Inhibition of acetylcholine release from presynaptic terminals of skate electric organ by calcium channel antagonists: a detailed pharmacological study

Release of acetylcholine (ACh) from the presynaptic terminals in skate electric organ was tested for its sensitivity to calcium channel antagonists. A pharmacological profile was established by measuring inhibition of K(+)-stimulated release of [3H]ACh from prelabelled tissue slices. Peptide antagonists of N-type (omega-conotoxins GVIA and MVIIA) and P-type (omega-agatoxin-IVA) channels had no effect, whereas both omega-conotoxins MVIIC and SVIB produced concentration-dependent inhibition and could completely block ACh release. omega-Conotoxin GVIA and omega-agatoxin IVA did not attenuate the block by omega-conotoxin MVIIC. The inorganic ions, Cd2+ and Ni2+, also produced a full inhibition of release (Cd2+ > > Ni2+) and Gd3+ a partial one. Drugs targeting L-type channels (diltiazem, nifedipine and verapamil) at low microM concentrations and a synthetic analogue of the polyamine toxin from funnel web spider venom (sFTX) at 1 mM were all non-inhibitory. Inhibition by omega-conotoxins MVIIC (IC50 25 nM) and SVIB (IC50 500 nM) was reversible and modulated by external concentrations of Ca2+. Inhibitory potency was increased by lowering and decreased by elevating external Ca2+. This "antagonistic" effect of Ca2+ was also seen with Cd2+ inhibition. The inhibitory potency of omega-conotoxin MVIIC was unaffected by predepolarisation. End plate potentials generated by release of endogenous ACh in electrically-stimulated slices were also reversibly blocked by Cd2+ and omega-conotoxins MVIIC and SVIB but were unaffected by omega-conotoxin GVIA and omega-agatoxin IVA. It is concluded that ACh release in skate electric organ depends on presynaptic calcium channels which have different pharmacological properties from established sub-types.     

Microsphere embolism-induced changes in presynaptic function of the cerebral cortex in rats

The present study was undertaken to elucidate pathophysiological changes in the cortical presynaptic function, K(+)-stimulated calcium influx, noradrenaline release and noradrenaline uptake, on the 1st and 3rd days after microsphere embolism in rats. Voltage-dependent calcium channels were characterized pharmacologically using three types of calcium channel blockers, L-type (nifedipine and diltiazem), N-type (omega-conotoxin GVIA), and P-type channel (omega-agatoxin IVA) blockers. K(+)-stimulated calcium influx of the normal rat synaptosome was inhibited by 100 nM omega-agatoxin IVA, but not by 10 microM nifedipine, 10 microM diltiazem and 100 nM omega-conotoxin GVIA. Calcium influx of the cortical nerve terminals of the right hemisphere was decreased on the 1st and 3rd days after the embolism. Noradrenaline release and uptake were also decreased on the 1st and 3rd days after the embolism. However, the percent release of noradrenaline was not altered. The results suggest that P-type channels are predominant in the cerebrocortical nerve terminals in rats and that calcium influx, noradrenaline release and uptake in the cerebrocortical nerve terminals are decreased by microsphere embolism. The decrease in noradrenaline release may be mainly due to a reduction in the activity of noradrenaline uptake in cerebrocortical nerve terminals of the microsphere-embolized rat.     

Recognition of acute cardiac allograft rejection from serial integrated backscatter analyses in human orthotopic heart transplant recipients. Comparison with conventional echocardiography

The distribution of voltage-sensitive elevations of the level of Ca2+ in untreated SH-SY5Y cells and cells that had been induced to differentiate with staurosporine was investigated by monitoring fura-2 fluorescence in cell suspensions, and by using microfluorometry and quantitative fluorescence imaging on cell bodies and on cellular processes. Cell bodies of both types of cells displayed small Ca2+ elevations, which were composed of transient and sustained components. Elevations were partially sensitive to the L- and N-channel blockers nifedipine (1 microM) and omega-conotoxin GVIA (100 nM) respectively. Up to ten times Ca2+ elevations were observed in varicosities of treated cells than in cell bodies of treated and cells. These elevations were insensitive to compounds known to release Ca2+ from intracellular stores. Elevations of Ca2+ were sustained, and they were insensitive to 5 microM nifedipine, 100 nM omega-agatoxin IVA and 100 nM omega-conotoxin GVIA, and partially sensitive to 2 microM omega-conotoxin GVIA, indicating predominance of non-L-type, non-N-type, non-P-type channel activity. The intracellular localization of neuropeptide Y, a marker of differentiation in these cells, was also investigated by fluorescence immunocytochemistry. Varicosities of treated cells displayed marked fluorescence when viewed in a confocal microscope. These findings show that the varicosities of staurosporine-treated cells exhibit some of the functional properties of nerve terminals. The varicosities resemble boutons en passant nerve endings and they seem to express Ca2+ channels different from those in the cell body.     

