Ceramide inhibits L-type calcium channel currents in GH3 cells

In this study, we investigated the effect of ceramide on the L-type Ca2+ channel (L-channel) in GH3 cells. We found that C6-ceramide, but not C6-dihydroceramide, the inactive analogue, had an inhibitory effect on BayK 8644-stimulated GH release. Using patch clamp analysis, C6- and C2-ceramide, but not C6-dihydroceramide, were found to inhibit the L-channel current. Increasing intracellular ceramide level with sphingomyelinase also inhibited the L-channel current. The inhibitory effect of ceramide on the L-channel current was attenuated by calphostin C, a myristolated pseudosubstrate protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor. Combined treatment with lavendustin A and the myristolated PKC inhibitor blocked the effect of ceramide on the L-channel current. These results indicate that ceramide, a lipid messenger of the sphingomyelin pathway, is an important regulator of the L-channel in GH3 cells and both tyrosine kinase and PKC are involved in this effect of ceramide.     

Control of intracellular calcium redistribution by guanine nucleotides and inositol 1,4,5-trisphosphate in permeabilized GH4C1 cells

In GH4C1 rat pituitary cells, a GTP-binding protein appears to be involved in signal transduction between the TRH receptor and phospholipase C. In certain other cell types, another role for GTP has been reported, namely regulation of Ca2+ translocation from one intracellular pool to another. Using digitonin-permeabilized GH4C1 cells, we have investigated whether an analogous process occurs in pituitary cells. In permeabilized GH4C1 cells, TRH, inositol 1,4,5-trisphosphate (IP3), and nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and 5'-guanylyl imidodiphosphate each increased free Ca2+ concentration [( Ca2+]). Unlike several other systems, GTP did not increase [Ca2+]. Guanosine 5'-O-(2-thiodiphosphate) inhibited Ca2+ release induced by both TRH and GTP gamma S. Heparin abolished IP3-induced Ca2+ release but did not prevent Ca2+ release induced by TRH or GTP gamma S, suggesting a mechanism for their actions that did not depend solely on IP3 production. Neomycin inhibited GTP gamma S-induced Ca2+ release, but it did not prevent TRH- or IP3-induced Ca2+ release. In the absence of ATP, GTP gamma S did not elevate [Ca2+], although TRH and IP3 did, suggesting that ATP-dependent sequestration of Ca2+ was necessary for the action of GTP gamma S in this system, but not for TRH and IP3. Repeated additions of IP3 resulted in an attenuation of the response to IP3- GTP gamma S, which itself increased [Ca2+] after IP3 attenuation, restored the attenuated Ca2+ response to IP3. We conclude that, in permeabilized GH4C1 cells, GTP gamma S as well as TRH cause intracellular Ca2+ release; however, their mechanisms of action are, at least in part, distinct. Furthermore, the IP3-depletable Ca2+ pool can be refilled from a GTP gamma S-sensitive compartment via Ca2+ transport through the cytosol.     

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

Aims/hypothesis

The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulin-secretory function of SYN-3, which is particularly abundant in SGs, is unclear.

Methods

Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM).

Results

Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG–SG fusion, which was confirmed by electron microscopy.

Conclusions/interpretation

In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG–SG fusion that contributes to biphasic GSIS.     

Recruitment of calcium from intracellular stores does not occur during the expression of large spontaneous calcium oscillations in GH(3) cells and lactotropic cells in primary culture

We used simultaneous electrophysiological and intracellular calcium microfluorometry recordings to directly test for the presence of a calcium-induced calcium release mechanism in individual GH(3) cells and cells of a lactotrope-enriched primary culture. In voltage-pulse experiments, extending the duration of a depolarizing voltage-pulse increased intracellular calcium concentration ([Ca(2+)](i)), but we did not observe any evidence for recruitment of intracellular calcium stores. Furthermore, depletion of intracellular calcium stores with thapsigargin or caffeine did not change the calculated calcium buffer capacity of the cells. In current-clamp experiments, we observed elevations in [Ca(2+)](i) in response to spontaneous action potentials. These [Ca(2+)](i) responses were not inhibited by thapsigargin or caffeine. We did find a significant correlation between the magnitude of spontaneous [Ca(2+)](i) increases and action potential duration. We conclude that intracellular calcium stores are not released during the spontaneous [Ca(2+)](i) oscillations observed in these cells, and that the magnitude of [Ca(2+)](i) oscillations is a direct result of extracellular calcium influx that is determined in part by action potential duration.     

