Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.     

Antioxidants prevent depletion of [Mg2+]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: possible relationship to alcohol-induced stroke.

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis, and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Mg(2+) ([Mg(2+)](i)) depletion associated with cerebral vascular injury induced by alcohol. Exposure of cultured canine cerebral vascular smooth muscle cells to alcohol (10-100 mM) for 24 h induced marked depletion in [Mg(2+)](i) (i.e., approximately 30-65%, depending upon alcohol concentration). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Mg(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the depletion of [Mg(2+)](i) induced by exposure to 10-100 mM ethanol. These results indicate that alpha-tocopherol and PDTC prevent decreases in [Mg(2+)](i) produced by ethanol. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in prevention and amelioration of cerebral vascular injury and stroke in alcoholics.     

Low concentrations of ethanol deplete type-2 astrocytes of intracellular free magnesium

The acute effects of low concentrations of ethanol on intracellular free magnesium ions ([Mg2+]i) in cultured type-2 astrocytes were studied by digital imaging microscopy using the Mg2+ fluorescent probe, mag-fura-2. In 0-mM ethanol, the basal level of [Mg+]i was 124.7+/-2.56 microM with a heterogeneous distribution within the cells. Treatment of the cells with 10 and 25 mM ethanol (10 min) resulted in rapid concentration-dependent reduction in [Mg2+]i; the greater the concentration of alcohol, the greater the depletion of [Mg2+]i. Exposure of cells to 10 and 25 mM resulted in approximately 27 and 50% reductions in [Mg2+]i, respectively. Reincubation in normal Mg2+-physiological buffer solution restored [Mg2+]i levels. These observations may suggest that acute "binge drinking" of ethanol, which often results in cerebral ischemia and stroke, may do so as a result of depletion of astrocytic [Mg2+]i, possibly producing disruption of the blood-brain barrier.     

Antioxidants prevent elevation in [Ca(2+)](i) induced by low extracellular magnesium in cultured canine cerebral vascular smooth muscle cells: possible relationship to Mg(2+) deficiency-induced vasospasm and stroke

Low serum concentrations of Mg(2+) ions have been reported, recently, in patients with coronary disease, atherosclerosis and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [alpha-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Ca(2+) ([Ca(2+)](i)) overload associated with cerebral vascular injury induced by low extracellular free Mg(2+) ([Mg(2+)](o)). Exposure of cultured canine cerebral vascular smooth muscle cells to low [Mg(2+)](o) (0.15-0.6 mM) vs. normal [Mg(2+)](o) (1.2 mM) for either 10 min or 2 h induced concentration-dependent rises in [Ca(2+)](i). Treatment of the cultured cells with either PDTC (0.1 microM) or alpha-tocopherol (15 microM) for 24 h, alone, failed to interfere with basal [Ca(2+)](i) levels. However, preincubation of the cells with either alpha-tocopherol or PDTC for 24 h completely inhibited the elevation of [Ca(2+)](i) induced by exposure to low [Mg(2+)](o), not only for 10 min, but also for 2 h. These results indicate that alpha-tocopherol and PDTC prevent rises in [Ca(2+)](i) produced by low [Mg(2+)](o), which probably result from low [Mg(2+)](o)-induced lipid peroxidation of cerebral vascular smooth muscle cell membranes. Moreover, these new results suggest that such protective effects of alpha-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in cerebral vascular injury associated with low [Mg(2+)](o) and accumulation of [Ca(2+)](i).     

Estrogen blocks 3-nitropropionic acid-induced Ca2+i increase and cell damage in cultured rat cerebral endothelial cells

