Plasma membrane voltage changes during nanosecond pulsed electric field exposure.

The change in the membrane potential of Jurkat cells in response to nanosecond pulsed electric fields was studied for pulses with a duration of 60 ns and maximum field strengths of approximately 100 kV/cm (100 V/cell diameter). Membranes of Jurkat cells were stained with a fast voltage-sensitive dye, ANNINE-6, which has a subnanosecond voltage response time. A temporal resolution of 5 ns was achieved by the excitation of this dye with a tunable laser pulse. The laser pulse was synchronized with the applied electric field to record images at times before, during, and after exposure. When exposing the Jurkat cells to a pulse, the voltage across the membrane at the anodic pole of the cell reached values of 1.6 V after 15 ns, almost twice the voltage level generally required for electroporation. Voltages across the membrane on the side facing the cathode reached values of only 0.6 V in the same time period, indicating a strong asymmetry in conduction mechanisms in the membranes of the two opposite cell hemispheres. This small voltage drop of 0.6-1.6 V across the plasma membrane demonstrates that nearly the entire imposed electric field of 10 V/mum penetrates into the interior of the cell and every organelle.

Simulations of transient membrane behavior in cells subjected to a high-intensity ultrashort electric pulse.

A molecular dynamics (MD) scheme is combined with a distributed circuit model for a self-consistent analysis of the transient membrane response for cells subjected to an ultrashort (nanosecond) high-intensity (approximately 0.01-V/nm spatially averaged field) voltage pulse. The dynamical, stochastic, many-body aspects are treated at the molecular level by resorting to a course-grained representation of the membrane lipid molecules. Coupling the Smoluchowski equation to the distributed electrical model for current flow provides the time-dependent transmembrane fields for the MD simulations. A good match between the simulation results and available experimental data is obtained. Predictions include pore formation times of about 5-6 ns. It is also shown that the pore formation process would tend to begin from the anodic side of an electrically stressed membrane. Furthermore, the present simulations demonstrate that ions could facilitate pore formation. This could be of practical importance and have direct relevance to the recent observations of calcium release from the endoplasmic reticulum in cells subjected to such ultrashort, high-intensity pulses.

Progesterone-induced calcium influx in cynomolgus monkey (Macaca fascicularis) spermatozoa.

For in vitro capacitation to occur in cynomolgus monkey (Macaca fascicularis) spermatozoa, there is an absolute requirement for exogenous stimulation with the sperm activators, caffeine (1 mM) and db-cyclic adenosine monophosphate (dbcAMP) (1 mM), which are known to induce capacitation-related hyperactivated motility. Tyrosine phosphorylation of sperm tail proteins is an integral component of this caffeine- and dbcAMP-stimulated hyperactivated motility. In both capacitated and noncapacitated human spermatozoa, progesterone (P4) has been reported to elicit an immediate, potent increase in intracellular calcium ion concentrations [Ca2+]i. The objective of this study was to examine the effects of progesterone on requisite events in macaque fertilization, including [Ca2+]i, hyperactivated motility, and the concomitant tyrosine phosphorylation of sperm tail (STTP) proteins after treatment with caffeine and dbcAMP. The effect of 1 microM of progesterone on [Ca2+]i was determined by spectrofluorometry with the fluorescent indicator, fura-2/AM, on hyperactivated motility using computer analysis (HTM-IVOS) with the sorting criteria lateral head amplitude (> or = 8.0 microm), curvilinear velocity (> or = 150 microm/s), linearity (< or = 60%), and on STTP by immunocytochemistry. The results showed that progesterone elicited a significant increase in [Ca2+]i in caffeine- and dbcAMP-activated macaque sperm with maximal stimulation at 30 minutes after activation. The response in nonactivated sperm was dramatically reduced compared with the response in activated sperm. Basal [Ca2+]i increased as a function of time in both activated and nonactivated control sperm although basal levels were significantly increased in activated sperm. Progesterone stimulation resulted in a small but significant increase in both hyperactivation and STTP when sperm were first pretreated with caffeine and dbcAMP. Our results provide evidence that macaque sperm activation with caffeine and dbcAMP is required for a progesterone-elicited response, which results in calcium influx, hyperactivated motility, and sperm tail tyrosine phosphorylation.

Rapid effects of estrogen and progesterone on tone and spontaneous rhythmic contractions of the rabbit bladder.

