The bradykinin receptor — a putative receptor-operated channel in PC12 cells: studies of neurotransmitter release and inositol phosphate accumulation

Bradykinin (BK) induced [³H]norepinephrine ([³H]NE) release and phosphatidylinositol turnover were investigated in PC12 cells. Induction of [³H]NE release by BK is mediated by activation of BK-B₂-receptors, as determined using type specific BK receptor antagonists. BK induces [³H]NE release with a half maximal effective concentration of30 ± 0.5nM, and reaches maximal net fractional release of9.0 ± 1% with 200 nM BK. The BK-induced release is Ca²⁺ dependent, reaching maximal release at 1.0 mM Ca²⁺, is pertussis toxin insensitive (1 μg/ml), slightly increased by a dibutyryl cAMP (1 mM) and not affected by inhibitors of the cyclooxygenase or lipoxygenase pathways. Voltage-sensitive Ca²⁺ channel blockers, verapamil (10 μM), nifedipine (10 μM), and ω-conotoxin (CgTx 10 nM), do not block the BK-induced release. However, a considerable inhibitory effect was obtained by divalent cations Co²⁺ (ED₅₀ = 0.2mM) and Ni²⁺ (ED_50²⁺ = 1mM). These results indicate the involvement of a Ca²⁺ channel in the BK-mediated release which is different from the L- or N-type voltage sensitive calcium channels. Whereas [Ca²⁺]_ex is essential for the BK-induction of catecholamine release, the rise in level of InsP's induced by BK in the presence or in the absence of [Ca²⁺]_ex is similar up to concentration of 1 μM. This indicates that the rise in InsP's induced by BK is not sufficient to cause neurotransmitter release. Moreover, subsequent addition of Ca²⁺ to BK-stimulated cells in Ca²⁺-free medium yields no release. Hence, no activity triggered by BK alone could be further stimulated by Ca²⁺ for induction of release. Protein kinase C inhibitors polymyxin B, K_252a, sangivamicin, and Ara-A, do not affect release induced by BK, indicating that also the diacylglycerol pathway activated by phospholipase C is not involved in the BK-mediated release. Since (a) the receptor-mediated release is absolutely calcium-dependent, with no release detected when Ca²⁺ is omitted from the extracellular medium, and (b) the receptor-triggered release of Ca²⁺ from intracellular stores is independent of [Ca²⁺]_ex⁷, it appears that calcium influx, and not Ca²⁺ released from intracellular stores, is the signal for stimulating release. Therefore, it is suggested that the primary signal stimulating release is Ca²⁺ influx via a specific calcium channel, and that the BK receptor may be coupled to this channel, which could be classified as a receptor-operated channel.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

The sodium channels of the neuroblastoma x glioma 108 CC 15 hybrid cell change their sensitivity for volatile and local anesthetics upon continuous passage

Summary We have studied the ion flux through the sodium channels of low passage number (<50p.) and high passage number (>150p.) neuroblastoma x glioma hybrid cells using [¹⁴C] guanidinium and specific neurotoxins to induce channel opening and closing. The sodium channels of low passage number hybrid cells could be opened by veratridine alone, suggesting the presence of voltage dependent channels in agreement with electrophysiological studies reported in the literature. The sodium channels of the high passage number hybrid cells, however, needed the synergistic action of veratridine and scorpion toxin for activation suggesting that these channels are silent. The [¹⁴C] guanidinium ion flux through the sodium channels of the high passage number hybrid cells was inhibited by significantly lower concentrations of the volatile anesthetics (halothane, isoflurane and enflurane) and the lcoal anesthetics (tetracaine and bupivacaine) than the comparable flux through the sodium channels of the low passage number hybrid cells.Dedicated to Prof. Dr. E. Wecker on the occasion of his 65th birthday.     

4-Aminopyridine and l-cis-diltiazem block the cGMP-activated K channels closed by light in the molluscan extra-ocular photoreceptors

The cGMP-activated K⁺ channels closed by light lead to the depolarizing photocurrent of photoreceptors in the Onchidium ganglion. Whole-cell current records showed that external application of 100–200 μM 4-aminopyridine or 200–400 μM l-cis-diltiazem completely blocked the macroscopic photocurrent at any depolarizing and hyperpolarizing potentials. Single-channel current recordings suggested that both 4-aminopyridine and l-cis-diltiazem act to block the cGMP-activated K⁺ channels in their open state from inside the cell.     

Cytoskeleton mediates inhibition of the fast Na+ current in respiratory brainstem neurons during hypoxia

Whole-cell Na+ currents (INa) were recorded in inspiratory neurons in a medullary slice preparation from neonatal mouse that contains the functional respiratory network. Hypoxia and metabolic poisoning with KCN rapidly inhibited INa by reducing the number of Na+ channels available for opening during depolarization. Application of agents specific for G-proteins, protein kinase C and A, intracellular Ca2+ and pH did not prevent the hypoxic inhibition of INa. The effects of hypo-osmolarity and hypoxia were additive, whereas hyperosmolarity partially prevented a subsequent hypoxic inhibition of INa. Cytochalasin B and colchicine decreased, and taxol or phalloidin increased INa and reduced its hypoxic inhibition. We conclude that cytoskeleton rearrangements during hypoxia are responsible for suppression of a fast INa in brainstem respiratory neurons, which could be mediated by the uncoupling of channel inactivation gates from cytoskeletal elements.     

