Increase of Intracellular free [CA2+] in Single Human Motile Spermatozoa Treated with Human Follicular Fluid

The intracellular free [Ca²⁺] concentration ([Ca²⁺]_i) in individual human sperm was measured using a fluorescent Ca²⁺ indicator. In 18 of 23 motile sperm (78.3%), [Ca²⁺]_i increased significantly and promptly after addition of 20% human follicular fluid (hFF), but in the others it did not increase. The mean resting [Ca²⁺]_i level of sperm in which [Ca²⁺]_i increased after addition of 20% hFF (the influx group) was significantly lower than those in which it did not increase (112.8 ± 40.1 nM vs. 156.9 ± 13.5 nM,p <. 05). After addition of 20% hFF, the mean [Ca²⁺]_i in the influx group reached a peak value of 210.7± 24.7 nM within 30 s and then decreased slowly; the mean [Ca²⁺]_i values 1, 5, 10, and 15 min after addition of 20% hFF were 179.3 ± 31.4, 174.3 ± 30.2, 172.5 ± 27.8, and 175.1 ± 27.2 nM, and all values were significantly higher than the resting level (p <. 01). The frequency distribution of [Ca²⁺]_i after addition of 20% hFF was shifted toward higher concentrations (p <. 01). However, the addition of 20% hFF did not increase the percentage of live acrosome reaction (before 3.8 ± 0.9% vs. after 2.9 ± 0.5%, respectively). Thus, hFF increased [Ca²⁺]_i in about 80% of the motile sperm. Relatively high [Ca²⁺]_i levels persisted for at least 10–15 min after its addition. However, hFF did not trigger a rapid response in acrosome reaction.     

Influence of aluminum and H+ on the electrolyte homeostasis in the unionidaeAnodonta anatina L. andUnio pictorum L.

Two species of freshwater clams,Anodonta anatina andUnio pictorum, were exposed to aluminum (300-900 g/L) and acid (pH 4–5 and 6.6–8.3) in hard (35 mg Ca/L) and soft (3.5 mg Ca/L) water. Long- and shortterm pH depressions of 2 and 3 weeks and intermittent, repetitive pulses of 3 days were used. The pattern of change in the hemolymph electrolyte balance was different inU. pictorum and inA. anatina. In general, an increase in hemolymph [Ca²⁺], and a decrease in [Na⁺], [Cl^–], [K⁺] and [Mg²⁺] as a result of acid exposure was seen in both species. Hemolymph [Ca²⁺] ofU. pictorum was reduced after 3 days of exposure to acid water, whereas an exposure of one week was needed to affect the other hemolymph ions. In circumneutral, hard water Al had no effect on the electrolyte balance. Intermittent pulses of low pH and Al produced a transitory increase in hemolymph [Ca²⁺], whereas [Na⁺] and [K⁺] were not affected.     

TRPV1-Mediated Sensing of Sodium and Osmotic Pressure in POMC Neurons in the Arcuate Nucleus of the Hypothalamus

The central melanocortin system conducted by anorexigenic pro-opiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARC) not only regulates feeding behavior but also blood pressure. Excessive salt intake raises the Na⁺ concentration ([Na⁺]) in the cerebrospinal fluid (CSF) and worsens hypertension. The blood–brain barrier is immature in the ARC. Therefore, both AgRP and POMC neurons in the ARC have easy access to the electrolytes in the blood and can sense changes in their concentrations. However, the sensitivity of AgRP and POMC neurons to Na⁺ remains unclear. This study aimed to explore how the changes in the extracellular Na⁺ concentration ([Na⁺]) influence these neurons by measuring the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the single neurons isolated from the ARC that were subsequently immunocytochemically identified as AgRP or POMC neurons. Both AgRP and POMC neurons responded to increases in both [Na⁺] and osmolarity in C57BL/6 mice. In contrast, in transient receptor potential vanilloid 1 (TRPV1) knockout (KO) mice, POMC neurons failed to respond to increases in both [Na⁺] and osmolarity, while they responded to high glucose and angiotensin II levels with increases in [Ca²⁺]_i. Moreover, in KO mice fed a high-salt diet, the expression of POMC was lower than that in wild-type mice. These results demonstrate that changes in [Na⁺] and osmolarity are sensed by the ARC POMC neurons via the TRPV1-dependent mechanism.     

Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia

Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na⁺, K⁺-ATPase and Ca²⁺-ATPase and the expression of Na⁺, K⁺-ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na⁺, K⁺-ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na⁺, K⁺-ATPase activity and the Na⁺, K⁺-ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca²⁺-ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p<0.05). In comparison, PMCA4 expression in the asthenozoospermia group was lower than in the normozoospermia group (p>0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.     

Effects of ethanol exposure on neuropeptide-stimulated calcium mobilization in N1E-115 neuroblastoma

The effects of acute and chronic (100 mM for 7 days) ethanol exposures on resting intracellular free calcium, [Ca²⁺]_i, as well as bradykinin and neurotensin mediated [Ca²⁺]_i mobilization were determined in intact N1E-115 neuroblastoma. [Ca²⁺]_i was monitored fluorometrically with the calcium indicator, fluo-3/AM. Acute exposure to ethanol resulted in an inhibition of bradykinin mediated [Ca²⁺]_i mobilization with significant effects observed only at 400 mM ethanol. Neurotensin mediated [Ca²⁺]_i mobilization was not significantly affected by any of the ethanol concentrations tested. Similarly, resting [Ca²⁺]_i (64 ± 2 nM) was unaffected by either chronic or acute ethanol as high as 400 mM. However, chronic exposure to ethanol significantly reduced the magnitude of bradykinin mediated [Ca²⁺]_i mobilization both in the absence and presence of extracellular [Ca²⁺]. In contrast, [Ca²⁺]_i mobilization in the presence of various concentrations of neurotensin was not significantly affected by chronic ethanol exposure. The results suggest that neuropeptide mediated [Ca²⁺]_i mobilization is relatively insensitive to the acute presence of ethanol. In addition, chronic ethanol exposure appears to have selective effects on receptor mediated [Ca²⁺]_i mobilization because this response to bradykinin, but not neurotensin, was significantly reduced in cells exposed to ethanol. The results also suggest that the reduction in bradykinin stimulated [Ca²⁺]_i mobilization in chronically exposed cells is due in part to an inhibition of the release of intracellularly bound [Ca²⁺].     

Effects of aqueous crude extract of Echeveria gibbiflora on mouse sperm function

The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C₄H₅O₅)₂?6H₂O] present in this plant extract, we evaluated its effect on Ca²⁺ influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca²⁺ influx. Furthermore, OBACE displayed an LD₅₀ of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity.

Abbreviations: ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP: average path velocity; VCL: track speed; VSL: straight line velocity     

Effects of Cd, Zn and Cd + Zn Combination on ATPase Activitiy in the Gill and Muscle of Tilapia (Oreochromis niloticus)

This study investigated the responses of Na⁺/K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase in the gill and muscle of a freshwater fish Oreochromis niloticus exposed to 1 μg/mL of Cd and Zn and their mixture for different periods (0, 7, 14, 21 and 28 days). At the end of experimental periods, the activities Na⁺/K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase in gill tissues and only Ca²⁺-ATPase activity in muscle tissues were measured. Gill Na⁺/K⁺-ATPase activity generally decreased following single metal exposures, whereas their combinations increased its activity. Gill Ca²⁺-ATPase activity decreased relative to the control at most exposure times for single exposures of Zn and Cd, as well as for the combined exposure. There was no gill Ca²⁺-ATPase activity after 28 days of exposure to Zn and Cd combined. Mg²⁺-ATPase activity was not affected significantly in gill tissue by exposure to Zn and Cd individually or in combination. Muscle Ca²⁺-ATPase activity also decreased significantly following metal exposure, but not as greatly as in the gill tissue. Tissue protein levels were mostly unaffected by metal exposures. This study showed that certain ATPases are highly sensitive to metal exposure whether the metals are essential or non essential, and suggests using gill tissue Na⁺/K⁺-ATPase and Ca²⁺-ATPase as sensitive biomarkers in metal contaminated waters.     

Effects of Magnesium on Cardiac Excitation-Contraction Coupling

Objective: Magnesium regulates a large number of cellular processes. Small changes in intracellular free Mg²⁺ ([Mg²⁺]_i) may have important effects on cardiac excitability and contractility. We investigated the effects of [Mg²⁺]_i on cardiac excitation-contraction coupling.

Methods: We used our ionic-metabolic model that incorporates equations for Ca²⁺ and Mg²⁺ buffering and transport by ATP and ADP and equations for MgATP regulation of ion transporters (Na⁺-K⁺ pump, sarcolemmal and sarcoplasmic Ca²⁺ pumps).

