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⁺].     

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⁺.     

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.     

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.     

d-Allulose Inhibits Ghrelin-Responsive,Glucose-Sensitive and Neuropeptide Y Neurons in the Arcuate Nucleus and Central Injection Suppresses Appetite-Associated Food Intake in Mice

d-allulose, a rare sugar, has sweetness with few calories. d-allulose regulates feeding and glycemia, and ameliorates hyperphagia, obesity and diabetes. All these functions involve the central nervous system. However, central mechanisms underlying these effects of d-allulose remain unknown. We recently reported that d-allulose activates the anorexigenic neurons in the hypothalamic arcuate nucleus (ARC), the neurons that respond to glucagon-like peptide-1 and that express proopiomelanocortin. However, its action on the orexigenic neurons remains unknown. This study investigated the effects of d-allulose on the ARC neurons implicated in hunger, by measuring cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single neurons. d-allulose depressed the increases in [Ca²⁺]_i induced by ghrelin and by low glucose in ARC neurons and inhibited spontaneous oscillatory [Ca²⁺]_i increases in neuropeptide Y (NPY) neurons. d-allulose inhibited 10 of 35 (28%) ghrelin-responsive, 18 of 60 (30%) glucose-sensitive and 3 of 8 (37.5%) NPY neurons in ARC. Intracerebroventricular injection of d-allulose inhibited food intake at 20:00 and 22:00, the early dark phase when hunger is promoted. These results indicate that d-allulose suppresses hunger-associated feeding and inhibits hunger-promoting neurons in ARC. These central actions of d-allulose represent the potential of d-allulose to inhibit the hyperphagia with excessive appetite, thereby counteracting obesity and diabetes.     

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.     

Naringenin stimulates cholecystokinin secretion in STC-1 cells

BACKGROUND/OBJECTIVES

Cholecystokinin (CCK), a hormone or neuropeptide, is secreted in response to intraluminal nutrients by enteroendocrine I-cells of the intestine and has important physiological actions related to appetite regulation and satiety. The stimulation on CCK secretion from the intestine is of potential relevance for body weight management. Naringenin (4'',5,7-trihydroxyflavanone) and its glycoside naringin (naringenin 7-rhamnoglucoside) have been reported to have many biological functions. In the current study, we investigated the question of whether naringenin and naringin could stimulate CCK secretion and then examined the mechanisms involved in CCK release.

MATERIALS/METHODS

STC-1 cells were used as a model of enteroendocrine cells. CCK release and changes in intracellular Ca²⁺ ([Ca²⁺]_i) were measured after incubation of cells with naringenin and naringin for 1 h.

RESULTS

Naringenin caused significant (P < 0.05) stimulation of CCK secretion, but naringin did not. In addition, regarding the secretory mechanisms, naringenin-induced CCK secretion involved increases in [Ca²⁺]_i, influx of extracellular Ca²⁺, at least in part, and activation of TRP channels, including TRPA1.

CONCLUSION

Findings of this study suggest that naringenin could have a role in appetite regulation and satiety.     

Modulation of oxidative stress,apoptosis, and calcium entry in leukocytes of patients with multiple sclerosis by Hypericum perforatum

Objectives

Hypericum perfortarum (HP, St John's wort) is a modulator of Ca²⁺ entry in neutrophils and it may modulate intracellular free Ca²⁺ ([Ca²⁺]_i) entry in leukocytes of patients with multiple sclerosis (MS). We investigated effects of HP on oxidative stress, apoptosis, and [Ca²⁺]_i concentrations in serum and leukocytes of patients with MS.

Methods

Neutrophils of nine newly diagnosed MS patients and nine healthy subjects within four subgroups were used in the study. The first group was a control; the second group was patients with MS. The neutrophils from patient group were incubated non-specific TRPM2 channel blocker (2-APB), voltage-gated calcium channel blockers, verapamil and diltiazem (V + D) with HP before N-formyl-L-methionyl-L-leucyl-L-phenylalanine stimulation, respectively.

Results

Neutrophil and serum lipid peroxidation, neutrophil apoptosis and [Ca²⁺]_i levels in patients with MS were higher than in control although their levels were decreased by HP, 2-APB, and V + D incubations. The modulator role of V + D in MS and MS + HP groups was higher than in the 2-APB group. Neutrophilic glutathione peroxidase (GSH-Px) and serum vitamin A and E concentrations were lower in the MS group than in control. However, the neutrophil GSH-Px activity was increased by HP incubation. The neutrophil reduced glutathione, serum vitamin C and β-carotene concentrations did not change in control and patients.