Calcium influx is not required for TRH to elevate free cytoplasmic calcium in GH3 cells

TRH stimulation of prolactin secretion is thought to be mediated by an elevation of free cytoplasmic Ca2+. However, whether TRH-induced influx of extracellular Ca2+ is required to elevate cytoplasmic Ca2+ remains controversial. We measured cytoplasmic free Ca2+ concentration in GH3 cells with an intracellularly trapped fluorescent indicator, Quin 2. In unstimulated cells incubated in medium containing 1.5 mM Ca2+, cytoplasmic free Ca2+ concentration was 118 +/- 18 nM (mean +/- SD). TRH (1 microM) caused a rapid transient elevation of free cytoplasmic Ca2+ to a level estimated to be at least 500 nM. High extracellular K+, which induces extracellular Ca2+ influx, caused an elevation of free cytoplasmic Ca2+ which was greater and longer in duration that that caused by TRH. When cells were incubated in medium containing 3 mM EGTA, the K+ depolarization-induced increase in free cytoplasmic Ca2+ was abolished. By contrast, the TRH-induced increase was not affected by incubating cells in medium with 3 mM EGTA, or high K+, or both; incubation of cells in medium with EGTA and high K+ abolishes the electrochemical driving force for Ca2+ influx. These data demonstrate that Ca2+ influx is not required for TRH-induced elevation of free cytoplasmic Ca2+ in GH3 cells. We conclude that in GH3 cells TRH induces an elevation of free cytoplasmic Ca2+ leading to stimulated prolactin secretion by mobilizing cellular Ca2+.     

Evidence that actin disassembly is a requirement for matrix metalloproteinase secretion by sinusoidal endothelial cells during cold preservation in the rat.

Cold preservation induces the secretion of matrix metalloproteinases (MMPs) by hepatic sinusoidal endothelial cells (SECs). These enzymes are important mediators of cold preservation injury. The purpose of this study was to determine if low temperature caused actin disassembly in SECs and whether actin disassembly was required for secretion of MMPs under these conditions. To establish the basis of interpreting the effect of low temperature, isolated SECs were exposed to cytochalasin B with or without pretreatment with phalloidin. Cytochalasin B produced actin disassembly and resulted in the secretion of MMPs. Both were retarded by phalloidin pretreatment. Low temperature (4 degrees C) also induced actin disassembly and MMP secretion and pretreatment with phalloidin again retarded actin disassembly and MMP secretion. Cycloheximide had no effect on these results. Actin disassembly began with 30 minutes of exposure of isolated SECs to cold and reached a final state at 8 hours, at which time no actin stress fibers were visible, and the normally fusiform SECs were fully rounded. Increased MMP activity in the supernatant was also present at 30 minutes and continued to rise sharply in the first hour; thereafter the rate of rise diminished. The study shows that secretion of MMPs during cold preservation is dependent on the induction of actin disassembly by low temperature. The rapid appearance of increased MMP activity after exposure to cold and the studies using cycloheximide indicate that the MMPs originate from preformed MMPs rather than newly synthesized MMPs.     

Benzodiazepines modulate voltage-sensitive calcium channels in GH3 pituitary cells at sites distinct from thyrotropin-releasing hormone receptors

Benzodiazepines (BZs) have been shown to modulate voltage-sensitive Ca2+ channels in a number of neuronal and nonneuronal cell types and to competitively antagonize TRH binding to receptors on cells of the nervous system and anterior pituitary gland. Because interaction of TRH with its receptor is known to cause enhanced influx of Ca2+ through voltage-sensitive channels in rat pituitary GH3 cells, it was determined whether BZs and TRH were interacting with the same binding site on these cells. The potencies of three BZs, Ro5-4864, diazepam (DZP), and chlordiazepoxide (CDE), were compared as modulators of Ca2+ channels and as inhibitors of TRH binding in GH3 cells. Modulation of Ca2+ channel activity was measured as the inhibition by BZs of K+ depolarization-induced Ca2+ influx using intracellularly trapped quin 2 or 45Ca2+ uptake. The three BZs caused dose-dependent inhibition of Ca2+ influx with an order of potency of Ro 5-4864 greater than DZP greater than CDE. In contrast, the order of potency of the three BZs to inhibit [3H]TRH binding was CDE greater than DZP much greater than Ro 5-4864. The concentrations of BZs needed to inhibit Ca2+ influx and TRH binding were in the micromolar range. These data show that BZs can modulate Ca2+ channel activity in endocrine cells and that these sites are distinct from those that modulate TRH binding on pituitary cells.     