Results: Model results indicate that variations in cytosolic Mg²⁺ level might sensitively affect diastolic and systolic Ca²⁺, sarcoplasmic Ca²⁺ content, Ca²⁺ influx through L-type channels, efficiency of the Na⁺/Ca²⁺ exchanger and action potential shape. The analysis suggests that the most important reason for the observed effects is a modified normal function of sarcoplasmic Ca²⁺-ATPase pump by altered diastolic MgATP levels.

Conclusion: The model is able to reproduce qualitatively a sequence of events that correspond well with experimental observations during cardiac excitation-contraction coupling in mammalian ventricular myocytes.     

Physiological responses to severe acid stress in four species of freshwater clams (unionidae)

Four species of freshwater clam,Anodonta anatina, A. cygnea, Unio pictorum, andU. tumidus were exposed for 2 weeks to acidified soft water (pH 4.0–4.5, Ca 4.6 mg/L) and for 4 weeks to acid in hard water conditions (Ca 18.5 mg/L). The exposures caused a decrease in Na⁺, K⁺, and Cl^– ion and a rapid increase of Ca²⁺ in the hemolymph. The elevation of the hemolymph Ca²⁺ was positively correlated with the decrease in the hemolymph pH in all species studied. Low ambient [Ca²⁺] level accelerated the pH decrease and Ca²⁺ increase in the hemolymph. Na⁺ and Cl^– ion concentrations changed less rapidly in the soft conditions. Although there were minor changes in the mineral composition of the calcium concretions in the gills, the amount of calcium in the concretions did not change during the exposure. There was no correlation between the thickness of the shell and the ionic response, but all four species responded to low ambient pH in the same way.     

Effects on ionic composition of blood and tissues ofAnodonta grandis grandis (Bivalvia) of an addition of aluminum and acid to a lake

To test the hypothesis that summer low pH, episodic events cause stress and mortality in aquatic organisms including mussels, alum (aluminum sulfate) was added near the point of inflow to Lake 114 in the Experimental Lakes Area, northwestern Ontario from 3 to 5 July 1984. Lake 114 was at pH 5.9 before the alum addition. The alum produced measured extremes of pH 4.5 and [Al] of 2,237 g/L near the point of addition. This study examined the effects of the alum addition on ionic concentrations of blood and tissue (gills, adductor muscle, foot and visceral mass) of the floater mussel,Anodonta grandis grandis. Mussels were collected from a second lake, 377, and introduced into Lake 114 at five locations five days before the alum addition. In response to transfer from the oligotrophic, unmanipulated Lake 377 to acidified Lake 114 (pH 5.9), blood of mussels showed a marked elevation of [Ca⁺⁺], decline in [Mg⁺⁺] and a temporary increase in [Cl^–] but no change in [Na⁺], [K⁺] or [SO₄ ⁼]. During the alum addition, in mussels near the point source of the alum addition, blood [Na⁺] and [Cl^–] declined and [Ca⁺⁺] became still more elevated. Mussels suffered no mortality associated with the alum addition and almost no mortality during 26 days in Lake 114. Gill increased in [Al], [Ca], [Mn], declined in [Na] and showed no change in [Cd] in mussels near the alum addition. Visceral mass and adductor muscle also had lower [Na] in mussels near the point of alum addition. We attribute the increase in blood [Ca⁺⁺] to the dissolution of the Ca stores in the shell and/or mantle of mussels. This would provide protection to the mussels during short-term declines in pH such as spring or summer episodic events. Never-theless, chronic exposure to small decreases in pH by mussels, already near the limits for obtaining sufficient Ca⁺⁺, might be intolerable. It follows that acidification to the pH of 5.9 of soft water lakes containing mussels would be expected to lead to the loss of A.g. grandis from these waters.     

