Chloride concentration in cultured hippocampal neurons increases during long-term exposure to ammonia through enhanced expression of an anion exchanger.

The effects of long-term exposure to ammonia on [Cl-]i in cultured hippocampal neurons were examined. Ammonia increased the [Cl-]i time- (>/=24 h) and concentration- (>/=2 mM) dependently, resulting in a depolarizing shift of the equilibrium potential of the GABAA receptor-Cl- channel opening (EGABA). Such an effect of ammonia was diminished by the inhibitors of Cl-/HCO3- exchangers, 0.1 mM 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a carbonic anhydrase inhibitor, 2 mM acetazolamide, but not by a Na+/K+/2Cl-cotransport inhibitor, 50 microM bumetanide, suggesting an enhanced Cl-/HCO3- exchange activity by ammonia. The ammonia-induced increase in [Cl-]i was also abolished by the inhibitors of protein kinase C (PKC), 0.1 microM calphostin C and 10 microM 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine dihydrochloride (H-7), and of transcription and de novo protein synthesis, 1 microM actinomycin D and 0.5 microg/ml cycloheximide, while a PKC activator, 0.1 h microM phorbor 12-myristate 13-acetate (PMA), increased the [Cl-]i. The mRNA level of the AE3 Cl-/HCO3- exchanger was increased by ammonia in a calphostin C- and H-7-sensitive manner. The AE3-like immunoreactivity was also increased by ammonia. These findings suggest that long-term exposure to ammonia increases the expression of AE3 through the activation of PKC, resulting in an increase in [Cl-]i in neurons and a reduction of inhibitory postsynaptic potentials.

Intracellular signaling pathway of substance P-induced superoxide production in human neutrophils.

We examined the intracellular mechanisms of substance P-induced superoxide anion (O2-) production in human neutrophils. Addition of substance P (30 microM) caused O2- production and biphasic increases in intracellular Ca2+ concentrations ([Ca2+]i) (early transient and subsequent sustained components) associated with the formation of inositol 1,4,5-trisphosphate (IP3). O2- and [Ca2+]i were assayed by using ferricytochrome C and fura 2-AM, respectively. These responses were abolished by tachykinin NK1 receptor antagonists, [D- Pro9[spiro-gamma-lactam],Leu10,Trp11]physalaemin-(1-11) (GR82334) or [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), and an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Inhibition of IP3 formation by GTP-binding protein (G-protein) inactivators such as guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and islet-activating protein (IAP), or a phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)- trien-17-yl]amino]hexyl]1 H-pyrrole-2,5-dione (U-73122), blocked the substance P-induced O2- production and biphasic increases in [Ca2+]i. An IP3 receptor antagonist, heparin, reduced both the substance P-induced O2- production and the transient increase in [Ca2+]i without any significant effects on the sustained increase in [Ca2+]i. Protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and calphostin C, only slightly suppressed O2- production, and abolished the sustained increase in [Ca2+]i without any significant effects on the transient increase in [Ca2+]i. A Ca2+ entry blocker, nicardipine, completely inhibited the sustained increase in [Ca2+]i without affecting O2- production and the transient increase in [Ca2+]i. These results suggest that the tachykinin NK1 receptor/G-protein-linked IP3 formation with the resulting IP3-induced transient increase in [Ca2+]i is the main signal transduction pathway for substance P-stimulated O2- production in neutrophils.

Distribution of neurone-specific clathrin light chain b between clathrin-coated vesicle subclasses.

The clathrin light chains (LCs) may serve to introduce diversity into the structure and/or function of clathrin-coated vesicles (CVs). To understand such involvement of LCs, it is advantageous to study the distribution of various LC subclasses among CV populations with specified structure and/or function. We have previously separated three populations of CV from rat brain, the small- and medium-sized populations originated from neurones and large-sized one from glial cells. In the present study, we examined whether the neurone-specific LCb is localized in either or both of those neuronal CV populations by immunogold electron microscopy, and showed the distribution of the LCb between both CVs. These findings suggest that the neurone-specific LC subclass does not specify morphologically distinct subtype of neuronal CVs but plays a role in constructing the CVs generally smaller than those from other tissues and/or in neurone-specific mechanisms associated with both of the neuronal CVs.

Atrial natriuretic peptide stimulates Cl- transport in retinal pigment epithelial cells.

