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.     

POTASSIUM INCREASES INTRACELLULAR CALCIUM SIMULATING PROGESTERONE ACTION IN HUMAN SPERM

Progesterone (P) and zona pellucida are known to induce acrosome reaction in human sperm by increasing cytosolic calcium. High concentrations of potassium ions (K⁺) improve the rate of acrosome reaction in human sperm in vitro. This article determined whether the effect of K⁺ on the acrosome in human sperm is mediated by increasing intracellular calcium ([Ca²⁺]i). The effect of K⁺ on [Ca²⁺]i was examined by using Fura 2 as the fluorescent indicator. The effect of K⁺ and P on [Ca²⁺]i in sperm and the involvement of ion channels was compared. Motile sperm were collected by the swim-up method from semen of healthy volunteers and capacitated overnight in BWW containing 0.5% BSA. Incubation of capacitated sperm with different concentrations of potassium chloride (1.25-20 mM) resulted in dose-dependent increase in [Ca²⁺]i similar to that observed with P. The increase in [Ca²⁺]i by K⁺ and P was blocked by the addition of EGTA, a Ca²⁺ chelator. K+-induced change in [Ca²⁺] was not altered by the addition of dihydropyridine derivatives. The combined treatment of K+ (20 mM) and P (0.75 mug/mL) caused an additive effect on the increase in [Ca²⁺]i. It would appear that human sperm plasma membrane possess different Ca2+ channels responsive to P and K⁺.     

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.     

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⁺].     

Acute Toxicity of Potassium to the Adult Zebra Mussel Dreissena polymorpha

The acute toxicity of potassium (K⁺) to adult zebra mussels, Dreissena polymorpha, and the efficacy of using K⁺ to enhance the toxicity of a commercial biocide was examined. Mussels, 15–20 mm in total shell length, collected from Lake Ontario, were exposed to static concentrations of K⁺ for 3, 6, 12, and 24 h, and to a sublethal concentration of K⁺ prior to and during exposure to Clam-Trol^? CT-2 for 6, 12, and 24 h. Tests were conducted at ambient lake temperatures of 12°C and 22°C and mussels were subjected to a 96 h recovery period. Valve closure was inhibited in mussels exposed to sublethal as well as lethal concentrations of K⁺, resulting in mussels that were nonresponsive to tactile stimulation. The median effective concentration (ED₅₀) of K⁺ to induce nonresponsive mussels increased as the length of the recovery period was extended from 24 to 96 h, indicating that some nonresponsive mussels were capable of recovering 96 h after exposure to the K⁺ treatments. A recovery period duration of 96 h was critical in assessing mortality in mussels exposed to high K⁺ levels and the use of tactile stimulation to test for valve responsiveness was insufficient to identify mortality. The 24 h median lethal concentration (LC₅₀) of K⁺ at 22°C (400 mg/L) was found to be sixfold higher than the LC₅₀ reported by other investigators utilizing shorter recovery periods. The LC₅₀ of the biocide to mussels treated with K⁺ was not reduced, suggesting that the use of K⁺ to inhibit valve closure may not be useful in methods to control mussel infestations. Received: 25 March 1997/Accepted: 15 October 1997     

Control of the Biofouling Mollusc,Dreissena polymorpha (Bivalvia: Dreissenidae), with sodium hypochlorite and with polyquaternary ammonia and benzothiazole compounds

Small adult zebra mussels (Dreissena polymorpha), 2–8 mm valve length, collected from Lake St. Clair were exposed to a range of concentrations of three biocides in static, acute toxicity tests in the laboratory. Laboratory conditions (22°C; pH 7.8; water hardness 100 mg/L) were representative of midsummer conditions in the nearshore of Lakes Erie and St. Clair. Mussels actively colonized styrene test substrates which were transferred to three replicate, 1-L test vessels. Sodium hypochlorite was an effective biocide at concentrations exceeding 1.00 mg/L and resulted in complete mortality of mussels by 157 and 264 h at concentrations of 5.00 and 2.50 mg/L, respectively. Poly [oxyethylene (dimethylimino) ethylene (dimethylimino) ethylene dichloride] at 1,2,4 and 8 mg/l and (2-(thiocyanomethylthio) benzothiazole) at 0.5,1,2 and 4 mg/L resulted in 100% mortality at all concentrations in times ranging from 144 to 250 h and 110 to 192 h, respectively. Biocide concentration significantly affected the mean time of death for all three of the compounds tested. Mussel valve length had a significant positive effect on time of death in (2-(thiocyanomethylthio) benzothiazole) but only explained a maximum 18% of the variance. Resistance of these actively colonizing mussels to biocides was greater than that found by other laboratory studies, perhaps because of lowered handling stress in our experimental manipulations.     

