Histamine H3 receptor inhibition of K+-evoked release of acetylcholine from rat cortex in vivo

Inflammation Research -     

Localized IP3-evoked Ca2+ release activates a K+ current in primary vagal sensory neurons

Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP(3), and the consequent release of Ca(2+), could trigger a Ca(2+)-activated K(+) current (I(IP3)). Photorelease of caged IP(3) evoked an I(IP3) that averaged 2.36 +/- 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at -50 mV. I(IP3) was abolished by intracellular BAPTA (2 mM), a Ca(2+) chelator. Changing the K(+) equilibrium potential by increasing extracellular K(+) ion concentration caused a predicted Nernstian shift in the reversal potential of I(IP3). These results indicated that I(IP3) was a Ca(2+)-dependent K(+) current. I(IP3) was unaffected by three common antagonists of Ca(2+)-activated K(+) currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 microM) included in the patch pipette. We have previously demonstrated that both IP(3)-evoked Ca(2+) release and Ca(2+)-induced Ca(2+) release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca(2+)-activated K(+) current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca(2+) stores, we showed that IP(3)-evoked Ca(2+) release occurs independently of CICR, but interestingly, that a component of I(IP3) requires CICR. These data suggest that IP(3)-evoked Ca(2+) release activates a K(+) current that is pharmacologically distinct from other Ca(2+)-activated K(+) currents in NGNs. We describe several models that explain our results based on Ca(2+) signaling microdomains in NGNs.     

Inhibition of K(+)-evoked glutamate release from rat neocortical and hippocampal slices by gabapentin

Gabapentin (Neurontin((R))) has preclinical and clinical efficacy as an anticonvulsant, antihyperalgesic, anxiolytic, and neuroprotective drug. Since L-glutamic acid (GLU) is involved in various CNS (central nervous system) disorders, gabapentin may attenuate the release of this neurotransmitter possibly by interacting with the auxiliary alpha(2)delta subunit of voltage-sensitive calcium channels (VSCC). The effects of gabapentin, pregabalin (S-(+)-3-isobutylgaba) and its enantiomer R-(-)-3-isobutylgaba, and N- and P/Q-type VSCC-targeting peptide ligands (omega-conotoxin MVIIA, omega-conotoxin MVIIC, omega-agatoxin TK) were assessed in vitro on K(+)-evoked (endogenous) GLU release from rat neocortical and hippocampal slices. Gabapentin and pregabalin decreased GLU release by 11-26% with R-(-)-3-isobutylgaba being less effective than pregabalin. The reference N- and P/Q-type VSCC-targeting ligands reduced GLU release by 19-55% to implicate these VSCC in this Ca(2+)-dependent process. The inhibitory effect of gabapentin and related compounds on GLU release may reflect a subtle modulation of VSCC function which normalizes pathological changes in neurotransmitter release.     

GABAergic mechanism in histamine H3 receptor inhibition of K+-evoked release of acetylcholine from rat cortex in vivo

Inflammation Research -     

A1 and A2A receptor activation by endogenous adenosine is required for VIP enhancement of K+ -evoked [3H]-GABA release from rat hippocampal nerve terminals

Vasoactive intestinal peptide (VIP) modulates GABA release from hippocampal nerve terminals and enhances hippocampal synaptic transmission through a pathway dependent on GABAergic transmission. Since VIP modulation of hippocampal synaptic transmission is dependent on the tonic actions of adenosine we investigated if endogenous adenosine could influence VIP enhancement of GABA release from isolated hippocampal nerve endings, and which adenosine receptors could be mediating this influence. When extracellular endogenous adenosine was removed using adenosine deaminase (ADA, 1 U/ml), the enhancement (57.2 ± 3.7%) caused by VIP on GABA release was prevented. Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10 nM) or of A_2A receptors with ZM241385 (50 nM) abolished the effect of VIP. In the presence of ADA, selective A_2A receptor-activation with CGS21680 (10 nM) readmitted most of the enhancement caused by VIP on GABA release (50.7 ± 5.3%). Also in the presence of ADA, A₁ receptor activation with N⁶-cyclopentyladenosine (CPA, 50 nM) partially readmitted that effect of VIP (32.6 ± 3.8%). In conclusion, the enhancement of GABA release caused by VIP in hippocampal nerve terminals is dependent on the tonic actions of adenosine on both A₁ and A_2A receptors, and this action of adenosine is essential to VIP modulation of GABA release.     