Previous studies indicate that bladder instability in man may be associated with increased spontaneous rhythmic contractile activity. Ca(2+) influx plays a central role in smooth muscle contractions, and recent evidence suggests that steroid hormones rapidly affect Ca(2+) influx. Therefore we tested the hypothesis that estrogen and progesterone modulates spontaneous rhythmic detrusor contractions. Tissues were secured to isometric force (F) transducers in tissue baths and length-adjusted until K(+)-depolarization produced maximum contractions (F(o)). Spontaneous rhythmic contractions (SRC) were sampled before and immediately after addition of estradiol or progesterone (10(-5) M) to tissue baths. The average frequency and amplitude of SRC were, respectively, 0.156 Hz and 0.053 F/F(o) (n = 24). Estradiol caused an immediate reduction in SRC, such that by 10 min, tone, frequency and amplitude were each reduced by, respectively, 36%, 46% and 47% (n = 7, P < 0.05). However, progesterone caused an immediate weak contraction, and at steady state (10 min), progesterone increased frequency of SRC by 152% but decreased SRC amplitude by 50% (n = 10, P < 0.05). Novel therapies using unique steroids that do not interact with genomic receptors may potentially reduce bladder smooth muscle activity, thereby reducing detrusor instability.

Differential effects of sex hormones and phytoestrogens on peak and steady state contractions in isolated rabbit detrusor.

PURPOSE: Recent evidence suggests that sex steroids may produce rapid inhibition of voltage operated Ca2+ channels (VOCCs). Detrusor smooth muscle is highly dependent upon Ca2+ influx for receptor-activated contractions. Thus, we examined the relative effectiveness of a select group of sex steroids and dietary phytoestrogens to relax detrusor contracted with the muscarinic receptor agonist, bethanechol (BE) and the purinergic P2X receptor agonist, alpha,beta-methylene ATP (alpha,beta-MeATP). MATERIALS AND METHODS: Isolated strips of rabbit detrusor were secured to isometric force transducers in a tissue bath and length-adjusted until maximum contractions were achieved. Peak (P) contractile responses were recorded for alpha,beta-MeATP (P(ATP)) and BE (P(BE)) and steady-state (SS) responses were recorded for BE (SS(BE)) in the presence and absence of selected sex steroids and phytoestrogens (10 microM, unless indicated). RESULTS: The L-type VOCC inhibitor, nifedipine (1 to 10 microM), completely inhibited P(ATP) but reduced SS(BE) by approximately 50%, whereas the VOCC and non-VOCC inhibitor, SKF 96365, inhibited SS(BE) by approximately 95%, suggesting that P(ATP) was entirely dependent on L-type VOCCs, but (BE)-induced contractions depended also on activation of non-VOCCs. 17Beta-estradiol (estradiol) and progesterone inhibited P(ATP) by approximately 60% and 20%, respectively, and 32 microM estradiol and ethinyl estradiol inhibited SS(BE) by approximately 80 and 95%, respectively. Inhibition by estradiol was potentiated, rather than blocked, by the nuclear estrogen receptor antagonist, tamoxifen. Moreover, tamoxifen alone nearly completely relaxed SS(BE). The inactive metabolite of estradiol, 17alpha-estradiol, inhibited both P(ATP) and P(BE) by approximately 40%. Testosterone had no effect on P(ATP) and P(BE). The phytoestrogen and tyrosine kinase inhibitor, genistein, inhibited SS(BE) by 44%, whereas daidzein, a phytoestrogen without tyrosine kinase inhibitory activity, produced only a 7% inhibition. None of the phytoestrogens examined inhibited P(BE), whereas all inhibited P(ATP) by approximately 20 to 35%. A comparison of inhibition of (BE) and alpha,beta-MeATP-induced contractions by selected estrogen isomers showed some distinct differences. For example, estrone did not inhibit P(BE) or SS(BE), but inhibited P(ATP) by approximately 20%, whereas DES inhibited SS(BE) by nearly 90%, but P(ATP) by a lesser degree (approximately 70%). CONCLUSIONS: Our data support the hypothesis that 17beta-estradiol, ethinyl estradiol, DES, tamoxifen and genistein may relax detrusor contractions by inhibition of both VOCCs and non-VOCCs. Moreover, our data show that genistein, a dietary phytoestrogen with tyrosine kinase inhibitory activity, selectively reduced alpha,beta-MeATP-induced peak and BE-induced steady-state contractions, sparing the maximum response to BE. Lastly, the inactive isomer, 17alpha-estradiol, inhibited both BE- and alpha,beta-MeATP-induced contractions. These data suggest that certain dietary phytoestrogens (for example, genistein) or sex steroids, especially those with weak activity at the nuclear steroid site (for example, 17alpha-estradiol), or tamoxifen may prove therapeutically useful in treating overactive bladder caused by elevated muscarinic and purinergic receptor activation.

trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets.