17 beta-Estradiol regulates prolactin secretion but not cell proliferation of GH3B6 cells in chemically defined medium

The effects of 17 beta-estradiol on proliferation and prolactin secretion by a clonal rat pituitary cell, GH3B6, have been investigated in a chemically defined medium (Ham's F12, transferrin, insulin and parathyroid hormone). Under these conditions, 17 beta-estradiol alone (5 X 10(-14)-5 X 10(-9) M) was not a mitogen for GH3B6 cells as shown by cell counts and DNA measurements. However, it slowed down the drop in prolactin production induced by culture in this chemically defined medium. After 3 and 7 days it stimulated PRL production up to 5 times in a dose-dependent manner. This secretory response was abolished by tamoxifen, and not mimicked by progesterone, testosterone or dexamethasone. The thyroliberin-induced stimulation of prolactin production occurred more rapidly (24 h) than and was additive to the 17 beta-estradiol response, suggesting that these two factors act through independent mechanisms. PRL neosynthesis in culture was attested to by the incorporation of [35S]methionine (5 h) into immunoprecipitable prolactin. Neither 17 beta-estradiol nor thyroliberin increases specifically prolactin radioactivity, although they strongly decreased the specific activity of prolactin in both cells and medium. This suggests that they mostly acted by decreasing PRL turnover rate.     

Changes in the actin cytoskeleton of cardiac capillary endothelial cells during ischaemia and reperfusion: the effect of phalloidin on cell shape

A reduction in capillary dimensions has been demonstrated in postischaemic reperfusion in the heart. The aim of this study was to demonstrate that in ischaemia and ischaemia followed by reperfusion, the change in shape of the constituent endothelial cells can be inhibited by phalloidin which stabilises the actin microfilament system. Isolated, perfused rat hearts were made globally ischaemic both with and without reperfusion and in the presence or absence of phalloidin. Changes in ischaemic endothelial cell dimensions were quantified by measuring whole capillary and luminal cross-sectional areas, abluminal and luminal membrane lengths. The distribution of beta-actin within the endothelial cells was determined by immunocytochemistry. In control hearts, beta-actin is distributed throughout the endothelium with a slight increase towards the luminal membrane. In ischaemia, this was more marked and other patterns of actin distribution were also observed. After reperfusion, a 'double ring' of actin could be distinguished. With phalloidin, the actin staining was more regular and the ring pattern was not observed. Morphometry showed that phalloidin was more effective in reducing endothelial cell shape change after reperfusion than after ischaemia alone. We conclude that endothelial cell shape change on reperfusion can be modified by agents which target the contractile proteins.     

Tetracaine, propranolol and trifluoperazine inhibit thyrotropin releasing hormone-induced prolactin secretion from GH3 cells by displacing membrane calcium: further evidence that TRH acts to mobilize cellular calcium

TRH stimulation of prolactin release from GH3 cells is associated with loss of cellular Ca2+. Chlortetracycline (CTC), a fluorescent probe of Ca2+ in biological membranes, was previously employed to monitor indirectly changes in membrane Ca2+ in GH3 cells. Tetracaine, propranolol and trifluoperazine, agents that are known to displace Ca2+ from biological membranes, were utilized to demonstrate more rigorously that TRH affects cellular membrane Ca2+ in GH3 cells. Tetracaine (1 mM), propranolol (1 mM), and trifluoperazine (0.03 mM) inhibited basal and TRH-stimulated prolactin release, decreased cellular 45Ca2+ content and decreased cell-associated CTC fluorescence. Most importantly, these agents abolished the decrease in CTC fluorescence induced by TRH. These data suggest that tetracaine, propranolol and trifluoperazine displace membrane Ca2+ in intact GH3 cells and offer further evidence that TRH acts to mobilize cellular Ca2+ from a membrane-bound pool(s) during stimulation of GH3 cells.     

