Spatial and dynamic changes in intracellular Ca measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca²⁺ ([Ca²⁺]_i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K⁺ (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca²⁺]_i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca²⁺-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca²⁺]_i in response to stimuli in neurones. Such heterogenic changes in [Ca²⁺]_i would better be studied by CLMS.     

Transient rhythmic network activity in the somatosensory cortex evoked by distributed input in vitro

The initiation and maintenance of physiological and pathophysiological oscillatory activity depends on the synaptic interactions within neuronal networks. We studied the mechanisms underlying evoked transient network oscillation in acute slices of the adolescent rat somatosensory cortex and modeled its underpinning mechanisms. Oscillations were evoked by brief spatially distributed noisy extracellular stimulation, delivered via bipolar electrodes. Evoked transient network oscillation was detected with multi-neuron patch-clamp recordings under different pharmacological conditions. The observed oscillations are in the frequency range of 2–5 Hz and consist of 4–12 mV large, 40–150 ms wide compound synaptic events with rare overlying action potentials. This evoked transient network oscillation is only weakly expressed in the somatosensory cortex and requires increased [K⁺]_o of 6.25 mM and decreased [Ca²⁺]_o of 1.5 mM and [Mg²⁺]_o of 0.5 mM. A peak in the cross-correlation among membrane potential in layers II/III, IV and V neurons reflects the underlying network-driven basis of the evoked transient network oscillation. The initiation of the evoked transient network oscillation is accompanied by an increased [K⁺]_o and can be prevented by the K⁺ channel blocker quinidine. In addition, a shift of the chloride reversal potential takes place during stimulation, resulting in a depolarizing type A GABA (GABA_A) receptor response. Blockade of -amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA), N-methyl-d-aspartate (NMDA), or GABA_A receptors as well as gap junctions prevents evoked transient network oscillation while a reduction of AMPA or GABA_A receptor desensitization increases its duration and amplitude. The apparent reversal potential of −27 mV of the evoked transient network oscillation, its pharmacological profile, as well as the modeling results suggest a mixed contribution of glutamatergic, excitatory GABAergic, and gap junctional conductances in initiation and maintenance of this oscillatory activity. With these properties, evoked transient network oscillation resembles epileptic afterdischarges more than any other form of physiological or pathophysiological neocortical oscillatory activity.     

Effectiveness in learning complex problem solving and salivary ion indices of psychological stress and activation

Following Hinton et al. (1992, Biol. Psychol. 33, 63–71) and Richter et al. (1995, Biol. Psychol. 39, 131–142) ionic concentration of [K⁺] in unstimulated saliva was predicted to rise with perceived challenge, while lowered [Na⁺] was expected when experiencing psychological stress (PS). Subjects had to learn an engaging complex problem-solving ‘game', via positive and negative feed-back on three ‘games' lasting 2.5–3.0 h overall. Comparisons were made between three groups: (1) high success; (2) partial success (‘strugglers'); and (3) total failure to learn. Saliva was sampled after resting and after each of three ‘games'. Successful learners had a significant rise in [K⁺] on the first ‘game' followed by a significant fall, consistent with task-challenge reaction followed by fast autonomic adaptation with successful learning. The ‘strugglers' [Na⁺] fell significantly over the ‘games', indicating mineralocorticoid-induced PS response of Na⁺ reabsorption. The ‘total failure' subjects had generally significantly higher [K⁺] than the successful ones, showing raised tonic sympathetic relative to parasympathetic activity — this outcome being interpreted from interference theories. The ‘failures' also had significantly higher tonic [Na⁺] on ‘games' — indicating low PS as predicted from McGrath's (1976) theory.     

The Na/K-ATPase as [K]o sensor: Role in cardiovascular disease pathogenesis and augmented production of endogenous cardiotonic steroids