Reversible and irreversible neuronal damage caused by excitatory amino acid analogues in rat cerebellar slices

Slice preparations of the developing rat cerebellum were used to investigate the light and electron microscopic correlates of reversible and irreversible neuronal injury caused by the neurotoxic excitatory amino acid receptor agonists, kainate and N-methyl-D-aspartate. The slices were examined after various periods of exposure to the agonists (up to 30 min) with or without a 90 min recovery period in agonist-free medium. N-Methyl-D-aspartate (100 microM) caused necrosis of deep nuclear neurons and differentiating granule cells, the exposure times necessary to induce non-recoverable damage (leading to necrosis), being, respectively, 10 min and 20-30 min. Exposure periods of only 2-4 min with kainate (100 microM) were needed for Golgi cells to subsequently undergo necrosis. Other cell types (Purkinje, granule and deep nuclear neurons) were altered histologically by kainate but most recovered fully from 30 min exposures. Before the recovery period, the worst affected of these cells (deep nuclear neurons) displayed increased cytoplasmic and nuclear electron density and microvacuolation due to swelling of Golgi cisterns but little or no chromatin clumping or mitochondrial expansion. The neurons which were injured irreversibly by the agonists within 30 min displayed, near the time of lethal injury, increased cytoplasmic and nuclear electron lucency, marked focal aggregation of chromatin and swelling of Golgi apparatus. Mitochondrial swelling did not appear to precede lethal injury and even after exposure times sufficient, or more than sufficient, to lead to necrosis, large numbers of mitochondria remained in a condensed configuration. The significance of the histological changes is discussed and they are compared with those occurring in other pathological conditions. The time scales required for the receptor agonists to induce irreversible cellular lesions would be consistent with this being a process which is responsible for acute neuronal necrosis in the brain.     

K(+)-evoked dopamine release depends on a cytosolic Ca2+ pool regulated by N-type Ca2+ channels.

Membrane depolarization evoked by 25-40 mM K+ elicited an immediate increase of somatic and neuritic [Ca2+]i in cultured dopaminergic neurons as measured by digital fluorescence microscope imaging. The rise of neuritic [Ca2+]i was inhibited by N-type but not L-type Ca2+ channel blockers, while the rise of somatic [Ca2+]i was prevented by both L- and N-type Ca2+ channel blockers. Similarly, depolarization-induced [3H]dopamine release was selectively attenuated by N-type Ca2+ channel blockers. The present results suggest that [3H]dopamine release from mesencephalic neuronal cell cultures relates to a Ca(2+)-dependent mechanism regulated by N-type channels located in the vicinity of the exocytotic sites within neuritic processes.     

Insecticidal properties of 3-aminopropyl(methyl)phosphinic acid and its effect on K(+)-evoked release of acetylcholine from cockroach synaptosomes

3-Aminopropyl(methyl)phosphinic acid (APMP), a potent agonist of mammalian GABAB receptors, caused prostration in houseflies (Musca domestica L.) on injection into their thoraces, with an ED50 value of 0.42 microgram/fly. The 48-h LD50 values of APMP were estimated to be 0.45 and 5.6 micrograms/fly in the presence and absence of piperonyl butoxide, a mixed-function oxidase inhibitor, respectively. Analogues of APMP, bearing a longer or shorter side chain by a CH2 unit, or a phenyl group in the place of the methyl group, were without effects. In perfusion assays with synaptosomes prepared from the thoracic/abdominal nerve cords of cockroaches (Periplaneta americana L.), 1 mM APMP reduced K(+)-evoked acetylcholine release to 10.4% of the control. These findings indicate that the--physiologically important site of action of APMP, which might be implicated in neurotransmitter release, is present in insect neurons.     