Changes in calcium, 25(OH) vitamin D3 and other biochemical factors during pregnancy

INTRODUCTION AND AIMS: Calcium and vitamin D play major roles in calcium homeostasis and skeletal development, especially during pregnancy. This study was conducted to determine changes in calcium, 25 hydroxy [25(OH)] vitamin D3 and other biochemical factors (PTH, osteocalcin, alkaline phosphatase, magnesium, phosphorus) related to calcium homeostasis and bone turnover during pregnancy and compare the values to those of non-pregnant women. MATERIALS AND METHODS: In a cohort study, 48 pregnant women, in their first trimester of pregnancy (12+/-2.7 weeks), from 5 prenatal care centers, and 47 non-pregnant women randomly selected from the Tehran Lipid and Glucose Study (TLGS) population were enrolled. These pregnant women were followed in their second (26+/-1.9 weeks) and third trimesters (37+/-3.2 weeks) of pregnancy. Samples were drawn from June 2002 to March 2003. Including criteria were healthy women with no background of disease. Women using photo protection and calcium and vitamin D supplementation were excluded. A questionnaire was used to obtain demographic information for both groups. Venous blood samples were taken after 12-14 h of overnight fasting to measure serum calcium, phosphorus, magnesium, alkaline phosphatase, PTH, 25 (OH) vitamin D3 and serum osteocalcin levels. The repeated measures analysis of variance and t-test were used for statistical analysis. Data were matched for age and weight in both the case (in the first trimester) and control groups. RESULTS: Significant differences were found in the mean serum levels of osteocalcin and alkaline phosphatase between the three trimesters of pregnancy (p< 0.001). Osteocalcin was significantly higher in the first trimester as compared to second and third trimesters of pregnancy. Alkaline phosphatase was significantly lower in the first trimester as compared to the second and third trimesters of pregnancy and their controls. There was also a significant difference in osteocalcin in the second and third trimesters and alkaline phosphatase in the first and third trimesters of pregnancy in comparison to the control group. The mean values of osteocalcin were 12.7+/-8.5, 8.1+/-6.9, 5.6+/-5.0 and 13.9+/-7.9 ng/ml, respectively, and mean values for alkaline phosphatase were 115+/-38, 125+/-37, 174+/-61 and 134+/-35.0 Iu/l, respectively. In the first trimester, alkaline phosphatase was lower and osteocalcin was higher than in the second and third trimesters. In the first trimester of pregnancy, 20 and 40% of women had 25(OH) vitamin D3 < 10 and < 20 ng/ml, respectively, and 19% of women had serum calcium levels < 8.6 mg/dl. CONCLUSION: 60% of women in the first trimester, 48% in the second and 47% in the third trimester had either severe or moderate vitamin D deficiency. It is recommended that the importance of calcium supplements with vitamin D in pregnant women be stressed for these individuals.     

Endoplasmic reticulum-through-Golgi transport assay based on O-glycosylation of native glycophorin in permeabilized erythroleukemia cells: role for Gi3.

An assay for endoplasmic reticulum (ER)-through-Golgi transport has been developed in streptolysin O-permeabilized murine erythroleukemia (MEL) cells. The reporter proteins are metabolically labeled native murine glycophorins, which display a distinctive shift in electrophoretic mobility after acquisition of O-linked oligosaccharides. The O-linked sugars are acquired at a site distal to a brefeldin A block, presumably in a cis Golgi compartment, and sialylation occurs in middle and/or trans Golgi compartments. In permeabilized cells supplemented with cytosolic proteins and an ATP-generating system, 20-50% of the radiolabeled precursor glycophorins can be converted to the mature, sialylated form. This maturation process is ATP- and cytosol-dependent and is blocked by guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). Electron microscopy of permeabilized MEL cells shows retention of ER elements, stacked Golgi cisternae, free polysomes, and other subcellular components. In the presence of GTP[gamma S], dilated vesicles accumulate around the Golgi stacks. Antisera to the carboxyl terminus of the Golgi resident alpha subunit of Gi3 inhibit maturation of glycophorin. To our knowledge, a transport assay utilizing O-glycosylation of an endogenous protein as a monitor of ER-through-Golgi traffic in permeabilized cells has not been reported previously. Furthermore, the data provide evidence for heterotrimeric GTP-binding protein involvement in Golgi function.     