Systemic administration of 3-nitropropionic acid (3-NPA, a mycotoxin) induces brain damage accompanied by disturbance in the blood-brain barrier (BBB). Since the endothelial cells are important components of the BBB and the first target of a systemic intoxication, in the present study, the effect of 3-NPA on primary cultured rat brain endothelial cells (rBECs) was examined by studying intracellular Ca(2+) ([Ca(2+)](i)) response using imaging techniques with fura-2. rBECs were prepared using a method of Kis et al. [Eur. J. Pharmacol. 368 (1999) 35-42] and Szabo et al. [Neurobiology 5 (1997) 1-16]. Almost all cells were immunoreactive to antibody against the factor VIII-related antigen (von-Willebrand factor). They showed a typical dose-dependent increase of [Ca(2+)](i) in response to ATP or bradykinin. Low concentrations of 3-NPA (1.7 mM, 3.4 mM) caused no changes, and a medium concentration (6.8 mM) increased the [Ca(2+)](i) gradually and progressively, and the increase was reversed incompletely back to the resting level after washing. A high concentration (13.6 mM) increased the [Ca(2+)](i) irreversibly. These elevations of [Ca(2+)](i) were absent in a Ca(2+)-free medium. In endothelial cells treated with 17beta-estradiol (above 10(-5) M) or with a selective estrogen receptor modulator, tamoxifen (5 x 10(-7) M), no elevation of [Ca(2+)](i) was observed with 3-NPA treatment. The response to ATP was impaired after application of 3-NPA, but it was preserved by cotreatment with 17beta-estradiol or tamoxifen. An estrogen receptor antagonist ICI 182,780 inhibited these effects by 17beta-estradiol or tamoxifen. Lysosomal neutral red uptake and TUNEL experiments revealed the necrotic but not apoptotic cell death at least in this acute stage. Data indicate that a medium to high concentration of 3-NPA induces damage on rBECs as revealed by an accumulation of [Ca(2+)](i), but the damage was protected by cotreatment with 17beta-estradiol or tamoxifen, suggesting that estrogen may be protective for the brain vascular damage via estrogen receptor.     

Sodium valproate at therapeutic concentrations changes Ca2+ response accompanied with its weak inhibition of protein kinase C in human astrocytoma cells

Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of gamma-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 microM) on the elevation of intracellular free calcium concentration ([Ca(2+)](i)) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 microM VPA for 2 min did not change the carbachol-induced [Ca(2+)](i) elevation. Treatment with 300 and 600 microM VPA for 48 h, however, reduced the elevation. Since we have shown that Li(+) reduced carbachol-induced [Ca(2+)](i) elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca(2+)response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17-22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 microM VPA for 24 or 48 h slightly increased the B(max) value, but not the K(d) value, in the binding of [(3)H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca(2+)](i) elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca(2+)](i) elevation may play a role in the treatment of mood disorder.     

Stimulated increase in free cytosolic Ca and protein kinase C activity in human cerebrocortical synaptosomes

Protein kinase C (PKC) is an important family of kinases regulated by lipid second messengers and cofactors that interact with cellular membranes. Both Ca(2+)-dependent and -independent isoforms of PKC have been described in rat cerebrocortical presynaptic nerve terminals (synaptosomes). In the present study, synaptosomes were prepared from human cerebral cortex obtained from standard temporal lobe specimens removed due to epilepsy. In order to measure free cytosolic Ca(2+) ([Ca(2+)](i)) and PKC activity continuously, the synaptosomes were loaded with the fluorescent probes fura-2 and fim-1. Membrane depolarisation by 4-aminopyridine (4-AP) 1 mM increased the [Ca(2+)](i) fluorescence by 14.4+/-2.2% and the PKC activity fluorescence by 16.7+/-1.6%. Partial depolarisation with 4-AP 0.3 mM increased the [Ca(2+)](i) fluorescence by 9.0+/-1.5% and the PKC activity fluorescence by 4.5+/-0.7%. When CaCl(2) was omitted from the media, PKC activity fluorescence increased by 7.9+/-1.2% subsequent to stimulation with 4-AP 1 mM. This method is thus well suited for studying presynaptic [Ca(2+)](i) and PKC activity involved in neurotransmission, both under physiological conditions and under the influence of neuropharmacological agents.     

Mechanisms of sodium azide-induced changes in intracellular calcium concentration in rat primary cortical neurons