To study the regulatory role of atrial natriuretic peptide (ANP) on the Cl- transport activity of retinal pigment epithelial (RPE) cells, RPE cells from rabbits were cultured and exposed to ANP and other reagents under perfusion. The changes in intracellular Cl- concentration ([Cl-]i) were continuously recorded using a Cl(-)-sensitive fluorescent dye. The cGMP content was estimated by radioimmunoassay. ANP increased the cGMP content and the [Cl-]i in RPE cells. A guanylate cyclase activator, nitric oxide, and a cell permeable cGMP precursor, 8-Br-cGMP, also increased the level of cGMP and the [Cl-]i. A guanylate cyclase inhibitor, LY83583, an inhibitor of cGMP-dependent protein kinase, KT5823, and an inhibitor of Na+/K+/2Cl- cotransporter, bumetanide, diminished or abolished the ANP-induced increase in [Cl-]i. ANP facilitates Cl- accumulation in RPE cells, which is mediated by guanylate cyclase, cGMP-dependent protein kinase, and the Na+/K+/2Cl- cotransporter.

Adrenergic receptor-mediated Cl- transport in rabbit corneal endothelial cells.

Adrenoceptor-mediated Cl- transport in cultured rabbit corneal endothelium was examined using a Cl(-)-sensitive fluorescent dye. The intracellular Cl- concentration ([Cl-]i) in the endothelial cells was estimated to be about 30 mM. Noradrenaline (0.001-0.1 mM) transiently decreased the [Cl-]i in a dose-dependent manner. Such a decrease in [Cl-]i was completely antagonized by pretreatment with the alpha-adrenoceptor antagonist phentolamine (0.1 mM). The selective alpha 2-adrenoceptor agonist UK 14304-18 (5-bromo-6-[(4H,5H-imidazol-2-yl)amino]quinoxaline, 0.1 mM) persistently decreased the [Cl-]i, but neither the alpha 1-adrenoceptor agonist phenylephrine (0.1 mM) nor the beta-adrenoceptor agonist isoproterenol (0.1 mM) had any effect. The alpha 2-adrenoceptor agonist/antagonist yohimbine (0.1 mM) persistently and more strongly decreased the [Cl-]i than UK 14304-18 did. The yohimbine-induced decrease in the [Cl-]i was not further altered by UK 14304-18 or phenylephrine, but partly reversed by noradrenaline, isoproterenol and an adenylate cyclase activator, forskolin (0.1 mM). The yohimbine-induced decrease in [Cl-]i was inhibited by the carbonic anhydrase inhibitor acetazolamide (1 mM), and Cl-/HCO3- exchange inhibitors, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by the H(+)-ATPase inhibitor N,N'-dicylohexylcarbodiimide. The forskolin-induced recovery in [Cl-]i was inhibited by the Na+/K+/Cl- cotransport inhibitor bumetanide (0.1 mM), but not by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of neurotrophic factor for retinal cholinergic neurons derived from cultured hippocampal neurons.

A neurotrophic factor that supports the development of cholinergic retinal neurons was purified from media conditioned by a primary culture of embryonic hippocampal neurons. Retinal choline acetyltransferase (ChAT), which is located exclusively in amacrine cells, served as a marker for the development of retinal cholinergic neurons. In a serum-free control culture, retinal neurons from 17-day-old rat fetus displayed little increase in the enzyme activity and a low proportion of neurite-bearing cells (15-20%) within 7 days. The conditioned media, when added to the retinal neuron culture, dose-dependently increased ChAT activity and the number of neurite-bearing cells (40-60%), the maximum ChAT activity being approximately sixfold higher than that in the control. The fraction with these stimulatory activities was purified by Sephadex G-15 column chromatography and two times reverse-phase HPLC. The final fraction showed approximately 3,000-fold higher purification as compared with that in the Sephadex G-15 fraction. Gas-phase protein sequencing analysis of the final fraction yielded a peptide sequence: Tyr-Leu-Leu-Pro-Ala-Gln-Val-Asn-Ile-Asp. A synthetic peptide with this sequence dose-dependently stimulated ChAT activity in the retinal cell culture and dissociated cell culture of the septal nucleus. These findings suggest that the developing hippocampal neurons produce a neurotrophic peptide that stimulates the development of cholinergic neurons.

A cytosolic peptide potentiates the GTP effect on beta-adrenergic response of adenylate cyclase.