Signal transduction and the regulation of actin conformation during myeloid maturation: studies in HL60 cells

Maturation of human myeloid cells is associated with quantitative and qualitative changes in protein kinase C (PKC) and increases in N-formyl- L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors, actin, and actin regulatory proteins. We have studied the actin responses and cell shape changes caused by FMLP and its second messenger pathways in HL60 cells undergoing neutrophilic maturation. In uninduced cells, the PKC activators 12-O-tetradecanoyl phorbol-13-acetate (TPA), bryostatin, and 1-oleyl-2-acetylglycerol (OAG) resulted in 15% to 30% decreases in F- actin, whereas FMLP had no effect. Ionomycin had no effect on actin but did cause a 10-fold increase in intracellular calcium. Cells grown for 24 hours in 1% dimethyl sulfoxide (DMSO) acquired the ability to polymerize actin in response to FMLP and ionomycin. TPA continued to cause a decrease in F-actin at 24 hours, but caused an increase in F- actin at 48 to 72 hours of maturation. The PKC inhibitor 1-5- isoquinolinesulfonyl 2-methylpiperazine (H7) partially blocked the F- actin increase caused by TPA in induced cells, but had no effect on the decrease in F-actin caused by TPA in uninduced cells or the increase in F-actin seen in FMLP-treated neutrophils. F-actin rich pseudopods developed following TPA or FMLP stimulation of induced HL60 cells; in uninduced cells neither agent caused pseudopod formation but TPA caused a dramatic loss of surface ruffles. The ability of FMLP and ionomycin to elicit a neutrophil-like actin response in HL60 cells within 24 hours after DMSO treatment shows that the actin regulatory mechanism is mature by that time. The inability of ionomycin to increase F-actin in uninduced cells supports the view that calcium increases alone are insufficient for actin polymerization. The longer maturation time required for HL60 cells to develop an actin polymerization response to TPA compared with FMLP, coupled with the inability of H7 to block the FMLP-mediated F-actin increase in neutrophils, suggests that the F- actin increase caused by FMLP is not mediated solely by PKC. Lastly, the TPA-induced F-actin decrease and shape changes in uninduced HL60 cells, and the longer time required for a "mature" response to TPA, may reflect immaturity in the PKC isoenzyme pattern rather than immaturity of the actin regulatory mechanism.     

The calcium-sensing receptor regulates plasma membrane calcium adenosine triphosphatase isoform 2 activity in mammary epithelial cells: a mechanism for calcium-regulated calcium transport into milk

The calcium-sensing receptor (CaR) regulates transepithelial calcium transport into milk by mammary epithelial cells. Using a genome-wide screening strategy, we identified the plasma membrane calcium ATPase isoform 2 (PMCA2) as a potential downstream target of the CaR. We show that PMCA2 expression in the mouse mammary gland increases during lactation and that PMCA2 is localized solely to the apical plasma membrane of mammary epithelial cells. In milk from deafwaddler mice, which have mutations in the gene encoding PMCA2, calcium concentrations were reduced, confirming its importance in calcium transport into milk. Furthermore, in cultured primary and EpH4 mouse mammary epithelial cells, CaR stimulation up-regulated calcium-dependent ATPase activity in plasma membrane preparations. By small interfering RNA-mediated gene knockdown of PMCA2, we show that PMCA2 accounts for the preponderance of calcium-ATPase activity. We also show that reduction of CaR expression with small interfering RNA eliminates the ability of extracellular calcium to elicit an increase in calcium-dependent ATPase activity in EpH4 cell membranes. These results demonstrate that activation of the CaR increases PMCA2 activity in mouse mammary epithelial cells, providing a mechanism for the regulation of transepithelial calcium transport by calcium in the lactating mouse mammary gland.     

Changes in intracellular free calcium in isolated myometrial cells: role of extracellular and intracellular calcium and possible involvement of guanine nucleotide-sensitive proteins