An intracellular calcium ([Ca(2+)](i)) increase is involved in sodium azide (NaN(3))-induced neurotoxicity, an in vitro model of brain ischemia. In this study the questions of possible additional sources of calcium influx, besides glutamate receptor activation, and of the time-course of NaN(3) effects have been addressed by measuring [Ca(2+)](i) in rat primary cortical cultures with the FURA-2 method. Basal [Ca(2+)](i) of neuronal populations was concentration-dependently increased 30 min, but not 24h, after a 10-min NaN(3) (3-30 mM) treatment; conversely, the net increase induced by electrical stimulation (10Hz, 10s) was consistently reduced. All the above effects depended on glutamate release and consequent NMDA receptor activation, since the NMDA antagonist MK-801 (1 microM) prevented them, and the spontaneous efflux of [(3)H]-d-aspartate from superfused neurons was concentration-dependently increased by NaN(3). In single neuronal cells, NaN(3) application progressively and concentration-dependently increased [Ca(2+)](i) (to 177+/-5% and 249+/-7% of the controls, 4 and 12 min after a 10mM-treatment, respectively). EGTA (5mM) pretreatment reduced the effect of 10mM NaN(3) (to 118+/-5% at 4 min, and to 148+/-10% at 12 min, respectively), while 1 microM cyclosporin A did not. Both MK-801 and CNQX (a non-NMDA glutamate antagonist, 10 microM) prevented NaN(3) effect at 4 min (to 147+/-8% and 153+/-5%, respectively), but not at 12 min after NaN(3) treatment. Conversely, 10 microM verapamil and 0.1 microM omega-conotoxin (L- and N-type calcium channel blockers, respectively) significantly attenuated NaN(3) effects at 12 min (to 198+/-8% and 164+/-5%, respectively), but not at 4 min; the P/Q-type calcium channel blocker, agatoxin, 0.3 microM, was ineffective. These findings show that the predominant source of calcium increase induced by NaN(3) is extracellular, involving glutamate receptor activation in a first step and calcium channel (mainly of the N-type) opening in a second step.     

Differential effects of bupivacaine enantiomers,ropivacaine and lidocaine on up-regulation of cell surface voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, (+/-)-bupivacaine inhibited veratridine-induced 22Na(+) influx (IC(50) 6.8 microM). The IC(50) of (+)-bupivacaine (2.8 microM) was 6.2-, 7.4-, and 17.1-fold lower than those of (-)-bupivacaine (17.3 microM), (-)-ropivacaine (20.6 microM), and lidocaine (47.8 microM). Chronic (i.e. 3-h) treatment of cells with (+/-)-bupivacaine increased cell surface [3H]saxitoxin ([3H]STX) binding capacity by 48% (EC(50) of 233 microM; t(1/2)=7.4 h), without changing the K(d) value. Treatment for 24 h with either (+)- or (-)-bupivacaine, or (-)-ropivacaine elevated [3H]STX binding, whereas 24-h treatment with lidocaine had no effect. The rise of [3H]STX binding by (+/-)-bupivacaine was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of cell surface vesicular exit from the trans-Golgi network; however, (+/-)-bupivacaine did not increase Na(+) channel alpha- and beta(1)-subunit mRNA levels. In cells subjected to (+/-)-bupivacaine treatment (1 mM for 24 h) followed by 3-h washout, veratridine-induced 22Na(+) influx was enhanced, even when measured in the presence of ouabain, an inhibitor of Na(+),K(+)-ATPase. Ptychodiscus brevis toxin-3 potentiated veratridine-induced 22Na(+) influx by 2.3-fold in the (+/-)-bupivacaine-treated cells, as in non-treated cells. These results suggest that lipophilic bupivacaine enantiomers or (-)-ropivacaine acutely inhibit Na(+) channel gating, whereas its chronic treatment up-regulates cell surface expression of Na(+) channels via translational and externalization events.     

Capsaicin modulates K+ currents from dissociated rat taste receptor cells

Chili pepper is one of most widely used spices. The main active component of chili pepper is the capsaicin. The effects of capsaicin on sensory nerve endings are well known; however, little is known regarding the direct effect of capsaicin on taste receptor cells (TRCs). In this study, patch clamp methods were used to study the effects of capsaicin on the K(+) currents in TRCs isolated from the rat circumvallate papilla. Fura-2 microspectrofluorimetry was also used to determine the effects of capsaicin on the intracellular Ca(2+) concentration ([Ca(2+)](i)). In the resting state, whole-cell experiments identified outward-rectifying K(+) currents, which were inhibited by 5 mM tetraethylammonium (TEA(+)) chloride. Voltage-dependent K(+) channels with a conductance of 55+/-4 pS (mean+/-S.E.M.; n=3), were observed in cell-attached patches. Capsaicin (500 nM) completely inhibited the outward-rectifying K(+) current in the whole-cell recordings. In cell-attached patches 500 nM capsaicin significantly reduced the open probability (P(o)) of the K(+) channels from 0.401+/-0.052 (n=3) in the resting state, to 0.018+/-0.002 (n=3, P<0.05 by unpaired t-test). In the fura-2-loaded TRCs, micromolar concentrations of capsaicin increased [Ca(2+)](i) in a dose-dependent manner, e.g., 100 microM capsaicin consistently increased the 340:380 fluorescence ratio from 1.04+/-0.05 in the resting state to 1.40+/-0.05 (n=28). These results suggest that capsaicin can enhance or modify the gustatory sensation by inhibiting the K(+) currents of the TRCs directly.     