D-aspartate release from cerebellar astrocytes: modulation of the high K-induced release by neurotransmitter amino acids

The properties of D-aspartate release were studied in cerebellar astrocytes (14-15 DIV) in primary cultures in the rat. The spontaneous release of D-aspartate from astrocytes was fast, being further enhanced in Na- and Ca-free (EDTA-containing) media. Kainate, quisqualate, D-aspartate and L-glutamate stimulated the release, whereas L-glutamatediethylester was inhibitory. The release was enhanced by veratridine and high K (50 mM). Substitution of chloride by acetate in the experimental medium did not change the basal release but slightly decreased the potassium-induced release, indicating that the high K-induced D-aspartate release is primarily due to depolarization of cells. The K-stimulated release was independent of extracellular Ca2+ and potentiated by kainate and quisqualate. The effect of kainate was reduced by kynurenate, and that of quisqualate by L-glutamatediethylester. Glycine, taurine and GABA were equally effective in depressing the stimulated release of D-aspartate. The inhibition of GABA could be blocked by GABA antagonists. The results suggest that inhibitory amino acids may be involved in the regulation of glutamate release from cerebellar astrocytes. A further implication is that cerebellar astrocytes possess functional glutamate receptors of kainate and quisqualate subtypes.     

Aging increases adenosine and inosine release by human fibroblast cultures

The effect of in vitro age and donor age on net release of adenosine and inosine was studied in cultures of normal human fibroblasts. Confluent cultures of low-(population doubling level [PDL] 23-25) and high- (PDL 43-45) passage human lung fibroblasts derived from a 16-week-old fetal donor (IMR-90) were incubated for 30 min in physiological saline and the release of adenosine and inosine into the saline was determined by HPLC. Release of adenosine and inosine into the saline bathing low-passage human skin fibroblasts derived from a 16-week-old fetal donor (GM6111) was also determined and compared with two strains of low-passage skin fibroblasts from aged (66-67 years) donors (GM3529 and GM3524). The release of adenosine and inosine by low-passage cultures of fetal lung fibroblasts was 911 and 225 pmol/30 min per mg protein, respectively. In high-passage cultures of lung fibroblasts, release of adenosine and inosine was significantly greater at 1403 and 351 pmol/30 min per mg protein, respectively. The release of adenosine and inosine by low-passage cultures of fetal skin fibroblasts was 250 and 179 pmol/30 min per mg protein, respectively. In low-passage skin fibroblasts from aged donors, release of adenosine and inosine was significantly greater at 583 and 652 pmol/30 min per mg protein, respectively. These results indicate that the net release of adenosine and inosine by cultured human fibroblasts into their extracellular environment is enhanced by in vitro aging of lung fibroblasts and is greater in skin fibroblast from aged donors.     

Hydrogen peroxide mediates damage by xanthine and xanthine oxidase in cerebellar granule neuronal cultures

The free radical-generating system of xanthine and xanthine oxidase is commonly used experimentally as a source of superoxide anion, which can produce oxidative stress, leading to cellular damage and death. Models of oxidative stress are important in elucidating pathologies associated with increased levels of reactive oxygen species, including stroke and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We therefore, examined the effect of the xanthine/xanthine oxidase system on the viability of postnatal cerebellar granule neurones obtained from 8-day old Sprague–Dawley rat pups. Xanthine (100 μM) and xanthine oxidase (0.02 U/ml) applied for 1 or 6 h reduced the viability of cells at 8 div assessed using the alamar blue assay, and induced morphological changes, such as shrinkage of the cell bodies and neurites. Heat-inactivation of xanthine oxidase resulted in complete loss of its activity. Superoxide dismutase (250 U/ml) failed to modify the damage by xanthine and xanthine oxidase, while catalase (250 U/ml) completely prevented it. When applied alone, xanthine oxidase significantly lowered cell viability, an effect that was blocked by allopurinol and catalase, but not by superoxide dismutase. The results indicate that xanthine and xanthine oxidase can produce predominantly hydrogen peroxide instead of the superoxide anion. Cerebellar granule cells in culture may also possess significant levels of endogenous xanthine.     

Nitric oxide enhances amino acid release from immature chick embryo retina

Nitric oxide (NO) was investigated for its ability to induce amino acid release from immature chick retina. The production of endogenous NO by activation of NO synthase after stimulation of N-methyl-d-aspartate (NMDA) subtype of glutamate receptor caused a significant increase in basal release of γ-aminobutyric acid (GABA) and glutamine, whereas a more modest increase in the glutamate release was also observed. The exposure of chick retina from 9-day-old embryos to NO-generating compounds, S-nitroso-N-acetylpenicillamine (SNAP) and sodium nitroprusside (SNP) produced a dose dependent increase in GABA, glutamine, and glutamate release. This effect was reduced by about 80% by haemoglobin. These results indicate that NO has a stimulatory effect on amino acid release from chick embryo immature retina. However, this effect does not appear to involve a cGMP-related mechanism because 8-bromo-cGMP, a stable analogue of cGMP, failed to affect spontaneous amino acid release and because zaprinast did not enhance NMDA-stimulated release. In conclusion, our present observations may account for a role of NMDA-mediated events in the biochemical maturation under depolarizing conditions.     