Properties of the voltage-gated calcium channels mediating dopamine and acetylcholine release from the isolated rat retina

We examined the properties of voltage-gated calcium channels mediating endogenous dopamine (DA) and acetylcholine (ACh) release in the isolated rat retina. Application of 30 mM KCl elicited the release of DA and ACh, and these releases were abolished in Ca(2+)-free medium. The high K(+)-evoked DA release was largely blocked by both of omega-agatoxin IVA and omega-conotoxin MVIIC, P- and Q-type calcium channel antagonists, and partly blocked by isradipine, and L-type calcium channel antagonist, and omega-conotoxin GVIA, an N-type calcium channel antagonist. omega-Agatoxin IVA at a small dose, sufficient to block P-type channels alone, was however without effect. On the other hand, the high K(+)-evoked ACh release was partly blocked by omega-agatoxin IVA and omega-conotoxin MVIIC, but was resistant to isradipine and omega-conotoxin GVIA. Flunarizine, a non-selective T-type calcium channel antagonist, did not inhibit the release of DA and ACh. Cd2+ markedly blocked the release of both DA and ACh, Co2+ and Ni2+ slightly blocked the release of DA, and the release of ACh was not blocked by these two divalent cations. These results suggest that the high K(+)-evoked release of retinal DA is largely mediated by omega-agatoxin IVA and omega-conotoxin MVIIC sensitive calcium channels (probably Q-type channels), while the release of retinal ACh is largely mediated by as yet uncharacterized Cd2+ sensitive calcium channels. The properties of voltage-gated calcium channels involved in the release of ACh in the rat retina differ from those of DA.     

High-voltage-activated calcium currents in neurons acutely isolated from the ventrobasal nucleus of the rat thalamus

We studied the high-voltage-activated (HVA) calcium currents in cells isolated from the ventrobasal nucleus of the rat thalamus with the use of the whole cell patch-clamp technique. Low-voltage-activated current was inactivated by the use of long voltage steps or 100-ms prepulses to -20 mV. We used channel blocking agents to characterize the currents that make up the HVA current. The dihydropyridine (DHP) antagonist nimodipine (5 microM) reversibly blocked 33 +/- 1% (mean +/- SE), and omega-conotoxin GVIA (1 microM) irreversibly blocked 25 +/- 5%. The current resistant to DHPs and omega-conotoxin GVIA was inhibited almost completely by omega-conotoxin MVIIC (90 +/- 5% at 3-5 microM) and was partially inhibited by omega-agatoxin IVA (54 +/- 4% block at 1 microM). We conclude that there are at least four main HVA currents in thalamic neurons: N current, L current, and two omega-conotoxin MVIIC-sensitive currents that differ in their sensitivity to omega-agatoxin IVA. We also examined modulation of HVA currents by strong depolarization and by G protein activation. Long (approximately 1 s), strong depolarizations elicited large, slowly deactivating tail currents, which were sensitive to DHP antagonists. With guanosine 5'-0-(3-thiotriphosphate) (GTP-gamma-S) in the intracellular solution, brief (approximately 20 ms), strong depolarization produced a voltage-dependent facilitation of the current (44 +/- 5%), compared with cells with GTP (22 +/- 7%) or guanosine 5'-O-(2-thiodiphosphate) (7 +/- 4%). However, the HVA current was inhibited only weakly by 100 microM acetylcholine (8 +/- 4%). Effects of the gamma-aminobutyric acid-B agonist baclofen were variable (3-39% inhibition, n = 12, at 10-50 microM).     

Heart rate variability in patients with atrial fibrillation is related to vagal tone

A "reduced retina" preparation, consisting of the photoreceptor layer attached to the pigment epithelium in the eyecup, was used to study the pharmacology of the calcium channels controlling glutamate release by photoreceptors in Xenopus. Glutamate release was evoked either by dark adaptation or by superfusion with elevated (20 mM) potassium medium. Both darkness- and potassium-induced release were blocked by cadmium (200 microM). The N-type calcium channel blocker, omega-conotoxin GVIA (500 nM), the P-type calcium channel blocker, omega-agatoxin IVA (20 nM), and the P- and Q-type channel blocker omega-conotoxin MVIIC (1 microM) had no effect on glutamate release. In contrast, the dihydropyridines, nifedipine (10 microM) and nitrendipine (10 microM), which affect L-type calcium channels, blocked both darkness- and potassium-induced release. Bay K 8644 (10 microM), which promotes the open state of L-type calcium channels, enhanced glutamate release. These results indicate that photoreceptor glutamate release is controlled mainly by dihydropyridine-sensitive calcium channels. A dependence of glutamate release on L-type calcium channels also has been reported for depolarizing bipolar cells of a fish retina. Thus, it appears that non-inactivating L-type calcium channels are appropriate to mediate transmitter release in neurons whose physiological responses are sustained, graded potentials.     