Potentiation of ATP calcium responses by A2B receptor stimulation and other signals coupled to Gs proteins in type-1 cerebellar astrocytes.

We have studied the interaction between P1 and P2 purinoceptors in purified type-1 astrocyte cultures from postnatal days 7-8 rat cerebella using single cell microfluorimetry with fura-2. The stimulation of astrocytes with ATP elicits rapid [Ca2+]i transients showing an EC50 value of 7.9 +/- 0.3 microM. Costimulation of type-1 astrocytes with adenosine and ineffective ATP concentrations (0.1 or 1 microM) evoked [Ca2+]i transients that correspond to 60% of the maximal ATP response. NECA (5'-N-ethylcarboxamidoadenosine) was the only agonist that mimicked the adenosine effect and showed an EC50 value of 0.17 +/- 0.01 microM. This value was identical to that obtained for the cAMP production stimulation, indicating that A2B receptors coupled to adenylate cyclase activation were involved. The presence of A2B adenosine receptors was also confirmed by immocytochemistry experiments. When astrocytes were costimulated with isoproterenol and ineffective ATP concentrations similar [Ca2+]i transients were observed. The treatment of astrocytes with cholera toxin potentiated ATP calcium signals, lowering the EC50 value for ATP to 1.5 +/- 0.2 microM. However, the pretreatment of cells with forskolin or a permeable cAMP analogue had no effect on ATP calcium responses. These results indicated that the potentiation mechanism was elicited before the adenylate cyclase activation. We could conclude that in type-1 astrocytes, the activation of A2B adenosine receptors or other signals positively coupled to adenylate cyclase stimulation strongly potentiate metabotropic calcium responses to ATP. The potentiation was parallel but independent on cAMP accumulation suggesting the involvement of beta gamma subunits released after Gs stimulation.     

Antioxidants prevent depletion of [Mg]i induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke

Low serum concentrations of Mg²⁺ ions have been reported, recently, in patients with coronary disease, atherosclerosis, and stroke as well as in patients with cerebral hemorrhage. The aim of the present study was to determine whether potent antioxidants [α-tocopherol and pyrrolidine dithiocarbamate (PDTC)] can prevent or ameliorate intracellular Mg²⁺ ([Mg²⁺]_i) depletion associated with cerebral vascular injury induced by alcohol. Exposure of cultured canine cerebral vascular smooth muscle cells to alcohol (10–100 mM) for 24 h induced marked depletion in [Mg²⁺]_i (i.e., 30–65%, depending upon alcohol concentration). Treatment of the cultured cells with either PDTC (0.1 μM) or α-tocopherol (15 μM) for 24 h, alone, failed to interfere with basal [Mg²⁺]_i levels. However, preincubation of the cells with either α-tocopherol or PDTC for 24 h completely inhibited the depletion of [Mg²⁺]_i induced by exposure to 10–100 mM ethanol. These results indicate that α-tocopherol and PDTC prevent decreases in [Mg²⁺]_i produced by ethanol. Moreover, these new results suggest that such protective effects of α-tocopherol and PDTC on cerebral vascular cells might be useful therapeutic tools in prevention and amelioration of cerebral vascular injury and stroke in alcoholics.     

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.     