Okadaic acid inhibits the release of TSH in response to TRH and K+ from rat anterior pituitaries.

The effects of okadaic acid, a non-phorbol-12-tetradecanoate-13-acetate (non-TPA)-type tumor promoter and a potent inhibitor of protein phosphatases, on thyroid-stimulating hormone (TSH) secretion from the rat anterior pituitary were examined. Preincubation of anterior pituitaries with okadaic acid caused a time- and concentration-related decrease in a subsequent thyrotropin-releasing hormone (TRH)-stimulated TSH secretion, whereas it did not cause any changes in basal secretion of TSH. In addition, okadaic acid inhibited a subsequent high K(+)-induced TSH secretion. In contrast, ionomycin-induced TSH secretion was not inhibited by pretreatment with okadaic acid. The present results suggest that okadaic acid may block the release of TSH by inhibition of Ca2+ influx through voltage-sensitive and/or receptor-operated Ca2+ channels.     

The selective A2 adenosine receptor agonist CGS 21680 enhances excitatory transmitter amino acid release from the ischemic rat cerebral cortex.

The effects of the selective adenosine A2 receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride (CGS 21680) on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four vessel occlusion. Pretreatment with CGS 21680 failed to alter basal excitatory amino acid levels, however, CGS 21680 at 10(-6) M significantly enhanced the ischemia-evoked release. Thus, aspartate and glutamate release during ischemia can be stimulated via the activation of A2 receptors, in addition to the suppression of excitatory amino acid release mediated by selective A1 receptor agonists.     

Development and fine structure of murine Purkinje cells in dissociated cerebellar cultures: neuronal polarity

 Cerebellar Purkinje cells (PC) display a highly distinctive form of polarity. We have cultured murine PCs from dissociated E16 cerebellar anlagen for 1 week to investigate the early stages of neuronal compartmentalization and synaptic interactions, features which are important for the establishment of neuronal polarity. To unequivocally identify the PCs we utilized light and electron microscopic immunocytochemistry with an antiserum to the cell class-specific marker L7/pcp2 gene product. The PCs typically show a single, long axon, numerous short appendages classified as filopodia and protospines, and a small number of protodendrites. The nucleus is positioned asymmetrically in both the horizontal and vertical axes of the soma. The Golgi apparatus, coated and uncoated vesicles, and mitochondria are prominent ultrastructural features, while the endoplasmic reticulum is highly fragmented. The cell body receives rudimentary synapses on its smooth surfaces and appendages and no consistent morphological differences were detected between these elementary contacts. The axon is clearly identifiable; it emanates from either the cell body or a protodendrite, bifurcates at predominantly right angles, forms beaded collaterals, and terminates with relatively large growth cones. The varicosities of the PC axon contain pleomorphic synaptic vesicles and form rudimentary synapses primarily with the dendritic shafts of immunonegative neurons. The protodendrites are short, quickly tapering and sparsely branched; they emit numerous filopodia and immature spines and terminate with small growth cones. Rudimentary synapses are received on the proximal dendritic shafts and filopodia, and more mature synapses occur frequently on protospines. With few exceptions, PCs lie atop an astrocytic bed layer and glial processes are apposed to the various aspects of the PC body left free by the afferent axons. By contrast, PC processes are largely free of glial sheaths. We conclude that the ”stellate stage” of PC development in situ is replicated rather faithfully in culture and that PCs have established polarity and have begun to form intercellular contacts by 1 week in vitro. Moreover, the PCs are already morphologically distinct from other cell types in the 1-week cultures, although they have yet to develop the differentiated features that distinguish mature PCs. Accepted: 30 June 1997     

Evidence for two mechanisms of amino acid osmolyte release from hippocampal slices