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.     

Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.     

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves

This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 microM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 microM) and capsaicin (10 microM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 microM) had no effect. Both cyclosporin A and capsaicin (10 microM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 microM), the N-type voltage-dependent Ca2+ channel blocker omega-conotoxin GVIA (1 microM) nor the P-type channel blocker omega-agatoxin IVA (0.2 microM) affected cyclosporin A (1 microM)-induced contraction. In contrast, the L-type Ca2+ channel blocker nicardipine (10 microM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca2+ channels, resulting in contraction of the rabbit iris sphincter muscle.     

Clinical study on five myeloperoxidase specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) positive Churg-Strauss syndrome cases

Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba2+ (holding potential, Vh=-80 mV) revealed the presence of only high-threshold voltage-dependent Ca2+ channels; T-type Ca2+ channels were not seen. By using supramaximal concentrations of selective Ca2+ channel blockers, the whole-cell IBa could be fractionated into various subcomponents. Thus, IBa had a 25% fraction sensitive to 1 microM nifedipine (L-type channels), 21% sensitive to 1 microM omega-conotoxin GVIA (N-type channels), and 60% sensitive to 2 microM omega-agatoxin IVA (P/Q-type channels). The activation of IBa was considerably slowed down, and the peak current was inhibited upon superfusion with 10 microM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of IBa was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca2+ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba2+. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca2+ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5-50%. The results also provide strong support for the hypothesis that Ca2+ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates.     

Specificity in the interaction of HVA Ca2+ channel types with Ca2+-dependent AHPs and firing behavior in neocortical pyramidal neurons

Intracellular recordings and organic and inorganic Ca2+ channel blockers were used in a neocortical brain slice preparation to test whether high-voltage-activated (HVA) Ca2+ channels are differentially coupled to Ca2+-dependent afterhyperpolarizations (AHPs) in sensorimotor neocortical pyramidal neurons. For the most part, spike repolarization was not Ca2+ dependent in these cells, although the final phase of repolarization (after the fast AHP) was sensitive to block of N-type current. Between 30 and 60% of the medium afterhyperpolarization (mAHP) and between approximately 80 and 90% of the slow AHP (sAHP) were Ca2+ dependent. Based on the effects of specific organic Ca2+ channel blockers (dihydropyridines, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC), the sAHP is coupled to N-, P-, and Q-type currents. P-type currents were coupled to the mAHP. L-type current was not involved in the generation of either AHP but (with other HVA currents) contributes to the inward currents that regulate interspike intervals during repetitive firing. These data suggest different functional consequences for modulation of Ca2+ current subtypes.     

The glutamate receptor/NO/cyclic GMP pathway in the hippocampus of freely moving rats: modulation by cyclothiazide, interaction with GABA and the behavioural consequences

Monitoring of extracellular cGMP during intracerebral microdialysis in freely moving rats permits the study of the functional changes occurring in the glutamate receptor/nitric oxide (NO) synthase/guanylyl cyclase pathway and the relationship of these changes to animal behaviour. When infused into the rat hippocampus in Mg2+-free medium, cyclothiazide, a blocker of desensitization of the AMPA-preferring receptor, increased cGMP levels. The effect of cyclothiazide (300 microM) was abolished by the NO synthase inhibitor L-NARG (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (100 microM). During cyclothiazide infusion the animals displayed a pre-convulsive behaviour characterized by frequent "wet dog shakes" (WDS). Neither L-NARG nor ODQ decreased the WDS episodes. Both cGMP and WDS responses elicited by cyclothiazide were prevented by blocking NMDA receptor function with the glutamate site antagonist CGS 19755 (100 microM), the channel antagonist MK-801 (30 microM) or Mg2+ ions (1 mM). The AMPA/kainate receptor antagonists DNQX (100 microM) and NBQX (100 microM) abolished the WDS episodes but could not inhibit the cyclothiazide-evoked cGMP response. DNQX or NBQX (but not MK-801) elevated, on their own, extracellular cGMP levels. The cGMP response elicited by the antagonists appears to be due to prevention of a glutamate-dependent inhibitory GABAergic tone, since infusion of bicuculline (50 microM) caused a strong cGMP response. The results suggest that (a) AMPA/kainate receptors linked to the NO/cGMP pathway in the hippocampus (but not NMDA receptors) are tonically activated and kept in a desensitized state by endogenous glutamate; (b) blockade of AMPA/kainate receptor desensitization by cyclothiazide leads to endogenous activation of NMDA receptors; (c) the hippocampal NO/cGMP system is under a GABAergic inhibitory tone driven by non-NMDA ionotropic receptors; (d) the pre-convulsive episodes observed depend on hippocampal NMDA receptor activation but not on NO and cGMP production.     