Discussion

We observed that HP-induced protective effects on oxidative stress and [Ca²⁺]_i concentrations by modulating transient receptor potential and voltage gated calcium channel in the patients with MS. Thus, it may provide useful treatment of neutrophil activity in the patients.     

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 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     

Continuous osmotic minipump infusion of alcohol into brain decreases brain [Mg2+] and brain bioenergetics and enhances susceptibility to hemorrhagic stroke: An in vivo 31P-NMR study

³¹P-NMR spectroscopic studies were performed in vivo on brains of rats chronically infused for 7 and 14 days with 30% ethanol (in the third cerebral ventricle). Peripheral blood alcohol concentration (BAC) rose to between 16.5–30.5 mg/dl. Brain intracellular free Mg²⁺ ([Mg²⁺]_i) fell 33–39%, brain mitochondrial cytosolic phosphorylation potential (CPP) fell 31–48%, and brain phosphocreatine (PCr) fell approximately 15%; however, neither brain intracellular free hydrogen ion concentration (pHi) nor brain intracellular inorganic phosphate (Pi) were affected significantly by the chronic release of ethanol from the brain implants. Correlations were found between [Mg²⁺]_i and [PCr] and between [Mg²⁺]_i and CPP. Although brain free [MgADP] was not affected, [MgATP] fell by almost 20% accompanied by a 35–40% rise in free [ADP]. Interestingly, 14-day surgical implantation of 0.9% sterile saline into the third cerebral ventricle was associated with a 20% fall in brain [Mg²⁺]_i and a 35% fall in CPP; however, PCr, ATP, or pHi was not significantly altered. Systemic administration of 4 g/kg ethanol into the 7- and 14-day chronic ethanol animals resulted in a 9- and 12-fold increase in hemorrhagic stroke mortality compared to naive, control rats. Eating habits, grooming, gait and arterial blood pressure were not affected by the chronic brain implantation of ethanol. These data lend support to the notion, primarily based on epidemiologic evidence, that chronic exposure to alcohol can pose a high risk for hemorrhagic stroke. Our alcohol pump-implanted rats also might provide a new model of slow, moderate alcohol intoxication.     

Human Follicular Fluid Stimulates the Motility of Washed Human Sperm

Human follicular fluid (hFF) was collected by laparoscopic oocyte pickup during IVF to evaluate the effect of hFF on human sperm motility with a transmembrane migration method. Freshly ejaculated human sperm were washed with phosphate buffered saline (PBS) and mixed with either PBS or hFF. Amplitude of motility increases were 38% and 72% in washed fertile sperm and washed asthenozoo-spermic sperm when individual control motility was considered to be 100%. The stimulatory effect of hFF was lost when preheated at 100°C for 30 minutes. hFF collected from mature follicles stimulated sperm motility better than that collected from intermediate or immature follicles. hFF did not stimulate the motility of unwashed sperm in freshly ejaculated human semen. A heat labile factor(s) in hFF may stimulate the motility of washed human sperm. Whether this factor could be used to improve the success rate of IVF and artificial insemination awaits further investigation.     

Ischemia-induced endothelial cell swelling and mitochondrial dysfunction are attenuated by cinnamtannin D1, green tea extract,and resveratrol in vitro

Abstract

Polyphenols possess antioxidant and anti-inflammatory properties. Oxidative stress (OS) and inflammation have been implicated in the pathogenesis of cytotoxic brain edema in cerebral ischemia. In addition, OS and pro-inflammatory cytokines also damage the endothelial cells and the neurovascular unit. Endothelial cell swelling may contribute to a leaky blood–brain barrier which may result in vasogenic edema in the continued presence of the existing cytotoxic edema. We investigated the protective effects of polyphenols on cytotoxic cell swelling in bEND3 endothelial cultures subjected to 5 hours oxygen-glucose deprivation (OGD). A polyphenol trimer from cinnamon (cinnamtannin D1), a polyphenol-rich extract from green tea, and resveratrol prevented the OGD-induced rise in mitochondrial free radicals, cell swelling, and the dissipation of the inner mitochondrial membrane potential. Monocyte chemoattractant protein (also called CCL2), a chemokine, but not tumor necrosis factor-α or interleukin-6, augmented the cell swelling. This effect of monochemoattractant protein 1-1 was attenuated by the polyphenols. Cyclosporin A, a blocker of the mitochondrial permeability transition pore, did not attenuate cell swelling but BAPTA-AM, an intracellular calcium chelator did, indicating a role of [Ca²⁺]_i but not the mPT in cell swelling. These results indicate that the polyphenols reduce mitochondrial reactive oxygen species and subsequent cell swelling in endothelial cells following ischemic injury and thus may reduce brain edema and associated neural damage in ischemia. One possible mechanism by which the polyphenols may attenuate endothelial cell swelling is through the reduction in [Ca²⁺]_i.     

