Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

Effect of caffeine on the Ca2+-transport function of sarcoplasmic reticulum vesicles in the rat myocardium

The effects of caffeine on active transport of Ca2 by heavy and light fractions of rat myocardial microsomes were investigated with the use of a Ca2+-selective electrode and nephelometry. It was found that under the effect of caffeine (5 mM) the rate of Ca2 transport in the presence of oxalate decreased by 30 to 40%. The caffeine-induced inhibition was prevented by ruthenium and tetracaine, thus suggesting the inhibitor specificity. Since caffeine is a specific blocker of Ca2 transport to the terminal cisterns of the skeletal muscle sarcoplasmic reticulum, it is assumed that the microsomal fraction of rat myocardium contains terminal cistern fragments.     

Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver

Sinusoidal membrane vesicles from rat liver were employed to study the characteristics of GSH transport. Saturable concentration dependent uptake was best described by the sum of a high and low Km transport. Preloading with GSH markedly stimulated the initial uptake of GSH. GSH transport was electrogenic; uptake was enhanced by an inwardly directed K+ gradient which could be blocked by the K+-channel blocker, Ba2+. The other cations such as Na+, Li+ were poor substitutes for K+. These results therefore show that net GSH transport involves movement of K+.     

HIGH AFFINITY CHOLINE TRANSPORT IN GUINEA PIG BRAIN AND THE EFFECT OF NOREPINEPHRINE

—The influence of 1-norepinephrine on the accumulation of [¹⁴C]choline by nuclei-free homogenates and synaptosomes of guinea-pig brain was studied. Kinetic analysis of choline accumulation by guinea-pig brain resulted in both high and low affinity Michaelis constants. Norepinephrine stimulated the high affinity choline transport process but not the low and the magnitude of its stimulation in 3 different brain regions was correlated with the choline acetyltransferase activity of those regions. Depletion of norepinephrine from the brainstem by pretreatment with the catecholamine depleter alpha-methyl-para-tyrosine significantly decreased the maximal velocity of choline transport. Both the alpha adrenergic receptor blocker phentolamine and the beta adrenergic receptor blocker propranalol inhibited norepinephrine induced stimulation of choline transport. Cocaine stimulated choline transport at low concentrations and pretreatment of animals with reserpine significantly antagonized cocaine's stimulation of choline transport. The results suggest that endogenous norepinephrine may modify the high affinity choline transport process in guinea-pig brain.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.     

Ionic and osmotic effects of NaCl-induced inactivation of photosystems I and II in Synechococcus sp

We report here that osmotic effects and ionic effects are both involved in the NaCl-induced inactivation of the photosynthetic machinery in the cyanobacterium Synechococcus sp. PCC 7942. Incubation of the cyanobacterial cells in 0.5 M NaCl induced a rapid and reversible decline and subsequent slow and irreversible loss of the oxygen-evolving activity of photosystem (PS) II and the electron transport activity of PSI. An Na(+)-channel blocker protected both PSII and PSI against the slow, but not the rapid, inactivation. The rapid decline resembled the effect of 1.0 M sorbitol. The presence of both an Na(+)-channel blocker and a water-channel blocker protected PSI and PSII against the short- and long-term effects of NaCl. Salt stress also decreased cytoplasmic volume and this effect was enhanced by the Na(+)-channel blocker. Our observations suggested that NaCl had both osmotic and ionic effects. The osmotic effect decreased the amount of water in the cytosol, rapidly increasing the intracellular concentration of salts. The ionic effect was caused by an influx of Na(+) ions through potassium/Na(+) channels that also increased concentrations of salts in the cytosol and irreversibly inactivated PSI and PSII.     

Purinergic P2Y6 receptors induce Ca2+ and CFTR dependent Cl- secretion in mouse trachea.