A cytosolic peptide-GTP complex that stimulates l-isoproterenol-responsive adenylate cyclase activity was identified in the rat liver. The peptide component was purified and characterized with regard to its interaction with GTP. The peptide was isolated as a complex form with GTP on a Sephadex G-25 column in 1 mM NaHCO3, and was purified as a dissociated form, with relative molecular weight (M(r)) approximately 3,000 and GTP-binding ability, in 200 mM ammonium acetate. The purified peptide alone displayed little stimulatory effect on adenylate cyclase activity, but its reassociated form with GTP clearly enhanced the effect of GTP on the enzyme activity. The isoproterenol competition curve using l-[3H]dihydroalprenolol as an antagonist ligand shifted to lower affinity by the addition of the peptide reassociated with GTP (16.5-fold shift), whereas the same concentration of GTP (1 microM) or the peptide alone had little or no effect (1.5- or 0.9-fold shift, respectively). Furthermore, the peptide enhanced the GTP effect in response to l-isoproterenol but not to glucagon, prostaglandin E1, or fluoride. These results suggest that the cytosolic peptide potentiates the effect of GTP on the agonist-beta-adrenergic receptor-stimulatory guanine nucleotide-binding regulatory component of the adenylate cyclase ternary complex.

Ethacrynic acid-induced convulsions and brain neurotransmitters in mice.

Intracerebroventricular injection of ethacrynic acid (50% convulsive dose; 50 micrograms/mouse) accelerated the synthesis/turnover of 5-hydroxytryptamine (5-HT) but suppressed the synthesis of gamma-aminobutyric acid and acetylcholine in mouse brain. These effects were completely antagonized by pretreatment with a glutamate/N-methyl-D-aspartate antagonist, aminophosphonovaleric acid. In ethacrynic acid-induced convulsions, these neurotransmitter systems may be differentially modulated, probably through activation of glutaminergic neurons in the brain.

Uneven distribution of intracellular Cl- in rat hippocampal neurons.

Electrophysiological observations of neurons suggest that perikarya and dendrites differ in local intracellular Cl- concentration ([Cl-]i), that has not been demonstrated yet. Regional [Cl-]i in cultured hippocampal neurons was estimated using a Cl(-)-sensitive fluorescent dye. Calibration showed that perikaryonic [Cl-]i was lower than dendritic [Cl-]i. Ethacrynic acid, an inhibitor of the outwardly directed Cl(-)-pump, increased the perikaryonic but not dendritic [Cl-]i. A decrease in [Cl-]i induced by furosemide or bumetanide, inhibitors of Na+/K+/2Cl- cotransporters, was more prominent in dendrites than in perikarya. These findings suggest that uneven distribution of Cl- is generated by the region-specific localization of these transporters.

An ATP-driven Cl- pump regulates Cl- concentrations in rat hippocampal neurons.

To investigate the role of Cl(-)-stimulated Mg(2+)-ATPase (Cl(-)-ATPase) in neurons, we examined the effects of ethacrynic acid (0.3 mM), which completely inhibits Cl(-)-ATPase on the intracellular Cl- concentrations of cultured rat hippocampal neurons, using Cl(-)-sensitive fluorescent probes. Ethacrynic acid and ATP consuming treatment increased the intracellular Cl- concentration, but elevation of the extracellular K+ concentration up to 10 mM, inhibition of Na+/K(+)-ATPase, or dissolution of H+ gradients had no effect. Furosemide (0.1 mM), an inhibitor of Na+/K+/Cl- co-transport, decreased the intracellular Cl- concentrations. These results indicate that an ethacrynic acid-sensitive and ATP-driven Cl- pump functions to reduce intraneural Cl- concentrations.

Ethacrynic acid-induced convulsions and brain noradrenaline in mice.

The intracerebroventricular injection of ethacrynic acid (a 50% convulsive dose; 50 micrograms/mouse) accelerated brain noradrenaline turnover and decreased noradrenaline contents. The decrease in noradrenaline contents was antagonized by 2-amino-5-phosphonovalerate but not by diazepam. Both 2-amino-5-phosphonovalerate and diazepam suppressed the incidence of ethacrynic acid-induced convulsions while reserpine, alpha-methyl-para-tyrosine or FLA-63 augmented it. The results suggest that stimulation by ethacrynic acid of excitatory amino acid neurons enhances-noradrenergic neuronal anticonvulsive activity.