immunoreactive for calbindin. They are the only calbindin-immunoreactive neurons in the plexus. Neurons with analogous shape and electrophysiology are found in submucosal ganglia, but, with antibodies used in previous studies, they lack calbindin immunoreactivity. An antiserum that is more effective in revealing calbindin in the guinea-pig enteric nervous system has been reported recently. In the present work, we found that this antiserum reveals the same population that was previously identified in myenteric ganglia, and does not reveal any further population of myenteric nerve cells. In submucosal ganglia, 9-10% of nerve cells were calbindin immunoreactive with this antiserum. The submucosal neurons with calbindin immunoreactivity were also immunoreactive for choline acetyltransferase, but not for neuropeptide Y (NPY) or vasoactive intestinal peptide (VIP). Small calbindin-immunoreactive neurons (average profile 130 microm2) were calretinin immunoreactive, whereas the large calbindin-immunoreactive neurons (average profile 330 microm2) had tachykinin (substance P) immunoreactivity. Calbindin immunoreactivity was seen in about 50% of the calretinin neurons and 40% of the tachykinin-immunoreactive submucosal neurons. It is concluded that, in the guinea-pig ileum, only one class of myenteric neuron, the AH/Dogiel type II neuron, is calbindin immunoreactive, but, in the submucosal ganglia, calbindin immunoreactivity occurs in cholinergic, calretinin-immunoreactive, secretomotor/vasodilator neurons and AH/Dogiel type II neurons.     

Vasa recta pericytes express a strong inward rectifier K+ conductance

Strong inward rectifier potassium channels are expressed by some vascular smooth muscle cells and facilitate K+-induced hyperpolarization. Using whole cell patch clamp of isolated descending vasa recta (DVR), we tested whether strong inward rectifier K+ currents are present in smooth muscle and pericytes. Increasing extracellular K+ from 5 to 50 and 140 mmol/l induced inward rectifying currents. Those currents were Ba2+ sensitive and reversed at the K+ equilibrium potential imposed by the electrode and extracellular buffers. Ba2+ binding constants in symmetrical K+ varied between 0.24 and 24 micromol/l at -150 and -20 mV, respectively. Ba2+ blockade was time and voltage dependent. Extracellular Cs+ also blocked the inward currents with binding constants between 268 and 4,938 micromol/l at -150 and -50 mV, respectively. Ba2+ (30 micromol/l) and ouabain (1 mmol/l) depolarized pericytes by an average of 11 and 24 mV, respectively. Elevation of extracellular K+ from 5 to 10 mmol/l hyperpolarized pericytes by 6 mV. That hyperpolarization was reversed by Ba2+ (30 micromol/l). We conclude that strong inward rectifier K+ channels and Na+-K+-ATPase contribute to resting potential and that KIR channels can mediate K+-induced hyperpolarization of DVR pericytes.     

Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions

Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.     

Reduction of calcium currents in identified neurons of Helix pomatia: intracellular injection of D890

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.     

The proliferation marker pKi-67 becomes masked to MIB-1 staining after expression of its tandem repeats

The P2X(2) subtype of purine receptor was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines of the guinea-pig, and nerve cells of submucosal ganglia in the intestine. Nerve cells with strong and with weak immunoreactivity could be distinguished. Immunoreactivity in both strongly and weakly immunoreactive neurons was absorbed with P2X(2) receptor peptide. In the myenteric plexus, strong immunoreactivity was in nitric oxide synthase (NOS)- and in calbindin-immunoreactive neurons. In all regions, over 90% of NOS-immunoreactive neurons were strongly P2X(2) receptor immunoreactive. The intensity of reaction varied in calbindin neurons; in the ileum, 90% were immunoreactive for the receptor, about one-third having a strong reaction. In the submucosal ganglia, all vasoactive intestinal peptide-immunoreactive neurons were P2X(2) receptor immunoreactive, but there was no receptor immunoreactivity of calretinin or neuropeptide Y neurons. Varicose nerve fibres with P2X(2) receptor immunoreactivity were found in the gastric myenteric ganglia. These fibres disappeared after vagus nerve section. It is concluded that the P2X(2) receptor is expressed by specific subtypes of enteric neurons, including inhibitory motor neurons, non-cholinergic secretomotor neurons and intrinsic primary afferent neurons, and that the receptor also occurs on the endings of vagal afferent fibres in the stomach.     

Activation of group II mGlu receptors inhibits voltage-gated Ca2+ currents in myenteric neurons

The enteric nervous system (ENS) contains functional ionotropic and group I metabotropic glutamate (mGlu) receptors. In this study, we determined whether enteric neurons express group II mGlu receptors and the effects of mGlu receptor activation on voltage-gated Ca(2+) currents in these cells. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group II mGlu receptor agonist, reversibly suppressed the Ba(2+) current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by L-glutamate and the group II mGlu receptor agonists, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV > L-glutamate > L-CCG-I, and was reduced by the group II mGlu receptor antagonist LY-341495. Pretreatment of neurons with pertussis toxin (PTX) reduced the action of mGlu receptor agonists, suggesting participation of G(i)/G(o) proteins. Finally, omega-conotoxin GVIA blocked current suppression by DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in the submucosal and myenteric plexus of the ileum. A subset of these cells displayed a glutamatergic phenotype as shown by the expression of vesicular glutamate transporter 2. These results provide the first evidence for functional group II mGlu receptors in the ENS and show that these receptors are PTX sensitive and negatively coupled to N-type calcium channels. Inhibition of N-type calcium channels produced by activation of group II mGlu receptors may modulate enteric neurotransmission.     

Calcium currents in bullfrog sympathetic neurons. II. Inactivation

Calcium currents in bullfrog sympathetic neurons inactivate slowly and partially during depolarizations lasting 0.5-1 s. There is also a slower (minutes) inactivation process with a broad voltage dependence. An irreversible loss of current (rundown) is prominent with low concentrations of intracellular Ca2+ buffers, with either Ca2+ or Ba2+ as the charge carrier. The extent and rate of the more rapid inactivation process are maximal near the voltage at which the peak inward current is generated, suggesting that inactivation might be Ca2+ dependent. However, inactivation occurs with either Ca2+ or Ba2+ as the charge carrier, is not prevented by strong buffering of intracellular Ca2+ with 10 mM BAPTA, and varies little as the peak current is changed 10-fold by changing the divalent ion concentration. That is, rapid inactivation is not explained by simple versions of voltage, Ca2+- or current-dependent inactivation models. A model in which ion binding within the channel allows a slower, rate-limiting inactivation process fits some but not all of the observed features of inactivation. A purely voltage-dependent three-state cyclic model fits the data if microscopic inactivation is favored by hyperpolarization.     

钡、锌和镉离子对离体蜗牛食道下神经节细胞放电的影响

本文应用了细胞内微电极电位记录技术和放射性同位素示踪方法研究了与钙通道具有不同关系的Ba~(2 )、Zn~(2 )和Ca~(2 )三种二价阳离子对爆放性放电的效应,并对其机理作了初步讨论.20mMBaCl_2或17mM ZnSO_4均可使具有自发放电的神经细胞产生爆放性放电.Ba~(2 )和Zn~(2 )引起的爆放性放电,在振幅、持续时间、去极化波振幅和峰电位数等参数上都具有统计学意义上的高度显著性差异.10mM CdCl_2对自发动作电位在振幅和频率上均显示抑制效应.~(45)Ca同位素示踪实验结果表明,在Ba~(2 )、Zn~(2 )和Cd~(2 )的分别作用下,~(45)Ca进入胞内量都是减少的.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)