In airways Cl- secretion is activated and Na+ absorption is inhibited when P2Y2 receptors are stimulated by ATP or UTP. Both nucleotides are subject to degradation to ADP and UDP by ecto-nucleotidases. Here we show that these metabolites change electrolyte transport by stimulation of P2Y6 receptors in mouse trachea. Immunohistochemistry confirmed luminal and basolateral expression of P2Y6 receptors. In Ussing chamber experiments luminal ADP, UDP or the P2Y6 receptor agonist INS48823 induced both transient and persistent increase in short circuit currents (ISC). Activation of ISC was inhibited by the P2Y6 receptor blocker PPADS. The transient response was inhibited by DIDS, whereas the persistent ISC was inhibited by glibenclamide and by the protein kinase A (PKA) blocker H-89. Moreover, sustained activation of ISC by luminal UDP was inhibited by blocking basolateral K+ channels with 293B. Possible effects of diphosphates on P2Y1 or adenosine receptors were excluded by the inhibitors MRS2179 and 8-SPT, respectively. Inhibition of amiloride sensitive Na+ absorption was only seen after blocking basolateral K+ channels with 293B. In contrast, Cl- secretion activated by basolateral ADP or UDP was only transient and was blocked by the sk4 K+ channel blocker clotrimazole. In summary, activation of luminal P2Y6 receptors in the airways shifts electrolyte transport towards secretion by increasing intracellular Ca+ and activation of PKA.     

3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+.     

Blocker-related changes of channel density. Analysis of a three-state model for apical Na channels of frog skin

Blocker-induced noise analysis of apical membrane Na channels of epithelia of frog skin was carried out with the electroneutral blocker (CDPC, 6-chloro-3,5-diamino-pyrazine-2-carboxamide) that permitted determination of the changes of single-channel Na currents and channel densities with minimal inhibition of the macroscopic rates of Na transport (Baxendale, L. M., and S. I. Helman. 1986. Biophys. J. 49:160a). Experiments were designed to resolve changes of channel densities due to mass law action (and hence the kinetic scheme of blocker interaction with the Na channel) and to autoregulation of Na channel densities that occur as a consequence of inhibition of Na transport. Mass law action changes of channel densities conformed to a kinetic scheme of closed, open, and blocked states where blocker interacts predominantly if not solely with open channels. Such behavior was best observed in "pulse" protocol experiments that minimized the time of exposure to blocker and thus minimized the contribution of much longer time constant autoregulatory influences on channel densities. Analysis of data derived from pulse, staircase, and other experimental protocols using both CDPC and amiloride as noise-inducing blockers and interpreted within the context of a three-state model revealed that Na channel open probability in the absence of blocker averaged near 0.5 with a wide range among tissues between 0.1 and 0.9.     

Molecular mechanisms of band 3 inhibitors. 2. Channel blockers

Band 3 is proposed to contain substrate channels that lead from the aqueous medium to a transport site buried within the membrane, and which can be blocked by inhibitors. The inhibitors 1,2-cyclohexanedione (CHD) and dipyridamole (DP) each inhibit the transport site 35Cl NMR line broadening, but neither competes with Cl- for binding. Thus these inhibitors do not occupy the transport site; instead they slow the migration of Cl- between the transport site and the medium. The simplest explanation for this behavior is that CHD and DP block one or more substrate channels. CHD is an arginine-specific covalent modification reagent, and its effectiveness as a channel blocker indicates that the channel contains arginine positive charges to facilitate the migration of anions through the channel. DP is a noncovalent channel blocker that binds with a stoichiometry of 1 molecule per band 3 dimer. DP binding is unaffected by CHD but is prevented by phenylglyoxal (PG), 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), or niflumic acid. Thus the DP and CHD binding sites are distinct, with DP binding sufficiently close to the transport site to interact with PG and DNDS. It is proposed that substrate channels may be a general feature of transport proteins.     

Effect of lysophosphatidic acid on the ovum transport in mouse oviducts.

The effects of lysophosphatidic acid (LPA) on ovum transport in mouse oviducts were studied. When excised oviducts were incubated at 37 degrees C under 5% CO2 in humidified air for 24 hours, addition of LPA at 10 microM to the medium significantly accelerated the rate of ovum transport, and 1 microM LPA slightly increased the ovum transport rate. These increases were not inhibited by 10 microM indomethacin, a cyclooxygense inhibitor, but were suppressed by 260 ng/ml of pertussis toxin or 10 microM verapamil, a voltage-sensitive calcium channel blocker. These data suggested that LPA stimulates mouse ovum transport by contracting oviductual smooth muscle via a voltage-sensitive calcium channel mediated by a pertussis toxin-sensitive G-protein-linked receptor.     