Should unipolar leads be implanted in the atrium? A Holter electrocardiographic comparison of threshold adapted unipolar and high sensitive bipolar sensing

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches). 2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba2+ currents at - 50 mV (Ki=30.6 nM). 3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (> or =30 microM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately -82 mV by application of 100 microM levcromakalim. 4. The 100 microM levcromakalim-induced inward current (symmetrical 140 mM K+ conditions, -50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 microM). In contrast, Bay K 8644 (1 microM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current. 5. When 100 microM nifedipine was included in the pipette solution during conventional whole-cell recording at -50 mV, application of levcromakalim (100 microM) caused a significant inward membrane current which was suppressed by 5 microM glibenclamide. On the other hand, inclusion of 5 microM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current. 6. The levcromakalim-induced K+ channel openings in cell-attached configuration were suppressed by subsequent application of 5 microM glibenclamide but not of 100 microM nifedipine. 7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K+ channel activity mainly through extracellular blocking actions on the K+ channel itself.     

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection. 2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7 +/- 0.9 nmol g-1 tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 microM), omega-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 microM), aspartate (100 microM), gamma-aminobutyric acid (100 microM) or carbachol (50 microM). 3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6 +/- 12.7 nmol g-1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100-400 microM) partially protected NO (15 microM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 microM), but not from inhibition by hydroquinone or duroquinone (both 100 microM). 4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 microM; 2 h) still exhibited release of ascorbate (2.5 +/- 0.4 nmol g-1 tissue) and urate (22.2 +/- 2.9 nmol g-1 tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K+ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles. 5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca2+ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca2+ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na+ channel activator veratridine. Contractions caused by either 45.9 mM KCl or veratridine (10 microM) were inhibited by spantide (10 microM), a tachykinin receptor antagonist, and capsaicin (10 microM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca2+ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. omega-Conotoxin GVIA (1 microM), an N-type Ca2+ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K 8644, an L-type Ca2+ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9 mM KCl, and enhanced contractions induced by 15.9 mM KCl and veratridine (2 microM). omega-Agatoxin IVA (0.3 microM), a P-type voltage-dependent Ca2+ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca2+ channel blockers or by Bay K 8644. These results suggest that, in the rabbit iris sphincter muscle. L- and N-type voltage-dependent Ca2+ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine.     

Neuronal synchronization and ionic mechanisms for propagation of excitation in the functional network of immortalized GT1-7 neurons: optical imaging with a voltage-sensitive dye

Immortalized gonadotropin releasing hormone (GnRH) neurons (GT1 cell line) in culture release GnRH in a pulsatile manner, suggesting that GT1 cells form a functional neuronal network. Optical imaging techniques and a voltage-sensitive fluorescent dye (RH795) were used to study the mechanism of neuronal synchronization and intercellular communication in cultured GT1-7 cells (one of the subclones of the GT1 cell line). The majority (79%) of GT1-7 cells in contact with one another revealed synchronized fluctuations in spontaneous neuronal activity. When a cell in contact with other cells was electrically stimulated, the evoked excitation was propagated to neighbouring cells. The ionic mechanisms involved in the propagation of electrical signals between interconnected GT1-7 cells were investigated using various blockers of Na+, Ca2+ and K+ channels. The propagation of stimulus-evoked excitation was prevented by the voltage-dependent Na+ channel blocker tetrodotoxin. It was also prevented by the voltage-dependent Ca2+ channel blockers, Ni+ (nonselective), nimodipine (L-type) and flunarizine (T-type > L-type), but not apparently affected by omega-agatoxin IVA (P- and Q-type) and omega-conotoxin MVIIA (N-type). The propagation was not influenced by the K+ channel blockers, quinine, tetraethylammonium and Ba2+, but in some cases, it was enhanced by 4-aminopyridine (4-AP) and prevented by apamin. These results suggest that voltage-dependent Na+ channels and L- and T-type Ca2+ channels are involved in the propagation of electrical signals in the GT1-7 neuronal network. Ionic mechanisms, through 4-AP- or apamin-sensitive K+ channels, also seem to be involved in the regulation of signal propagation. These mechanisms may underlie the functioning of the neuronal network formed by immortalized GnRH neurons.     