Effects of inhibitors of Na,K-ATPase on the membrane potentials and neurotransmitter efflux in rat brain slices

The potassium potential EK, of rat brain slices was estimated by determining the uptake of 86Rb+. The ERb was the same for slices prepared from five rostral brain regions, the average value being 66.4 mV. The ERb values in the presence of 20 microM ouabain were only slightly lower than the resting values; increasing concentrations of ouabain above 20 microM resulted in a graded depolarization in all five brain regions. High concentrations (1 mM) of two other inhibitors of Na+,K+-ATPase, dihydro-ouabain and strophanthidin, produced no more depolarization than did 20 microM ouabain. Competitive binding studies indicated that the differential effects were due to the relative binding to brain slices. Erythrosin B, an inhibitor of Na+,K+-ATPase, had no measurable effect on ERb. Intermediate concentrations of the Na+/H+ ionophore monensin slightly hyperpolarized striatal slices, whereas the same monensin concentrations plus 20 microM ouabain, 1 mM strophanthidin or 70 microM erythrosin B resulted in marked depolarization. Measurement of the membrane potential via uptake of methyltriphenylphosphonium cation indicated that ERb was indeed a valid estimation of the membrane potential. EK was measured directly by monitoring 42K+ uptake in striatal slices and was found to be essentially identical to ERb. Uptake of 22Na+ was consistent with the values for ERb or EK. Several conditions that resulted in little or no measurable depolarization of striatal slices did induce efflux of exogenously loaded GABA and dopamine; these conditions included 20 microM ouabain, 1 mM dihydro-ouabain or strophanthidin, and 70 microM erythrosin B. Neurotransmitter efflux in the absence of general cell depolarization was not accompanied by altered rates of respiration or decreased ATP levels.     

Delta9-tetrahydrocannabinol protects hippocampal neurons from excitotoxicity

Excitotoxic neuronal death underlies many neurodegenerative disorders. Because cannabinoid receptor agonists act presynaptically to inhibit glutamate release, we examined the effects of Win 55212-2, a full agonist at CB(1) receptors, and Delta(9)-tetrahydrocannabinol (THC), a partial agonist, on the survival of neurons exposed to an excitotoxic pattern of synaptic activity. Reducing the extracellular Mg(2+) concentration ([Mg(2+)](o)) to 0.1 mM evoked an aberrant pattern of glutamatergic activity that produced synaptically mediated death of rat hippocampal neurons in culture. Neuronal viability was quantified with a multiwell fluorescence plate scanner equipped to detect propidium iodide fluorescence. Win 55212-2 (100 nM) and THC (100 nM) significantly reduced 0.1 mM [Mg(2+)](o)-induced cell death by 77 +/- 11% and 84 +/- 8%, respectively. Interestingly, the protection afforded by THC was not significantly different from that produced by Win 55212-2, suggesting that attenuation without a complete block of excitatory activity is sufficient for neuroprotection. The effect of prolonged drug exposure on the neuroprotection afforded by cannabinoid receptor agonists was also studied. When cultures were pretreated for 24 h with Win 55212-2 (100 nM) or THC (100 nM), inhibition of 0.1 mM [Mg(2+)](o)-induced toxicity was significantly reduced to 39 +/- 19% and 45 +/- 13%, respectively. Thus, desensitization of CB(1) receptors diminishes the neuroprotective effects of cannabinoids. This study demonstrates the importance of agonist efficacy and the duration of treatment on the neuroprotective effects of cannabinoids. It will be important to consider these effects on neuronal survival when evaluating pharmacologic treatments that modulate the endocannabinoid system.     

Astrocytes in 17beta-estradiol treated mixed hippocampal cultures show attenuated calcium response to neuronal activity

Glial cells in the brain are capable of responding to hormonal signals. The ovarian steroid hormone 17beta-estradiol, in addition to its actions on neurons, can directly affect glial cells. Estrogen receptors have been described on both neurons and astrocytes, suggesting a complex interplay between these two in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration ([Ca(2+)](i)). Using simultaneous electrophysiology and calcium imaging techniques, we monitored neuronal activity evoked astrocyte ([Ca(2+)](i)) changes in mixed hippocampal cultures loaded with fluo-3 AM. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca(2+)](i)) elevations in astrocytes, which could be blocked by 200 microM MCPG and also 1 microM TTX. We compared astrocytic ([Ca(2+)](i)) transients in control and 24-hour estradiol treated cultures. The amplitude of the ([Ca(2+)](i)) transient, the number of responsive astrocytes, and the ([Ca(2+)](i)) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca(2+)](i)) rise in astrocytes in response to local application of the metabotropic glutamate receptor (mGluR) agonist t-ACPD was attenuated in estradiol treated cultures, suggesting functional changes in the astrocyte mGluR following 24-h treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca(2+)](i)) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling.     