Current evidence demonstrates that augmented production of endogenous cardiotonic steroids (CTS) such as ouabain and marinobufagenin is involved in the pathogenesis of hypertension and other cardiovascular diseases associated with volume expansion. It is also well documented that the development of hypertension and the cardiovascular complications of this disease are provoked by hypokalemia and suppressed by high-K⁺ diet. We hypothesized that altered extracellular K⁺ (K⁺)_o handling contributes to the pathogenesis of hypertension via modulation of interaction of endogenous CTS with Na⁺/K⁺-ATPase. To examine this hypothesis, experiments were performed with C7-Madin-Darby canine kidney epithelial cells at [K⁺]_o detected in plasma under control conditions (4.5 mM), severe hypokalemia (2 mM), and hyperkalemia (7 mM). Elevation of [K⁺]_o from 2 to 7 mM increased the threshold of modulation of intracellular (Na⁺)_i and (K⁺)_i content by ouabain from 1 to 10 nM, which corresponds to the range of endogenous CTS detected in plasma from patients with volume-expanded disorders. In control medium, 30% activation of cell proliferation was observed with 3 nM ouabain, whereas the addition of 0.3 nM ouabain was sufficient to induce about the same increment of cell proliferation in K⁺-depleted medium. [K⁺]_o elevation up to 7 mM completely abolished the proliferative effect of ouabain. At [K⁺]_o = 2, 4.5 and 7 mM, the death of ouabain-treated cells was indicated in the presence of 10, 30 and 300 nM ouabain, respectively. In conclusion, our results showed that modulation of [K⁺]_o in a pathophysiologically reasonable range sharply affected efficacy of endogenous CTS in the elevation of the [Na⁺]_i/[K⁺]_i ratio and in triggering (Na⁺)_i,(K⁺)_i-independent signaling resulting in cell proliferation and death. We propose that Na⁺/K⁺-ATPase may be considered as a [K⁺]_o sensor involved in the crosstalk of (K⁺)_o handling with the pathogenesis of cardiovascular diseases.     

Studies on mitochondrial ATPase of Leishmania donovani using digitonin-permeabilized promastigotes

Mitochondrial ATPase of Leishmania donovani was characterized using digitonin-permeabilized promastigotes and the results were compared with those from isolated mitochondria. Maximum mitochondrial ATPase activity was obtained in promastigotes permeabilized with digitonin at a final concentration of 20 μM and the specific activity of the enzyme was 46% and 57% higher than that of homogenized and sonicated promastigotes, respectively. At concentrations above 20 μM digitonin inhibited ATPase activity and the degree of inhibition increased with increasing concentrations of the detergent. The ATPase activity of promastigotes remained DCCD-sensitive when permeabilized with digitonin at concentrations up to 120 μM but the enzyme became increasingly resistant to this inhibitor as digitonin concentrations were increased to 140 μM and more, indicating the loss of functional activity of the enzyme. The pH and temperature optima for mitochondrial ATPase were determined to be 7.5 and 30°C, respectively. Mg²⁺ ions were essential for ATPase activity but free Mg²⁺ ions were found to be inhibitory. A Mg²⁺/ATP ratio of 1:3 supported the optimum ATPase activity. Sulfite and hexanol activated the enzyme but failed to prevent the inhibition by free Mg²⁺ ions. The results indicate that digitonin-permeabilized promastigotes provide an ideal system for studying the mitochondrial ATPase of L. donovani.     

Lack of an interaction between orexinergic and opioid/nociceptinergic systems in rat cerebrocortical slices

We have recently reported that orexins (OXs) selectively evoke norepinephrine release from rat cerebrocortical slices. In the present study, we have examined orexin–opioid interactions in OXA (100 nM) and K⁺ (40 mM)-evoked norepinephrine release. OXA-evoked norepinephrine release was reversed 90% by SB-334867 (OX₁-receptor antagonist) (10 μM) but not naloxone (10 μM). [D-Pen²,D-Pen⁵]-enkephalin (DPDPE) (DOP-agonist) and nociceptin/orphanin-FQ (N/OFQ) also failed to affect OXA-evoked release. [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO) (MOP-agonist) and spiradoline (KOP-agonist) significantly reduced OXA-evoked release with the concentration producing 50% of the maximal inhibition (EC₅₀) [maximal inhibition (E_max)] of 3.2 μM [41.8%] and 4.3 μM [54.9%] respectively. The effects of DAMGO and spiradoline were naloxone (10 μM)-insensitive. In contrast, naloxone significantly antagonized the inhibitory effects of DAMGO and spiradoline on K⁺-evoked release. We conclude that opioid receptors (DOP and KOP) are involved in K⁺ but not OXA-evoked release. Moreover, we have failed to demonstrate an interaction between orexinergic and opioid/N/OFQ-ergic systems in this system.     

Regulation of intracellular Ca concentration by interleukin-1β in rat cortical synaptosomes: an age-related study

The pro-inflammatory cytokine interleukin-1β (IL-1β) is released by cells during injury and stress, and increased neuronal expression of IL-1β is a feature of age-related neurodegeneration. We have recently reported that IL-1β has a biphasic effect on the K⁺-induced rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cortical synaptosomes, exerting an inhibitory effect on the K⁺-induced rise in [Ca²⁺]_i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K⁺-induced rise in [Ca²⁺]_i at higher (100 ng/mL) concentrations. In the present study, we observed that the K⁺-induced rise in [Ca²⁺]_i was inhibited to a similar extent by the lower concentration of IL-1β in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1β, resulting in a marked elevation in [Ca²⁺]_i. We propose that the age-related increase in neuronal concentration of IL-1β promotes a dramatic elevation in [Ca²⁺]_i following membrane depolarization, thereby altering Ca²⁺ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.     

Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [³H]noradrenaline from prelabelled 3–7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [³H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na⁺ (veratridine) and Ca²⁺ (high [K⁺]) channels or direct, channel-independent promotion of Ca²⁺ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na⁺ deprivation, high Mg²⁺) or drugs known to block the activity of receptoroperated channels (imipramine, cocaine), voltage-dependent Na⁺ (tetrodotoxin) or Ca²⁺ (nitrendipine) channels.

Inhibition by nitrendipine, a potent Ca²⁺ antagonist, of the secretory responses to both nicotine and high [K⁺] indicates a preferential Ca²⁺ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [³H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K⁺], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na⁺ deprivation abolished [³H]noradrenaline release produced by nicotine, it seems that Na⁺ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view.

However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na⁺ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland. Propagated action potentials will activate voltage-sensitive Ca²⁺ channels, producing a massive Ca²⁺ entry and an explosive ejection of catecholamines simultaneously from many cells; during stressful situations this could constitute an efficient mechanism for the amplification of the secretory signal initiated by depolarization of chromaffin cells at restricted subsynaptic areas by acetylcholine liberated at splanchnic nerve-chromaffin cell junctions.     

Characterization of the norovirus 3C-like protease

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 °C, respectively. Increased concentration (100 mM) of monovalent cations such as Na⁺ and K⁺ was inhibitory to the activity. Hg²⁺ and Zn²⁺ remarkably inhibited the protease activity, while Mg²⁺ and Ca²⁺ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.     

Effects of treatment with 17beta-estradiol on the hypercholesterolemic rabbit middle cerebral artery

Objective: The effects of acute and long-term treatment with 17β-estradiol on the vasomotor responses of rabbit middle cerebral artery (RMCA) were investigated. Methods: For 8 weeks, male rabbits consumed standard chow (control group), standard chow+1% cholesterol (cholesterol group) or 1% cholesterol chow+17β-estradiol (i.m. injection 700 μg per week) (estradiol group). The RMCA was precontracted with high K⁺ solution and exposed to agonists. Results: Acute exposure to 17β-estradiol strongly induced relaxation of the RMCA isolated from either control or cholesterol groups. This effect was endothelium independent. Incubation with 17β-estradiol shifted the calcium contraction curve to the right. High cholesterol diet impaired the relaxation induced by acetylcholine and did not alter relaxation to sodium nitroprusside or to papaverine. Chronic treatment with 17β-estradiol restored this impaired relaxation to acetylcholine. This protective effect of estradiol was significantly reduced in the presence of N^ω nitro- -arginine methyl ester, a constitutive nitric oxide-synthase inhibitor and was not modified in the presence of aminoguanidine, an inducible nitric oxide-synthase inhibitor. Neither tetrabutylammonium, a blocker of calcium-activated K⁺ channels, nor glibenclamide, a blocker of ATP-sensitive K⁺ channels, affected concentration–response to acetylcholine in the RMCA of the estradiol group, whereas 4-aminopyridine, a blocker of voltage-dependent K⁺ channels strongly inhibited this relaxation. Conclusions: These results suggest that acute effects of 17β-estradiol in the RMCA is mediated through blockade of calcium entry into vascular smooth muscle cells, while chronic treatment with this hormone seems to be mediated by release of nitric oxide which activates voltage-dependent potassium channels.     

Calcitonin gene-related peptide-mediated increase in K(+)-induced [(3)H]-dopamine release from rat caudal striatal slices

The calcitonin-gene receptor peptide (CGRP) receptor is present in high levels in the caudal striatum of the rat. Previous behavioural experiments have highlighted a possible correlation between CGRP-mediated effects and the dopaminergic system. In this study, we examined the effect of CGRP on K⁺-evoked [³H]-dopamine release in a slice preparation of the rat caudal striatum. The unstimulated release of [³H]-dopamine was not affected by CGRP. However, CGRP increased the release of [³H]-dopamine evoked by K⁺ (30 mM) in a concentration-dependent manner. The stimulatory effect of CGRP was blocked by the CGRP1 antagonist hCGRP(8–37) (without effect on its own). The stimulatory effect of 1 μM CGRP was blocked by dizocilpine (MK-801), suggesting that excitatory transmission is involved in mediating the facilitated release. This study suggests that the peptide CGRP, modulates dopamine release in the rat caudal striatum probably indirectly via glutamatergic transmission.     