Differential Effects of Methanol on Rat Aortic Smooth Muscle

LI, W., B. T. ALTURA AND B. M. ALTURA. Differential effects of methanol on rat aortic smooth muscle. Alcohol 16(3) 221–229, 1998.—The effects of methanol on isolated segments of rat aorta were investigated. In the absence of any vasoactive agent, methanol (5–675 mM) failed to alter basal tension. In rat aortic rings precontracted with high K⁺ (30 mM), methanol elicited a concentration-related relaxation at concentrations of from 5 to 675 mM. The K⁺-induced contraction in the presence of endothelium was more strongly inhibited by methanol than in the absence of endothelium. The effective concentration producing approximately 50% of the maximal relaxation response (ED₅₀) to methanol was about 96 mM. Methanol-induced relaxations could not be abolished either by 5 × 10⁻⁵ M N-nitro-l-arginine methyl ester (l-NAME) or N^G-nitro-l-arginine (l-NNA), both selective inhibitors of nitric oxide (NO) formation; these relaxations were not potentiated by addition of excess l-arginine. An inhibitor of prostanoid synthesis, indomethacin (10⁻⁵ M), had no effects on methanol-induced relaxation. Removal of extracellular Ca²⁺ ([Ca²⁺]_o) resulted in almost complete inhibition of the relaxant effects of methanol on rat aortic ring segments. Marked attenuation of the relaxation responses of intact arteries to methanol was obtained after buffering intracellular Ca²⁺ ([Ca²⁺]_i) with 10 μM BAPTA-AM. In 5-hydroxytryptamine (5-HT, 2.5 μM)- or phenylephrine (PE, 0.1 μM)-precontracted rat aortic rings, methanol amplified contractile responses to 5-HT and PE; these increased responses were concentration dependent. No significant differences in these methanol potentiated responses were found between aorta with or without endothelial cells. The amplified rat aortic smooth muscle responses induced by methanol after PE could be modified only by phentolamine, an antagonist of PE, while responses to 5-HT could be inhibited by methysergide (an antagonist of 5-HT) and by phentolamine, diphenhydramine, and haloperidol. Pretreatment with 50, 200, and 500 mM methanol increased rat aortic contractile responses induced by 5-HT and PE. Our results suggest that: (a) acute methanol exposure relaxes rat aortic smooth muscle contractile responses induced by high K⁺, leading to vessel relaxation. This relaxation effect of methanol is endothelium-dependent, clearly Ca²⁺ dependent, and independent of endogenous vasodilators such as acetylcholine, histamine, catecholamines, serotonin, or PG. (b) Methanol seems to increase potassium current by shifting the potential towards more negative values in depolarized vascular muscle cell membranes, probably inducing hyperpolarization of the cell membranes leading to a repolarization. (c) In contrast to the relaxant responses, methanol protentiates contractile response of rat aorta to 5-HT and PE.     

Ethanol impairs calcium homeostasis following CCK-8 stimulation in mouse pancreatic acinar cells

Alcohol consumption has long been associated with cell damage, and it is thought that it is involved in approximately 40% of cases of acute pancreatitis. In the present study, we have investigated the early effects of acute ethanol exposure on cholecystokinin octapeptide (CCK-8)-evoked calcium (Ca²⁺) signals in mouse pancreatic acinar cells. Cells were loaded with fura-2 and the changes in fluorescence were monitorized using a spectrofluorimeter. Our results show that stimulation of cells with 1 nM CCK-8 led to a transient increase in [Ca²⁺]_c, which consisted of an initial increase followed by a decrease of [Ca²⁺]_c toward a value close to the prestimulation level. In the presence of 50 mM ethanol, CCK-8 lead to a greater Ca²⁺ mobilization compared to that obtained with CCK-8 alone. The peak of CCK-8-evoked Ca²⁺ response, the “steady-state level” reached 5 min after stimulation, the rate of decay of [Ca²⁺]_c toward basal values and the total Ca²⁺ mobilization were significantly affected by ethanol pretreatment. Thapsigargin (Tps) induced an increase in [Ca²⁺]_c due to its release from intracellular stores. After stimulation of cells with CCK-8 or Tps in the presence of 50 mM ethanol, a greater [Ca²⁺]_c peak response, a slower rate of decay of [Ca²⁺]_c, and higher values of [Ca²⁺]_c were observed. The effects of ethanol might result from a delayed or reduced Ca²⁺ extrusion from the cytosol toward the extracellular space by plasma membrane Ca²⁺adenosine triphosphatase (ATPase), or into the cytosolic stores by the sarcoendoplasmic reticulum Ca²⁺-ATPase. Participation of mitochondria in Ca²⁺ handling is also demonstrated. The actions of ethanol on CCK-8 stimulation of cells create a situation potentially leading to Ca²⁺ overload, which is a common pathological precursor that mediates pancreatitis.     