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.     

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.     

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.     

Characterization of the RAGE-binding protein,Strongyloides venestatin,produced by the silkworm-baculovirus expression system

The receptor for advanced glycation end products (RAGE) recognizes Ca⁺⁺-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca⁺⁺-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca⁺⁺ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca⁺⁺-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes.     

Erythrocyte membrane (Na+, K+) and (Mg++, Ca++) activated adenosine triphosphatases in women using oral contraceptives--a preliminary report.

(Na⁺, K⁺) and (Mg⁺⁺, Ca⁺⁺) activated adenosine triphosphatases (Na⁺, K⁺ and Mg⁺⁺, Ca⁺⁺ ATPase, respectively) were studied in women who had used oral contraceptives (OC) for a period of 13 to 15 months. Erythrocyte and plasma Na⁺ and K⁺ were also determined in them. The values were compared with those obtained in age matched non-pregnant women and those in the third trimester of pregnancy. No significant differences could be seen in the activity of (Na+, K+) and (Mg++, Ca++) ATPases between the three groups. Erythrocyte as well as plasma Na+ and K+ contents (mEg/10¹² cells and mEq/litre plasma) in the women using OC were also in the normal range.     

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.     

Use of Quick,Easy, Cheap,Effective, Rugged & Safe (QuEChERS) and molecular imprinted polymer followed by gas chromatography with tandem mass spectrometry for the quantitative analysis of polycyclic aromatic hydrocarbons (PAH4) in complex health supplements

A gas chromatography-tandem mass spectrometry sensitive and selective method based on a combination of QuEChERS and subsequent molecularly imprinted polymer (MIP) technology was validated for the analysis of polycyclic aromatic hydrocarbons in complex dry extracts of Eleutherococcus senticosus, Salvia officinalis, Camellia sinensis, Zingiber officinale, Uncaria tomentosa, Humulus lupulus, Pinus sylvestris L., Spirulina maxima, propolis and royal jelly. The method has been optimized using gas chromatography coupled to tandem mass spectrometry. An additional sample treatment of the dry extracts, based on the combined use of MIP-SPE and QuEChERS, was required because of the strong matrix effect observed related to interferences affecting analyte quantification. Estimation of the method detection limit and quantification limit was carried out for validation. The LOQs were 0.2 ng g⁻¹ for benz[a]anthracene, 0.3 ng g⁻¹ for benzo[a]pyrene and 0.4 ng g⁻¹ for benzo[b]fluoranthene and chrysene; and the LODs were 0.07, 0.09 and 0.1 ng g⁻¹ respectively. Recoveries were in the range of 88.5 % for B[b]F, to 114 % for B[a]A, and % RSD was < 14 % in all cases. The method was applied in routine analysis to a wide variety of dry extracts from EU and non-EU manufacturers.     

Comparative Studies on the Toxicokinetics of Benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene in <Emphasis Type="Italic">Pinctada martensii</Emphasis> and <Emphasis Type="Italic">Perna viridis</Emphasis>

Research on the kinetics of Benzo[a]pyrene (B[a]P) bioaccumulation in the clam Pinctada martensii and mussel Perna viridis showed that the initial rate of uptake was directly related to the PAH concentrations in the ambient environment. The uptake and depuration rate constants were different at the four B[a]P exposure levels, which indicated that the toxicokinetic rate constants mainly depended on the exposure levels of pollutants to the environment. In addition, the uptake rate constants of B[a]P were higher than the depuration rate constants in the entire experiment. The comparison demonstrated that mussels release B[a]P more rapidly than clams. The bioconcentration factors (BCFs) of B[a]P varied from 3335 to 12892 in the clam and 2373–6235 in the mussel. These findings on the bioaccumulation kinetics for petroleum hydrocarbons, in association with the critical body residue, will be valuable when choosing sensitive organisms to assess the potential ecotoxicological risk to the marine environment.     