Intracellular free calcium concentrations were measured directly in rat myometrial cells loaded with fura-2. The basal concentrations of calcium were 148 +/- 5.0 and 137 +/- 3.7 nM in the presence and absence of 1 mM extracellular calcium, respectively. Oxytocin, carbachol, and norepinephrine rapidly and transiently increased intracellular free calcium, with half-maximal effects at 0.19, 9.9, and 5.3 microM, respectively. The maximal effects of these agents were reduced by 57%, 32%, and 36%, respectively, when the extracellular calcium was replaced by 2 mM EGTA. Pretreatment with pertussis toxin partially (47-57%) inhibited the contractant-induced increase in intracellular free calcium in the presence of 1 mM extracellular CaCl2 and produced an even greater inhibition (76-98%) in the absence of extracellular calcium. Pretreatment with D600 (30 microM) or amiloride (50 microM) and reduction of extracellular sodium did not affect the oxytocin-induced calcium increase. However, adenosine and the A2-receptor agonist N-ethylcarboxamidoadenosine did attenuate the effect of oxytocin in a dose-dependent manner. These data represent the first direct evidence that oxytocin, carbachol, and norepinephrine increase the intracellular free calcium concentration in the rat myometrium. The data suggest that contractants mobilize calcium from both extracellular and intracellular sources, the latter involving a pertussis toxin-sensitive mechanism.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Changes in cytosolic free calcium ion concentrations in individual rat granulosa cells: effect of luteinizing hormone-releasing hormone

Changes in the cytosolic free calcium ion concentrations ([Ca2+]i) induced by LHRH were studied in individual rat granulosa cells using fura-2 microspectrofluorimetry. The resting [Ca2+]i concentration was 96.7 +/- 2.9 nM (n = 115). The majority of the granulosa cells responded to LHRH at doses between 10(-7) and 10(-5) M; the latter dose was the highest concentration required to initiate alterations in [Ca2+]i in these cells. The alterations in [Ca2+]i induced by LHRH were transient and returned to resting levels within 84 +/- 3 sec (n = 64). A potent LHRH antagonist completely blocked the effect of LHRH on [Ca2+]i. Within a single granulosa cell, three consecutive injections of the same dose of LHRH, delivered 5 min apart, induced three discrete peaks of [Ca2+]i of similar amplitudes. Sustained perfusions of LHRH, however, resulted in a desensitization of the [Ca2+]i response to LHRH, but not to the calcium ionophore Br-A23187. These results, which were obtained from individual cells, provide strong support for the hypothesis that acute changes in [Ca2+]i are involved in the early cellular transduction of the LHRH signal in the ovary.     

Changes in circulating Foxp3+ regulatory T cells and interleukin-17-producing T helper cells during HBV-related acute-on-chronic liver failure

AIM: To longitudinally investigate cytokine gene expression and protein levels in Th17 and Treg cells, to observe T-cell phenotypes during hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACHBLF) and to analyze changes in Th17 and Treg phenotypes during disease progression.METHODS: We measured the expression of seven Th17/Treg differentiation-related genes and serum concentrations of the corresponding cytokines in 18 ACHBLF, 18 chronic hepatitis B (CHB) disease controls and 10 healthy controls (HCs) by real-time quantitative PCR and enzyme linked immunosorbent assay. Peripheral Th17 and Treg cell frequencies were analyzed by flow cytometry.RESULTS: From the onset of ACHBLF, patients presented with a conductive Th17 differentiation cytokine environment accompanied by high Th17 frequency and high serum IL-17 levels, which were sustained throughout the disease course. The Treg-related cytokine IL-2 and Foxp3 were also up-regulated from disease onset, and Foxp3 gene expression showed a gradually increasing trend during ACHBLF. The circular phenotype of Treg and Th17 cells showed changes from the onset of ACHGLF. At disease onset, Th17 frequency increased significantly compared with both CHB and HCs, but Treg cell frequency decreased significantly compared with CHB. During the ACHBLF event, Th17 frequency remained higher compared with HCs, but decreased sharply from the peak point to the recovery point; Treg cell frequency increased gradually during the ACHBLF event. Treg and Th17 cell counts correlated with ACHBLF development; in all patients, serum IL-17 levels significantly correlated with patient serum ALT levels. In survivors, Th17 frequency at the onset point and the Treg to Th17 ratio at the peak point correlated with the patient’s model for end stage liver disease (MELD) plus sodium (MELD-Na) score. The Treg to Th17 ratio and the Th17 frequency at onset were significant predictors of patient survival. Low Treg/Th17 cell ratios at the onset predicted poor survival. Survivors exhibited an initial decrease in the circulating Treg/Th17 ratio from the onset to the peak time, and subsequently displayed a continuous increase.CONCLUSION: Treg and Th17 cells showed changes in genes, protein levels and T cell phenotypes during ACHBLF events. An increased Treg/Th17 ratio was associated with the survival of ACHBLF patients.