Low-molecular-weight-chitosan ameliorates cadmium-induced toxicity in the freshwater crab, Sinopotamon yangtsekiense

Cadmium (Cd) has been shown to induce oxidative stress. Low-molecular-weight-chitosan (LMWC) has been demonstrated to exhibit potent antioxidant effects. We investigated the regulation role in Cd²⁺-induced oxidative damage in the hepatopancreas of the freshwater crab Sinopotamon yangtsekiense and the protective effect of LMWC. The results showed that Cd²⁺ significantly increased the hepatopancreatic metallothionein (MT) mRNA levels and protein kinase C (PKC) activity while decreasing the activities of Na⁺,K⁺-ATPase and Ca²⁺-ATPase in crabs relative to the control group. Co-treatment with LMWC suppressed the levels of MT and PKC but raised the activities of Na⁺,K+-ATPase and Ca²⁺-ATPase in hepatopancreatic tissues compared with the crabs exposed to Cd²⁺ alone. We postulate that LMWC may exert its protective effect through regulating the expressions of MT, PKC, Na⁺,K⁺-ATPase and Ca²⁺-ATPase, thereby enhancing antioxidant defense. These observations suggest that LMWC may be beneficial because of its ability to alleviate the Cd²⁺-induced damages to the crabs.     

Effect of Major Cations and pH on the Acute Toxicity of Cadmium to the Earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>: Implications for the Biotic Ligand Model Approach

The purpose of this study was to characterize the individual effect of different cations (Ca²⁺, Mg²⁺, Na⁺, K⁺, and H⁺) on the acute toxicity of Cd to the earthworm Eisenia fetida. Higher activities of the considered cations increased the 48-h LC₅₀ (expressed as Cd²⁺ activities) linearly to different extents in simulated soil solution. The conditional constants for the binding of H⁺ (log K = 5.41), Cd²⁺ (log K = 4.0), Ca²⁺ (log K = 3.35), Mg²⁺ (log K = 2.82), Na⁺ (log K = 1.57), and K⁺ (log K = 2.31) to the biotic ligand (BL) of E. fetida were derived from the toxicity data based on the biotic ligand model (BLM). Furthermore, it was calculated that on average 72% of the BL sites needed to be occupied by Cd²⁺ to induce 50% lethal effect. Autovalidation of the model with the results of the test sets showed that 48-h LC₅₀ could be predicted within a factor of two.     

Glutamate and cysteinylglycine effects on NMDA receptors: Inhibition by ethanol

Glutamate, the endogenous neurotransmitter at the NMDA receptor, and cysteinylglycine are formed as byproducts of glutathione (GSH) metabolism by γ-glutamyltranspeptidase. Glutamate and cysteinylglycine were investigated in Fura-2-loaded whole-brain neonatal (< 24 h) dissociated neurons to determine 1) if cysteinylglycine might act as a glycine site coagonist, 2) the inhibitory effects of ethanol on glutamate-stimulated increases in cytosolic calcium concentration (Glu-[Ca 2+]_i), and 3) the effects of cysteinylglycine on ethanol's inhibition of Glu-[Ca²⁺]_i. Glu-[Ca²⁺]_i (EC₅₀ = 0.7 μM) in these cells was highly specific for NMDA receptor-operated calcium channels as they were dependent on extracellular calcium, enhanced by glycine, and blocked by magnesium, APV, and ethanol. However, because cysteinylglycine did not potentiate Glu-[Ca²⁺]_i nor reverse ethanol inhibition of Glu-[Ca²⁺]_i, it does not appear to act as a glycine coagonist or change the inhibitory sensitivity of ethanol to Glu-[Ca 2+]_i.     

ETHANOL DECREASES BASAL CYTOSOLIC-FREE CALCIUM CONCENTRATION IN CULTURED SKELETAL MUSCLE CELLS

We analysed the effect of ethanol on basal cytosolic-free calciumconcentration ([Ca²⁺]₁) in cultured rat myocytes. Ethanol causeda dose-dependent decrease of the resting [Ca²⁺]₁). Removal ofethanol was followed by a transitory increase of [Ca²⁺]₁ abovethe basal level. In cells chronically exposed to ethanol, [Ca²⁺]₁normalized to the previous level.     