Confirmatory tests for human T-cell leukemia virus type 1 (HTLV-1): Western blot compared with RIPA on African sera

Confirmation of human T-Cell leukemia virus type 1 (HTLV-1) seropositivity calls for reactivity against at least 2 proteins encoded by 2 different genes, revealed by Western blot (WB) and /or radioimmuno-precipitation assay (RIPA). To evaluate the use of WB as a basis for applying these criteria, we conducted a study of two types of WB and compared them with RIPA patterns. The first part of the work, performed with 40 African sera, used Dupont de Nemours commercialized WB and an ‘in-house’ WB. Both WB detected antibody to proteins encoded by 2 different genes: antibody to gag products were revealed equally by both WB, but commercialized WB detected antibody to tax protein whereas the ‘in-house’ WB detected antibody to env protein (gp46) more efficiently.

The second part of the work, conducted with 158 African sera, compared results of an ‘in-house’ virus lysate WB and RIPA. Our data show a perfect concordance between the two procedures when sera were clearly positive by WB (gag + env reaction). Sera reacting to p19 and p24 (both gag) by WB were confirmed positive by RIPA in 75% of the cases. The majority of the indeterminate WB profiles not confirmed by RIPA presented isolated gag reactivity (p15 or p19 or p24).     

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of −60 to −70 mV elicited a slow inward calcium (Ca²⁺) current and two outward potassium (K⁺) currents: an A current and a slower, Ca²⁺-dependent K⁺ current. Noradrenaline (NA) (20 μM) depressed the amplitude of the K⁺ currents without affecting the Ca²⁺ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K⁺ currents by binding to β-receptors.     

Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats

Several lines of experimental evidence support an association between altered Ca²⁺ regulation and aging. It has been supposed that free cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca²⁺]_i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca²⁺]_i was determined using the fluorescent Ca²⁺ chelator fura-2. Net KCl-dependent changes (ΔK) in [Ca²⁺]_i were determined by the difference between stimulatory (100 μM Ca²⁺/60 mM KCl) and resting (100 μM Ca²⁺/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca²⁺]_i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca²⁺]_i or KCl-stimulated [Ca²⁺]_i.     

Alterations in electrical and mechanical activity in Langendorff-perfused guinea pig hearts exposed to decreased external sodium concentration with or without hypotonic insult

In order to examine electrical and mechanical effects of hyponatremia and hypotonicity, relevant to those in patients with ‘water intoxication’ syndrome, Langendorff-perfused guinea pig hearts were exposed to reduced NaCl concentrations (hypotonic [NaCl]₀-reduction) under the monitoring of left ventricular developed pressure (LVDP) and epicardial ECG. In some hearts, hyponatremia (from 140 to 80 mEq/l) was compensated for by adding mannitol to maintain osmolarity at a constant level (isotonic [NaCl]₀-reduction) or tetraethylammonium chloride to maintain both osmolarity and chloride concentrations at a constant level (isotonic [Na⁺]₀-reduction). Progressive isotonic [NaCl]₀-reduction increased LVDP, which was abolished in the presence of KB-R7943, a novel inhibitor of Na⁺/Ca²⁺-exchange. LVDP was reduced in hypotonic [NaCl]₀-reduction in which myocardial water content was increased. PQ interval and QRS duration were prolonged with both hypotonic and isotonic [NaCl]₀-reduction and these changes tended to be more pronounced with hypotonic than with isotonic [NaCl]₀-reduction. Similar ECG changes were also evident with isotonic [Na⁺]₀-reduction. Gd³⁺ (1–5 μM), a blocker of stretch-activated nonspecific cation channels, had no substantial effects on the electrical or mechanical changes seen with hypotonic [NaCl]₀-reduction. In conclusion, isotonic [NaCl]₀-reduction produced a positive inotropism by modulating Na⁺/Ca²⁺-exchange, whereas hypotonic [NaCl]₀-reduction led to negative inotropism, due in part to hypotonic myocardial swelling. In addition, [Na⁺]₀-reduction, irrespective of the concomitant [Cl⁻]₀ or osmotic changes, depressed atrioventricular as well as intraventricular conduction.     