A 30% decrease in osmolarity stimulated 3H-taurine, 3H-GABA and glutamate (followed as 3H-D-aspartate) efflux from rat hippocampal slices. 3H-taurine efflux was activated rapidly but inactivated slowly. It was decreased markedly by 100 microM 5-nitro-(3-phenylpropylamino)benzoic acid (NPPB) and 600 microM niflumic acid and inhibited strongly by tyrphostins AG18, AG879 and AG112 (25-100 microM), suggesting a tyrosine kinase-mediated mechanism. Hyposmolarity activated the mitogen-activated protein kinases (MAPK) extracellular-signal-related kinase-1/2 (ERK1/ERK2) and p38, but blockade of this reaction did not affect 3H-taurine efflux. Hyposmosis also activated phosphatidylinositide 3-kinase (PI3K) and its prevention by wortmannin (100 nM) essentially abolished 3H-taurine efflux. 3H-taurine efflux was insensitive to the protein kinase C (PKC) blocker chelerythrine (2.5 microM) or to cytochalasin E (3 microM). The release of 3H-GABA and 3H-D-aspartate occurred by a different mechanism, characterized by rapid activation and inactivation, insensitivity to NPPB, niflumic acid, tyrphostins or wortmannin. 3H-GABA and 3H-D-aspartate efflux was not due to external [NaCl] decrease, cytosolic Ca2+ increase or depolarization, or to reverse operation of the carrier. This novel mechanism of amino acid release may be mediated by Ca2+-independent exocytosis and modulated by PKC and actin cytoskeleton disruption, as suggested by its inhibition by chelerythrine and potentiation by 100 nM phorbol-12-myristate-13 acetate (PMA) and cytochalasin E. GABA and glutamate osmosensitive efflux may explain the hyposmolarity-elicited increase in amplitude of inhibitory and excitatory postsynaptic potentials in hippocampal slices as well as the hyperexcitability associated with hyponatraemia.     

Calcium release in skeletal muscle: from K+ contractures to Ca2+ sparks

Journal of Muscle Research and Cell Motility -     

Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K+ channels

A swelling-activated, background K⁺ current in the corneal epithelium is characteristically activated by fenamates and inhibited by diltiazem. Fatty acids also stimulate this current, indicating that its origin is a lipid-sensitive mechano-gated 2P domain K⁺ channel. In the present study, modulation of TREK-1, TREK-2, and TRAAK channels by fenamates and diltiazem was examined. TREK-1, TREK-2, and TRAAK currents transiently expressed in COS-7 cells were recorded by the perforated-patch configuration. As previously reported, arachidonic acid (20 M) stimulated all of these channels, and a volatile anesthetic, halothane (1 mM) augmented TREK-1 and TREK-2 but not TRAAK. Flufenamic acid (FA, 100 M), niflumic acid (NA, 100 M), and mefenamic acid (MA, 100 M) markedly stimulated TREK-1, TREK-2, and TRAAK. The potency sequence for the activation of TREK-1 and TREK-2 was FA > NA = MA, and the potency sequence for the activation of TRAAK was FA = NA > MA. Diltiazem (1 mM) inhibited TREK-1 and TREK-2, but not TRAAK. In conclusion, fenamates are openers of the lipid-sensitive mechano-gated 2P domain K⁺ channels, and diltiazem may be a specific blocker for TREK. These novel findings could help to further understand channel functions of the mechano-gated 2P domain K⁺ channels.     

K(+)-evoked [(3)H]-norepinephrine release in human brain slices from epileptic and non-epileptic patients is differentially modulated by gabapentin and pinacidil

The modulation of K(+)-evoked [(3)H]-norepinephrine ([(3)H]-NE) release by gabapentin (GBP) and pinacidil (PIN), a known K(ATP) agonist, was examined in human brain slices. We compared the pharmacological effects on NE-release in human epileptic neocortex and epileptic hippocampus to non-epileptic neocortex. GBP (100 microM) decreased [(3)H]-NE release by 22% in non-epileptic neocortical slices, whereas this inhibition was absent in slices from epileptic hippocampus and epileptic neocortex. PIN (10 microM) also reduced [(3)H]-NE release by 30% in non-epileptic neocortical slices and only by 5% in epileptic hippocampal slices. The blockade of voltage-gated calcium channels by omega-conotoxins MVIIA and MVIIC (0.1 microM) reduced [(3)H]-NE release in epileptic and non-epileptic neocortical slices to the same extend. The data show a marked reduction in K(+)-evoked [(3)H]-NE release by GBP and PIN in epileptic hippocampus and neocortex, suggesting an alteration of K(ATP) channel function, whereas the effects of the calcium channel modulators omega-conotoxins MVIIA and MVIIC are similar in both epileptic and non-epileptic neocortex.