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.     

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.     

Alcohol-induced vascular damage of brain is ameliorated by administration of magnesium

Ethanol ingestion can cause irreversible neuronal and vascular damage in the brain and stroke-like events. Using an intact in vivo rat brain (pial) model, TV image-intensification, cultured cerebral vascular muscle cells, digital-image analysis, and a novel Mg²⁺ ion-selective electrode to measure extracellular ionized Mg²⁺, studies were designed to determine whether: 1) perivascular or systemic administration (IV or intra-arterial) of magnesium aspartate HCl (MgA) exert vasodilator effects on arterioles (65–130 μm o.d.) and venules (60–135 μm); 2) nonvasodilator doses of MgA could modify vascular spasms induced by BaCl₂ and ethanol; 3) nonvasodilator doses of MgA could ameliorate or prevent the cerebral vascular damage induced by high doses of ethanol; and 4) ethanol depletes cerebral vascular muscle of intracellular Mg ions ([Mg²⁺]_i). Perivascular application of MgA (0.01–100 μmol) produced dose-dependent vasodilatation of cerebral arterioles and venules; arterioles yielded greater vasodilator responses compared to venules. Nonvasodilator doses of Mg (1.0, 4.0 μmol/min), administered IV or intra-arterially, into a branch of the internal carotid artery, prevented: 1) the spasmogenic actions of ethanol and Ba²⁺; and 2) the vasculotoxic actions (rupture of postcapillary venules and focal hemorrhages) of ethanol. In addition, ethanol depleted cerebral vascular muscle cells of [Mg²⁺]_i; blood levels of ionized Mg²⁺ rose after IP ethanol. Despite the fact that systemic infusion of low nonvasodilator doses did not result in dilatation of the pial arterioles and venules, plasma total and ionized Mg rose 18–230%, depending upon doses of MgA and time of plasma sampling. These data support the idea that Mg²⁺ can act as a local vasodilator on brain microvessels and possess antispasmodic properties on brain arterioles and venules. In addition, our results indicate that Mg may posses some unique cerebral vascular protective properties against the vasculotoxic effects of ethanol. Lastly, these findings suggest ethanol-induced cerebrovasospasm and vascular damage appear to be associated with a rapid loss of [Mg²⁺]_i from cerebral vascular muscle cells.     

Subcellular distribution of Cd and Pb in earthworm Eisenia fetida as affected by Ca2+ ions and Cd-Pb interaction

A semi-static solution culture method was used to study the effects of Ca²⁺ supply and interaction of Cd–Pb on the subcellular distribution of Cd and Pb in earthworm Eisenia fetida. The subcellular distribution of Cd and Pb was shown to be metal specific. About 80% of the Cd was distributed in the cytosol (fraction G), and only about 20% of total Cd was found in the tissue and cell membrane (fraction E) and the microsomes (fraction F). Nearly 50% of the Pb was rich in the tissue and cell membrane (fraction E). The supply of Ca²⁺ ions significantly decreased Cd concentration in the cytosol (fraction G) and the whole tissue of the E. fetida. At the subcellular level, the addition of Pb²⁺ ions significantly decreased the Cd percentage associated with fraction G from 83.7% to 58.4% and increased fraction E from 10.7% to 34.0%, respectively.     

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.     

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells

Several recent studies have demonstrated that the direct precursor of vitamin D₃, the calcifediol [25(OH)D₃], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D₃ by binding the VDR could have a vitamin’s D₃ activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca²⁺ mobilization in response to 25(OH)D₃. Our result reveals the ability of 25(OH)D₃ to activate rapid, non-genomic pathways, such as an increase of intracellular Ca²⁺ levels, similar to what observed with the biologically active form of vitamin D₃. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca²⁺ concentration as a result of exposure to 10⁻⁵ M of 25(OH)D₃. In this work, we show for the first time the in vitro ability of 25(OH)D₃ to induce a rapid increase of intracellular Ca²⁺ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D₃ and its role in endocrine system.