Batrachotoxin blocks saltatory organelle movement in electrically excitable neuroblastoma cells

Batrachotoxin has been reported to inhibit fast axonal transport. We have examined the effect of batrachotoxin on saltatory organelle movements in N115 neuroblastoma cells (a model of fast axonal transport) using time-lapse video intensification microscopy. Batrachotoxin (0.1–1.0 μM) inhibits saltatory organelle movement. Contrary to previously published hypotheses, this inhibition of intra-axonal movement depends upon the action of batrachotoxin on action potential Na⁺ channels. Evidence for this conclusion is: (1) the range of concentrations of batrachotoxin which inhibit saltatory organelle movement is consistent with the dose-response curve for the activation of action potential Na⁺ channels by batrachotoxin in N18 neuroblastoma cells¹⁰; (2) tetrodotoxin, which blocks action potential Na⁺ channels, prevents the inhibition of organelle movements by batrachotoxin; (3) batrachotoxin has no effect on saltatory movement in cells, including some neuroblastoma cell lines, which lack action potential Na⁺ channels; and (4) in Na⁺-free or low Na⁺ media, batrachotoxin does not block organelle movement. We suggest that changes in internal ion concentrations which follow the influx of Na⁺ are responsible for the inhibition of fast axonal transport by batrachotoxin.     

Effects of funnel web spider toxin on Ca currents in neurohypophysial terminals

Funnel web spider toxin (FTX) is reportedly a specific blocker of P-type Ca²⁺ channels. The effects of FTX on the Ca²⁺ currents of isolated neurohypophysial nerve terminals of the rat were investigated using the ‘whole-cell’ patch-clamp technique. Both the transient and long-lasting Ca²⁺ current components were maximally elicited by depolarization from a holding potential equal to the normal terminal resting potential (−90 mV). Externally applied FTX inhibited the high-voltage-threshold, transient component of the Ca²⁺ current in a concentration-dependent manner, with a half-maximal inhibition at a dilution of approximately 1: 10000. FTX also shifted the peak current of the I–V relationship by + 10 mV. The long-lasting Ca²⁺ current component, which is sensitive to L-type Ca²⁺ channel blockers, was insensitive to FTX. The transient current, which is sensitive to ω-conotoxin GVIA, was completely blocked by FTX. These results suggest that there could be a novel, inactivating Ca²⁺ channel in the rat neurohypophysial terminals which is affected by both N-type and P-type Ca²⁺ channel blockers.     

Inhibition of GABA-gated chloride channel function by arachidonic acid

The effects of arachidonic acid and its metabolites on gamma-aminobutyric acid (GABAA) receptor function were determined in rat cerebral cortical synaptoneurosomes. Incubation of synaptoneurosomes with phospholipase A2 decreased muscimol-induced 36Cl- uptake. Arachidonic acid, the major unsaturated fatty acid released by phospholipase A2, also inhibited muscimol-induced 36Cl uptake. Similar inhibition was obtained with other unsaturated fatty acids (docosahexaenoic, oleic) but not with saturated fatty acids (stearic, palmitic). The effect of arachidonic acid on muscimol responses was inhibited by bovine serum albumin (BSA), and BSA enhanced muscimol responses directly, indicating the generation of endogenous arachidonic acid in the synaptoneurosome preparation. The generation of endogenous arachidonic acid was also indicated by the ability of 2 inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguaiaretic acid (NDGA), to inhibit muscimol-induced 36Cl uptake. We conclude that arachidonic acid probably has both direct and indirect actions on muscimol responses since both enzyme inhibitors inhibited muscimol responses but did not prevent the effect of exogenously added arachidonic acid. In additional experiments, arachidonic acid metabolites generated by cyclooxygenase, prostaglandins D2, E2 and F2 alpha, each decreased muscimol responses; prostaglandins F2 alpha was the most potent inhibitor. Since the unsaturated fatty acids and their metabolites are most susceptible to peroxidation, a generating system of superoxide radicals was tested on muscimol responses. A combination of xanthine and xanthine oxidase inhibited muscimol-induced 36Cl uptake in a concentration-dependent manner. We propose that the inhibition of GABAA neurotransmission by arachidonic acid and its metabolites can lead to increased neuronal excitability. This mechanism may play an important role in the development of neuronal damage following seizures or cerebral ischemia.     

Two modes of activity of nicotinic acetylcholine receptor channels in sympathetic neurons

Spectral analysis of acetylcholine (ACh) noise was performed in voltage-clamped neurons of the isolated rabbit superior cervical ganglion at 34–37°C and at membrane potential −80 mV. Two modes of activity were found in the ionic channels of nicotinic ACh receptors, with mean channel life-times of τ_f = 1.1 ± 0.1 ms for fast-operating channels and τ_s = 5.6 ± 0.6 ms for slow-operating channels. Excitatory postsynaptic current (EPSC) decays exponentially with a time constant which is very close to τ_s, indicating that the slow-operating channel activity determines the duration of EPSC. The mean value of conductance of single nicotinic ACh-receptor channel is 36 ± 3 pS.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.