Role of brain [Mg2+]i in alcohol-induced hemorrhagic stroke in a rat model: A 31P-NMR in vivo study

One hundred percent of anesthetized rats administered 6.6 gm/kg of ethanol IP died within 10–35 min of alcohol injection; upon autopsy of the brain all demonstrated profound subarachnoid and intracranial bleeding, clear signs of hemorrhagic stroke. Pretreatment of rats with 4 μmol/min MgCl₂, but not saline, via IV administration (for 30–45 min), prevented hemorrhagic stroke in all animals so treated with 6.6 gm/kg ethanol. Administration of the stroke dose of alcohol resulted in rapid (within 3–5 min) and marked deficits in whole brain intracellular free Mg ([Mg²⁺⁺]_i) as observed by in vivo ³P-NMR spectroscopy. Intracellular pH (pH_i) and the phosphocreatine [PCr]/[ATP] ratio also fell following a significant fall in brain [Mg²⁺]_i). Brains of rats that exhibited strokelike events, upon death and autopsy, demonstrated continued and marked intracellular acidosis with progressive fall in the [PCr]/[ATP] ratio and elevation of inorganic phosphate (P_i) and [H⁺]i; these events were not accompanied by any rises in systemic arterial blood pressure. Rats pretreated with MgCl₂ exhibited relatively stable brain [Mg²⁺]_i, and essentially unchanged pHi, [PCr], [ATP], or [P_i] following alcohol administration, although such animals exhibited threefold alterations in plasma Mg²⁺, as measured by ion selective electrodes. These observations suggest that high alcohol ingestion can result in severe vasospasm, ischemia, and rupture of blood vessels probably as a consequence of depletion of brain [Mg₂₊]_i, events that can be prevented by Mg²⁺ pretreatment.     

Responses of the Antioxidant and Osmoregulation Systems of Fish Erythrocyte Following Copper Exposures in Differing Calcium Levels

Freshwater fish Oreochromis niloticus were exposed to Cu in differing Ca²⁺ levels (15, 30 and 90 mg/L), using acute (0.3 µM, 3 d) and chronic (0.03 µM, 30 d) exposure protocols and enzyme activities related to the antioxidant (catalase, CAT, EC 1.11.1.6; superoxide dismutase, SOD, EC 1.15.1.1; glutathione peroxidase, GPx, EC 1.11.1.9) and osmoregulation (Total, Na⁺/K⁺-ATPase, EC 3.6.3.9, Mg²⁺-ATPase, EC 3.6.3.2) systems in the erythrocytes were measured. Activities of antioxidant enzymes generally decreased significantly following either Ca²⁺ alone or Ca²⁺+Cu combinations in both acute and chronic exposures. Na⁺/K⁺-ATPase activity significantly decreased in chronic exposures, though there was no clear trend in acute exposures. Mg²⁺-ATPase activity increased significantly in acute exposures, but not in chronic ones. There were more significant alterations in acute exposure compared to chronic ones. There was no clear trend regarding Cu toxicity and its relationship with Ca²⁺, which may possibly be prompted by the compensatory mechanisms of the enzymes. It may be concluded that freshwater fish erythrocytes may face different degrees of more physiological stress from different waters.     

Acrosome Reaction of Sperm in the Mud Crab Scylla serrata as a Sensitive Toxicity Test for Metal Exposures

In order to test the sensitivity of the sperm cell of the mud crab Scylla serrata to heavy metals, the toxic effects of Ag⁺, Cd²⁺, Cu²⁺, and Zn²⁺ on the acrosome reaction (AR) were studied by artificially inducing the AR of sperm exposed to heavy metals, counting the AR rates by light microscopy, and observing structural changes in sperm by transmission electron microscopy. The AR in S. serrata occurs at two stages. The first stage (ARI) is the eversion of the subacrosomal material. The second stage (ARII) is the ejection of the acrosomal filament. The results showed the EC₅₀ values of the AR based on (ARI + ARII)% for Ag⁺, Cd²⁺, Cu²⁺, and Zn²⁺ were 10.02, 2.14, 13.69, and 2.21 μg/L, and the EC₅₀ values based on ARII % of Ag⁺, Cd²⁺, Cu²⁺, and Zn²⁺ were 1.96, 0.20, 1.46, and 0.34 μg/L. The order of toxicity is Cd²⁺ > Zn²⁺ > Cu²⁺ > Ag⁺ based on the percentage of reacted sperm at the second stage. Sperm cells exposed to heavy metals showed an increased rate of swelling, shape irregularities, and the acrosomal filament of some sperm cells was, crooked, ruptured, and even dissolved. The AR of the sperm cell from S. serrata is more sensitive to the tested heavy metals compared to sea urchin sperm cell toxicity tests.