Photosynthetic utilization of bicarbonate in Zostera marina is reduced by inhibitors of mitochondrial ATPase and electron transport

When Zostera marina was irradiated after a period of darkness, initiation of photosynthetic O2 evolution occurred in two phases. During a lag phase, lasting 4 to 5 min, photosynthesis was supported by a diffusive entry of CO2. Photosynthesis then rapidly increased to its full rate. Tris buffer, at a concentration of 50 mm, completely inhibited this increase without affecting CO2-supported photosynthesis during the lag phase. These results verify that the increase in photosynthesis after the lag phase depended on an activation of bicarbonate (HCO3-) utilization through acid zones generated by proton pumps located to the outer cell membrane. In similar experiments, 6.25 microm of the mitochondrial ATPase blocker oligomycin inhibited photosynthetic HCO3(-) utilization by more than 60%. Antimycin A, a selective blocker of mitochondrial electron transport, caused a similar inhibition of HCO3(-) utilization. Measurements at elevated CO2 concentrations verified that neither oligomycin nor antimycin interfered with linear photosynthetic electron transport or with CO2 fixation. Thus, a major part of the ATP used for the generation of acid zones involved in HCO3(-) utilization in Z. marina was derived from mitochondrial respiration.     

Effect of caffeine on active Ca2+ ion transport in a homogenate of skeletal muscles and myocardium

A Ca2-selective electrode was used to study active transport of Ca2+ by sarcoplasmic reticulum fragments of rabbit skeletal muscle and myocardium homogenates. The specific Ca2+ transport activities (mumol Ca2+/min/mg tissue) are 40 = 60 and 3 = 5 units for fast and slow muscles and the myocardium, respectively. Caffeine (5 mM) exerts a powerful inhibitory influence on Ca2+ transport in skeletal muscle homogenates. For fast muscles, the degree of inhibition exceeds 50%. The rate of Ca2+ transport in the myocardium homogenate increases in the presence of creatine phosphate. The latter produces no effect on Ca2+ transport in skeletal muscle homogenates. The high sensitivity of Ca2 transport to caffeine, a specific blocker of Ca2+ transport to the terminal cisterns of the sarcoplasmic reticulum, suggests that the terminal cisterns, apart from being a reservoir for Ca2+ needed for contraction trigger, may play an essential role in muscle relaxation.     

Chronic elevation of extracellular glutamate due to transport blockade is innocuous for spinal motoneurons in vivo

Glutamate-mediated excitotoxicity has been considered to play an important role in the mechanism of spinal motoneuron death in amyotrophic lateral sclerosis (ALS), and some reports suggest that this excitotoxicity may be due to a decreased glutamate transport and the consequent elevation of its extracellular level. We have previously shown that short lasting increments in extracellular glutamate due to administration of the non-selective glutamate transport blocker l-2,4-trans-pyrrolidine-dicarboxylate (PDC) by microdialysis in the rat spinal cord do not induce motoneuron damage. In the present work we examined the potential involvement of chronic glutamate transport blockade as a causative factor of spinal motoneuron death and paralysis in vivo. Using osmotic minipumps, we infused directly in the spinal cord for up to 10 days PDC and another glutamate transport blocker, dl-threo-β-benzyloxyaspartate (TBOA), and we measured by means of microdialysis and HPLC the extracellular concentration of glutamate and other amino acids. We found that after the infusion of both PDC and TBOA the concentration of endogenous extracellular glutamate was 3–4-fold higher than that of the controls. Nevertheless, in spite of this elevation no motoneuron degeneration or gliosis were observed, assessed by histological examination and choline acetyltransferase and glial fibrillary acidic protein immunocytochemistry. In accord with this lack of toxic effect, no motor deficits, assessed by three motor activity tests, were observed.Because we had previously shown that under identical experimental conditions the infusion of α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) induced progressive motoneuron death and paralysis, we conclude that prolonged elevation of extracellular glutamate due to its transport blockade in vivo is innocuous for spinal motoneurons and therefore that these results do not support the hypothesis that glutamate transport deficiency plays a crucial role as a causal factor of spinal motoneuron degeneration in ALS.     

Chloride transport in human fibroblasts is activated by hypotonic shock

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.     

Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms

Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine (METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by (32)PO(4) metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK(1) cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.     