Effects of season,stock, and laboratory protocols on survival of zebra mussels (Dreissena polymorpha) in bioassays

A series of experiments were conducted to determine the effects of maintenance method (fed or starved), stock location, season, mussel size, and rate of acclimation to temperature on the responses (mortality) of zebra mussels in bioassays. Mussels maintained on a diet of crushed Chlorella are more tolerant to Bayer 73^® and more sensitive to sodium hypochlorite than starved mussels. Variability in LC50s of zebra mussels is high during the first 60 days in the laboratory, after which the resistance of mussels to both hypochlorite and Bayer 73^® declines with reductions in body condition. Zebra mussels collected during the early summer and late fall are more tolerant to both hypochlorite and Bayer 73^®. There is significant variation in tolerances to biocides depending on the stock, such that stocks from locations with more degraded water quality have increased tolerances. Acclimating mussels from 4 to 20°C at rates of 2 and 10°C d^–1 does not significantly affect tolerance to biocides. In general, LC50s of mussels vary by only 2–3×, suggesting that mussels from any location, any season, and maintained under any maintenance protocol can be used in range-finding tests. Comparisons of results among studies requires knowledge of mussel stock, collection season, and laboratory maintenance protocols.     

Quantitative Biomonitoring of PAHs Using the Barnes Mussel (<Emphasis Type="Italic">Elliptio complanata</Emphasis>)

The elimination rate constants (k ₂) of nine polycyclic aromatic hydrocarbons (PAHs) were examined for the freshwater mussel Elliptio complanata. The concentrations of fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, and indeno[1,2,3-c,d]pyrene revealed a significant inverse relationship with time and their k ₂ values ranged from 0.10 to 0.22 day⁻¹. The k ₂ values of these significantly cleared PAHs were similar to k ₂ values observed for nonmetabolized organochlorines in mussels previously reported in the literature. The inverse relationship between k ₂ and K _ow provides evidence that the nine PAHs were being passively eliminated from the mussels and that they can be used to calibrate the mussel as a quantitative biomonitor. A general expression relating elimination rate constants and chemical K _ow is derived for hydrophobic contaminants in E. complanata. The k ₂ versus log K _ow regression equation for mussels developed herein was similar to other studies documenting the elimination of PCBs and PAHs in a number of bivalve species. Received: 13 August 2001/Accepted: 25 April 2002     

Effect of aflatoxin B1, ochratoxin and rubratoxin B on a protozoan,Tetrahymena pyriformis HSM

Summary Rubratoxin B is more inhibitory to the growth ofT. pyriformis than is either aflatoxin B₁ or ochratoxin. Only a marginal effect on cell respiration was exerted by these mycotoxins. The presence of the divalent ions, Mg⁺⁺, Ca⁺⁺ and Fe⁺⁺, did not reduce the toxicity of rubratoxin B. A bioassay sensitive to 1 to 5 g/ml rubratoxin B has been developed employingT. pyriformis.     

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.     

Cadmium Concentration and Subcellular Distribution in Organs of the Mussel Crenomytilus Grayanus from Upwelling Regions of Okhotsk Sea and Sea of Japan

Cadmium (Cd) accumulation and subcellular distribution in the digestive gland and kidney of mussel Crenomytilus grayanus from naturally Cd elevated areas was studied. Mussels were collected from three sites of Okhotsk Sea and Sea of Japan: control area (site 1), seasonal upwelling region (site 2), and stationary upwelling region (site 3). Mussel from site 3 was shown to accumulate a significantly increased Cd concentration in the digestive gland and an extremely high Cd concentration (1780 ± 732 μg g⁻¹ dry weight) in the kidney. Cd was mainly sequestered into the kidney cytosol of the mussels from both upwelling regions and control sites (73% to 96%). However, digestive gland cytosol bound about 55% of Cd for the mussels from sites 1 and 2 and only 22.4% of Cd for the mussel from site 3. In the organs of the mussels from upwelling regions, the most of cytosolic Cd was associated with the protein fraction corresponding to molluscan metallothioneins. MTLP was isolated from kidney cytosol of the mussels from site 3 (powerful upwelling regions) by gel filtration chromatography, whereas in the organs studied of the mussels from other sites, MTLP content was below the detection limit.     

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.