Inhibition of N-type calcium channels: the only mechanism by which presynaptic alpha 2-autoreceptors control sympathetic transmitter release

Alpha 2-Adrenoceptors are known to inhibit voltage-dependent Ca2+ channels located at neuronal cell bodies; the present study investigated whether this or alternative mechanisms, possibly downstream of Ca2+ entry, underlie the presynaptic alpha 2-adrenergic modulation of transmitter release from chick sympathetic neurons. Using chick sympathetic neurons, overflow of previously incorporated [3H]noradrenaline was elicited in the presence of extracellular Ca2+ by electrical pulses, 25 mM K+ or 10 microM nicotine, or by adding Ca2+ to otherwise Ca(2+)-free medium when cells had been made permeable by the calcium ionophore A23187 or by alpha-latrotoxin. Pretreatment of neurons with the N-type Ca2+ channel blocker omega-conotoxin GVIA and application of the alpha 2-adrenergic agonist UK 14304 reduced the overflow elicited by electrical pulses, K+ or nicotine, but not the overflow caused by Ca2+ after permeabilization with alpha-latrotoxin or A23187. In contrast, the L-type Ca2+ channel blocker nitrendipine reduced the overflow due to K+ and nicotine, but not the overflow following electrical stimulation or alpha-latrotoxin- and A23187-permeabilization. The inhibition of electrically evoked overflow by UK 14304 persisted in the presence of nitrendipine and the L-type Ca2+ channel agonist BayK 8644, which per se enhanced overflow. In omega-conotoxin GVIA-treated cultures, electrically evoked overflow was also enhanced by BayK 8644 and almost reached the value obtained in untreated neurons. However, UK 14304 lost its effect under these conditions. Whole-cell recordings of voltage-activated Ca2+ currents corroborated these results: UK 14304 inhibited Ca2+ currents by 33%, nitrendipine caused a 7% reduction, and BayK 8644 increased the currents by 30%. Moreover, the dihydropyridines failed to abolish the inhibition by UK 14304, but pretreatment with omega-conotoxin GVIA, which reduced mean amplitude from 0.95 to 0.23 nA, entirely prevented alpha 2-adrenergic effects. Our results indicate that the alpha 2-autoreceptor-mediated modulation of noradrenaline release from chick sympathetic neurons relies exclusively on the inhibition of omega-conotoxin GVIA-sensitive N-type Ca2+ channels. Mechanisms downstream of these channels and voltage-sensitive Ca2+ channels other than N-type appear not to be important.     

Calcium oxalate crystal attachment to cultured kidney epithelial cell lines

Lamprey spinal neurons exhibit a fast afterhyperpolarization and a late afterhyperpolarization (AHP) which is due to the activation of apamin-sensitive SK Ca2+-dependent K+ channels (KCa) activated by calcium influx through voltage-dependent channels during the action potential (Hill et al. 1992, Neuroreport, 3, 943-945). In this study we have investigated which calcium channel subtypes are responsible for the activation of the KCa channels underlying the AHP. The effects of applying specific calcium channel blockers and agonists were analysed with regard to their effects on the AHP. Blockade of N-type calcium channels by omega-conotoxin GVIA resulted in a significant decrease in the amplitude of the AHP by 76.2+/-14.9% (mean +/- SD). Application of the P/Q-type calcium channel blocker omega-agatoxin IVA reduced the amplitude of the AHP by 20.3+/-10.4%. The amplitude of the AHP was unchanged during application of the L-type calcium channel antagonist nimodipine or the agonist (+/-)-BAY K 8644, as was the compound afterhyperpolarization after a train of 10 spikes at 100 Hz. The effects of calcium channel blockers were also tested on the spike frequency adaptation during a train of action potentials induced by a 100-200 ms depolarizing pulse. The N- and P/Q-type calcium channel antagonists decreased the spike frequency adaptation, whereas blockade of L-type channels had no effect. Thus in lamprey spinal cord motor- and interneurons, apamin-sensitive KCa channels underlying the AHP are activated primarily by calcium entering through N-type channels, and to a lesser extent through P/Q-type channels.