Actions of neuropeptide Y on basal, cyclic AMP-induced and neurally evoked ion transport in porcine distal jejunum

Neuropeptide Y (NPY) and its homolog, peptide YY, are present respectively in neurons and endocrine cells within the mammalian small intestine. In this study, we examined the actions of NPY on ion transport in the porcine distal jejunum mucosa-submucosa in vitro. Peptide YY and NPY were equieffective in producing rapid and sustained decreases in basal short-circuit current (Isc), a bioelectrical measure of active ion transport, eliciting half-maximal decreases at respective serosal concentrations of 0.8 and 30 nmol/l. NPY-induced changes in Isc were due to increased mucosa-to-serosa and net Cl fluxes and were not affected by the absence of extracellular HCO3 ions. NPY activity was correlated with the magnitude of the basal Isc and appeared to depend on the spontaneous production of eicosanoids. The peptide also decreased Isc stimulated by forskolin and 8-bromo-cyclic AMP, but the ionic bases for this effect were complex and differed from those determined under basal conditions. NPY attenuated increases in Isc produced by electrical stimulation of enteric neurons with an IC50 = 5 nmol/l. The actions of the peptide on basal and cyclic AMP-induced ion transport were abolished by the neuronal conduction blocker tetrodotoxin, but not by the opiate antagonist naloxone. The alpha-adrenoceptor blocker phentolamine diminished the effects of NPY on basal, but not cyclic AMP-induced Isc. These results indicate that NPY is capable of modulating NaCl transport in the porcine jejunal mucosa under several different conditions. Furthermore, the effects of the peptide are mediated in part through noradrenergic nerves as well as enteric neurons of unknown chemical identity.     

Electrical parameters of the toad skin: effects of forskolin.

Forskolin stimulated short-circuit current (SCC) and transepitelial electrical conductance (G) in the isolated skin of the toad Bufo arenarum in a concentration-dependent manner, between 1.0 x 10(-6) and 2.4 x 10(-5) M. At the latter concentration, glandular secretion appeared to be stimulated also. The increase in G was considerably greater in skins bathed in Ringer solution than in solutions containing no chloride. The increased SCC was abolished by amiloride, a specific blocker of sodium transport in amphibian membranes, irrespective of the anion present in the solution bathing the skin. G was also decreased by amiloride to control values in skins bathed in solutions without chloride, but remained elevated in the presence of Cl-. The increase in SCC following exposure to forskolin, 4.4 x 10(-6) M, was not altered when furosemide, a specific blocker of chloride transport, was present in the Ringer solution bathing the dermal side of the skin. The response to forskolin, 2.4 x 10(-5) M, however, was significantly decreased by dermal furosemide; the inhibitor was ineffective in the absence of chloride. The data indicate that forskolin acts on at least two sites: stratum granulosum cells (the main pathway for sodium transport, and an alternate site, responsible for the increase in permeability to chloride. In addition, at high concentration of the agent, glandular secretion is also stimulated. The data suggest that the adenylate cyclase-cyclic AMP system is involved in the regulation of the permeability of the toad skin to sodium and chloride, probably by separate cell types.     

Lidoflazine Combined with Nucleotide Precursors Increases ATP Content and Adenosine Production in Cardiomyocytes

We have previously identified that the nucleoside transport blocker dipyridamole increases adenosine production but may cause depletion of the nucleotide pool in cardiomyocytes during extended exposure and that this effect was abolished by co-administration of adenine and ribose. The present study aimed to establish whether lidoflazine, a newer generation of nucleoside transport inhibitor with calcium antagonist properties, would cause a similar effect. We conclude that lidoflazine did not affect the nucleotide pool while the combined application of lidoflazine with precursors of nucleotide resynthesis increased ATP concentration and further enhanced adenosine production.     

川芎嗪增加大鼠远端结肠阴离子分泌的基侧膜机制

本研究用短路电流技术来观察在川芎嗪作用下,电解质在大鼠远端结肠上皮细胞的转运及其细胞机制。在新鲜分离的结肠上皮的基侧膜加入川芎嗪,能产生较大的短路电流。用粘膜下神经元阻断剂——河豚毒素预作用于结肠上皮,不影响随后的川芎嗪所产生的短路电流,前列腺素合成抑制剂indomethacin预作用可使随后的川芎嗪产生的短路电流减少55．2％。在结肠上皮的顶膜加入Cl^-通道阻断剂DPC和glibenclamide,能完全阻断川芎嗪产生的短路电流。Bumetanide,基侧膜钠、钾、氯共转运体阻断剂能抑制川芎嗪引起的短路电流的85．2％,而结肠上皮细胞基侧膜的非选择性钾通道阻断剂Ba^2 能阻断90％以上的短路电流,说明基侧膜的钠、钾、氯共转运体和钾通道在川芎嗪引起的短路电流中起着重要的作用。上述结果表明,川芎嗪刺激大鼠远端结肠上皮细胞分泌Cl^-是通过上皮细胞顶膜Cl^-通道和基侧膜的钠、钾、氯共转体和K^ 通道介导的。