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.     

Ethanol and voltage- or receptor-mediated increases in cytosolic Ca2+ in brain cells.

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. Different mechanisms for stimulating increased free intracellular Ca2+ concentrations [( Ca2+]i) were examined in the absence and presence of ethanol. KCl, carbachol, and kainate concentration-dependently increased [Ca2+]i. Quisqualate also elevated [Ca2+]i but did not produce clear concentration-dependent increases. KCl, carbachol, and quisqualate responses reached peak levels within 10-30 s and then desensitized within 90 s. However, kainate-stimulated increases in [Ca2+]i plateaued and did not decline after 90 s. Of these different [Ca2+]i-mediated processes, only 60 mM KCl stimulation was significantly inhibited by 100 mM ethanol, while lower KCl concentrations were not affected. Carbachol-induced release of intracellular Ca2+ and activation of non-NMDA (i.e., kainate, quisqualate) excitatory amino acid receptor-operated cation channels were also not significantly inhibited by 100 mM ethanol. Thus, in acutely dissociated brain cells from newborn rats, only Ca2+ influx via voltage- and, as reported previously, NMDA-operated Ca2+ channels were sensitive to ethanol inhibition.     

Low doses of bisphenol A and diethylstilbestrol impair Ca2+ signals in pancreatic alpha-cells through a nonclassical membrane estrogen receptor within intact islets of Langerhans

Glucagon, secreted from pancreatic alpha-cells integrated within the islets of Langerhans, is involved in the regulation of glucose metabolism by enhancing the synthesis and mobilization of glucose in the liver. In addition, it has other extrahepatic effects ranging from lipolysis in adipose tissue to the control of satiety in the central nervous system. In this article, we show that the endocrine disruptors bisphenol A (BPA) and diethylstilbestrol (DES), at a concentration of 10(-9) M, suppressed low-glucose-induced intracellular calcium ion ([Ca2+]i) oscillations in alpha-cells, the signal that triggers glucagon secretion. This action has a rapid onset, and it is reproduced by the impermeable molecule estradiol (E2) conjugated to horseradish peroxidase (E-HRP). Competition studies using E-HRP binding in immunocytochemically identified alpha-cells indicate that 17beta-E2, BPA, and DES share a common membrane-binding site whose pharmacologic profile differs from the classical ER. The effects triggered by BPA, DES, and E2 are blocked by the G alpha i- and G alpha o-protein inhibitor pertussis toxin, by the guanylate cyclase-specific inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, and by the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester. The effects are reproduced by 8-bromo-guanosine 3',5'-cyclic monophosphate and suppressed in the presence of the cGMP-dependent protein kinase inhibitor KT-5823. The action of E2, BPA, and DES in pancreatic alpha-cells may explain some of the effects elicited by endocrine disruptors in the metabolism of glucose and lipid.     

Inhibition of Ca(2+) channels in mouse spermatogenic cells by male antifertility compounds from Tripterygium wilfordii Hook. f

The male antifertility effect of a water-chloroform extract (GTW) from the root xylem of Tripterygium wilfordii has attracted worldwide interest. In the present study, by using whole-cell recording, the effects of GTW and two isolated monomers from GTW, demethylzeylasteral and L-epicatechin, on the T-type Ca(2+) channels in mouse spermatogenic cells were investigated. The results showed that each of them concentration-dependently and partially reversibly inhibited T-type Ca(2+) current in the cells. The IC(50) of GTW and demethylzeylasteral were approximate, while L-epicatechin inhibited the channels at a much higher concentration. The voltage dependence of the inhibitory effect and the changes in activation and inactivation time constants after application of these compounds were also examined. These data suggest that the inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of these compounds.