Regulation by equilibrative nucleoside transporter of adenosine outward currents in adult rat spinal dorsal horn neurons

A current response induced by superfusing adenosine was examined in substantia gelatinosa (SG) neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. In 78% of the neurons examined, adenosine induced an outward current at -70 mV [18.8 +/- 1.1 pA (n = 98) at 1mM] in a dose-dependent manner (EC(50) = 177 microM). A similar current was induced by A(1) agonist N(6)-cyclopentyladenosine (1 microM), whereas A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) reversed the adenosine action. The adenosine current reversed its polarity at a potential being close to the equilibrium potential for K(+), and was attenuated by Ba(2+) (100 microM) and 4-aminopyridine (5mM) but not tetraethylammonium (5mM). The adenosine current was enhanced in duration by equilibrative nucleoside-transport (rENT1) inhibitor S-(4-nitrobenzyl)-6-thioinosine (1 microM) and adenosine deaminase (ADA) inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (1 microM), and slowed in falling phase by adenosine kinase (AK) inhibitor iodotubercidine (1 microM). We conclude that a Ba(2+)- and 4-aminopyridine-sensitive K(+) channel in SG neurons is opened via the activation of A(1) receptors by adenosine whose level is possibly regulated by rENT1, adenosine deaminase and adenosine kinase. Considering that intrathecally-administered adenosine analogues produce antinociception, the regulatory systems of adenosine may serve as targets for antinociceptive drugs.     

Sex steroid hormones exert biphasic effects on cytosolic magnesium ions in cerebral vascular smooth muscle cells: possible relationships to migraine frequency in premenstrual syndromes and stroke incidence

Clinically, it is known that: (1) magnesium (Mg) supplementation relieves premenstrual problems (e.g., migraine, bloating and edema) occurring in the late luteal phase of the menstrual cycle; and (2) migraine syndromes, particularly in women, are associated with deficits in brain and serum ionized Mg levels. We investigated whether concentrations of sex steroid hormones, found in the serum during the menstrual cycle of women, are associated with changes in the levels of cytosolic free magnesium ions ([Mg2+]i in single cultured canine cerebral vascular smooth muscle cells. The resting level of [Mg2+]i in these cells was 645 +/- 89 microM before exposure to sex steroid hormones. Exposure of these vascular cells to a low concentration of estrogen (10 pg/ml) failed to interfere with the levels of [Mg2+]i. However, exposure to estrogen, at concentrations ranging from 40 to 200 pg/ml, induced significant loss of [Mg2+]i in a concentration-dependent manner. At a concentration of 200 pg/ml estrogen, the level of [Mg2+]i decreased approximately 30% in comparison with controls. Progesterone produced biphasic effects on the levels of [Mg2+]i, depending on its concentration. Exposure of the cultured cells to a low concentration of progesterone (0.5 ng/ml) resulted in an increased level of [Mg2+]i (from 690 +/- 50 microM to 753 +/- 56 microM, p < 0.05). However, when these cells were exposed to higher concentrations of progesterone (i.e., from 5.0 to 20 ng/ml), the cellular levels of [Mg2+]i were decreased significantly. The higher the estrogen or progesterone concentration, the lower the levels of [Mg2+]i. In contrast, testosterone, a male hormone, didn't produce any significant alteration in [Mg2+]i levels in these cerebral vascular smooth muscle cells. These data indicate that low, physiological concentrations of female sex hormones, estrogen and progesterone, help cerebral vascular smooth cells sustain normal concentrations of [Mg2+]i, which are beneficial to vascular function, whereas high levels of estrogen and progesterone deplete, significantly, [Mg2+]i in cerebral vascular smooth muscle cells, possibly resulting in cerebrovasospasms and reduced cerebral blood flows related to premenstrual syndromes, migraine and stroke risk. Our findings could provide new insight into the mechanism whereby migraine occurs frequently in the late luteal phase in the premenstrual syndrome. In addition, our results demonstrate that female sex steroids but not testosterone (in physiologic concentrations) can exert direct effects on [Mg2+]i in cerebral vascular cells.