Effects of free Ca on the [Ca + Mg-Dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts

Differences between Alzheimer and control fibroblasts [Ca²⁺ + Mg²⁺]-dependent ATPase activity at free Ca²⁺ concentration considerably higher than physiologic concentrations were observed. At 50 μM free Ca²⁺, Alzheimer and control fibroblast homogenates exhibited maximum velocity values ranging from 8 to 25 nmoles phosphate released/min/mg protein. Higher free Ca²⁺ (350 μM) inhibited control fibroblast ATPase activity approximately 77%; whereas, Alzheimer fibroblasts retained greater than 75% starting activity. Although the pathophysiological significance of these findings is at present unclear, these data suggest the Ca²⁺ pump of Alzheimer fibroblasts behaves differently in the presence of high free Ca²⁺. Such behavior may be of potential diagnostic value.     

Effects of extracellular ATP on hair cells isolated from the guinea-pig semicircular canals

Externally applied ATP (100 μM) induced membrane currents in type I and type II vestibular hair cells enzymatically isolated from guinea-pig semicircular canals. In whole-cell voltage-clamp and with 140 mM K⁺ in the pipette solution, ATP evoked an inwardly directed current in 58% of the cells when held at potentials below −40 mV. In the remainder, external ATP produced an outward current. After block of the K currents, an inward current activated by ATP was revealed at −50 mV. Intracellular Ca²⁺ levels were monitored using the Ca²⁺ indicator Fura-2 and were found to rise in both hair cell types in response to ATP. These results strongly suggest that ATP directly controls the entry of Ca²⁺ into crista hair cells which can then further modulate K⁺ currents.     

Prenatal naloxone affects survival and morphine sensitivity of rat offspring

Excised middle cerebral and basilar arteries from dogs were incubated in Krebs-Ringer bicarbonate solution and exposed to normal, high and low concentrations of magnesium [Mg] in the medium. Sudden withdrawal of extracellular Mg²⁺ ([Mg²⁺]_o) from the medium produced contraction of arterial smooth muscle, whereas elevated concentrations of [Mg²⁺]_o decreased the basal tensions in a concentration-dependent manner.     

Disruption of ionic and cell volume homeostasis in cerebral ischemia: The perfect storm

The mechanisms of brain tissue damage in stroke are strongly linked to the phenomenon of excitotoxicity, which is defined as damage or death of neural cells due to excessive activation of receptors for the excitatory neurotransmitters glutamate and aspartate. Under physiological conditions, ionotropic glutamate receptors mediate the processes of excitatory neurotransmission and synaptic plasticity. In ischemia, sustained pathological release of glutamate from neurons and glial cells causes prolonged activation of these receptors, resulting in massive depolarization and cytoplasmic Ca²⁺ overload. High cytoplasmic levels of Ca²⁺ activate many degradative processes that, depending on the metabolic status, cause immediate or delayed death of neural cells. This traditional view has been expanded by a number of observations that implicate Cl⁻ channels and several types of non-channel transporter proteins, such as the Na⁺,K⁺,2Cl⁻ cotransporter, Na⁺/H⁺ exchanger, and Na⁺/Ca²⁺ exchanger, in the development of glutamate toxicity. Some of these ion transporters increase tissue damage by promoting pathological cell swelling and necrotic cell death, while others contribute to a long-term accumulation of cytoplasmic Ca²⁺. This brief review is aimed at illustrating how the dysregulation of various ion transport processes combine in a ‘perfect storm’ that disrupts neural ionic homeostasis and culminates in the irreversible damage and death of neural cells. The clinical relevance of individual transporters as targets for therapeutic intervention in stroke is also briefly discussed.     

Effect of zinc on lysis of sheep erythrocytes by guinea pig C4-deficient serum

The divalent cation Zn²⁺ enhanced the lysis of sheep erythrocytes (ShE) by guinea pig C4-deficient (C4D) serum. Enhancement by Zn²⁺ (0.1 mM) was demonstrated in the presence of cobra venom factor (CoVF, 2 units/ml) and optimal Mg²⁺ (1.0 mM). Moreover, Zn²⁺ (0.1 mM) in the presence of Mg²⁺ (1.0 mM) caused the lysis of ShE by C4D serum without the addition of CoVF. Further studies were therefore performed in the absence of CoVF. Although addition of Zn²⁺ or Mg²⁺ alone to some pools of C4D serum caused lysis of ShE, the greatest extent of lysis occurred when Zn²⁺ and Mg²⁺ were both added to the reaction mixtures. When C4D serum was resin-treated, both Mg²⁺ and Zn²⁺ were required for lysis. Addition of Ca²⁺ had no effect. This pathway required high concns of serum and was inhibited by pretreating the serum with zymosan (2 mg/ml serum, 37°C, 1 hr) or by heating the serum at 56°C for 20 min. K76-COOH monocarboxylic acid also inhibited this pathway. These results suggest that Zn²⁺ together with Mg²⁺ activates the alternative pathway on the nonactivator surface (ShE).