1. The phytoestrogenic compound trans-resveratrol (trans-3,5, 4'-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. 2. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. 3. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 microM. trans-Resveratrol at 0.1, 1.0 and 10.0 microM produced 20+/-6, 37+/-6 and 57+/-4% inhibition respectively of the effect of thrombin (0.01 u ml(-1)) to increase [Ca2+]i. 4. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 microM trans-resveratrol producing 10+/-5, 30+/-5 and 50+/-7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. 5. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. 6. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. 7. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to inhibit platelet aggregation induced by thrombin.

The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.

The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level.

Extragenomic actions of progesterone in human sperm and progesterone metabolites in human platelets.

Progesterone rapidly increased intracellular free calcium ([Ca2+]i) in human sperm, removal of extracellular Ca2+ prevented the increase in [Ca2+]i. The Ca2+ influx was not blocked by the T-type Ca2+ channel blocker mibefradil. However T-type calcium channels do appear to be present in human sperm because the neoglycoprotein mannose-albumin, an inducer of the acrosome reaction, was able to promote Ca2+ influx, which was blocked by mibefradil and more potently inhibited by Ni2+ than Cd2+. The receptor for progesterone that promotes the Ca2+ influx was located on the plasma membrane using FITC-progesterone-albumin. It is concluded that progesterone stimulates Ca2+ influx in human sperm via a unique Ca2+ channel possibly similar to a store-operated channel (SOC) or a receptor-operated channel (ROC). We have found that progesterone metabolites, such as pregnanolone and pregnanediol, promote a rapid rise in [Ca2+]i and aggregation in human platelets, similar to that observed with thrombin. The increase in [Ca2+]i was prevented when extracellular Ca2+ was removed or by the SOC inhibitor SKF-96365. The phospholipase C inhibitor U-73122 also prevented the increase in [Ca2+]i, suggesting that these metabolites interact with a cell surface receptor on the platelet to activate phospholipase C to produce inositol-P3, which mobilizes intracellular Ca2+, thereby activating the SOC in the plasma membrane. Progesterone and estradiol conjugated to albumin, also produced a rapid increase in [Ca2+]i, which was prevented by Ca2+ removal from the medium or when SKF-96365 or U-73122 were added. It is proposed that human platelets possess cell surface receptors for steroids.

Involvement of Shc in the signaling response of human prostate tumor cell lines to epidermal growth factor.

Autocrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors, implicating a role for EGFR in the progression of prostate cancer. To investigate early signaling mechanisms used by the EGFR in prostate tumor cells, we have characterized the involvement of the Shc (src homology 2/x-collagen related) adapter protein in EGFR signaling in several human prostate tumor cell lines. In androgen-responsive lymph node-prostate cancer (LNCaP) cells and androgen-insensitive PC3, DU145 and PPC-I cells, Shc was identified as one of the most prominent phosphotyrosine proteins to be elevated in response to EGF. Equivalent levels of the 46- and 52-kDa Shc isoforms were detected in all of the tumor cell lines tested. However, levels of the 66-kDa isoform were variable among the cell lines. In all of the tumor cell lines, EGF caused an association between Shc and Grb2, another adapter protein linked to cellular ras activation. Additionally, several phosphotyrosine proteins, including a 115-120-kDa protein in EGF-treated LNCaP cells, co-associated with Shc. The profile of these Shc-associating proteins, however, differed among the tumor cell lines. Our results indicate that Shc is a common downstream element of EGFR signaling in prostate tumor cells and suggest multiple functions for Shc in prostate tumorigenesis.

Attenuation of LPA-mediated calcium signaling and inositol polyphosphate production in rat-1 fibroblasts transformed by the v-src oncogene.

Alterations in cellular signaling underlie the transforming actions of many oncogenes. The vsrc oncogene tyrosine kinase, pp60vsrc, is known to alter multiple signal transduction pathways, including those involving phosphatidylinositol (PI) metabolism. In this study, we investigated the effects of vsrc-transformation on lysophosphatidic acid (LPA) receptor coupling to intracellular free calcium [Ca2+]i and PI turnover in rat-1 fibroblasts. In normal rat-1 cells, LPA rapidly elevated [Ca2+]i (EC50 = 10nM). In contrast, the ability of LPA to mobilize calcium was markedly attenuated in rat-1-vsrc cells. Further study revealed that the LPA-mediated generation of inositol (1,4,5)P3 and other inositol polyphosphates was also markedly attenuated in the vsrc-transformed cells. Although LPA caused a transient reduction in the level of PI(4,5)P2 in normal rat-1 cells, the agonist elevated the level of PI(4,5)P2 in the vsrc-transformed cells. These findings demonstrate that vsrc-transformation alters the coupling of LPA receptors to PI turnover and calcium signaling in rat-1 cells, and point to G protein-coupled receptor systems as targets for modulation by the vsrc kinase.

Calcium signaling in prostate cancer cells: evidence for multiple receptors and enhanced sensitivity to bombesin/GRP.

BACKGROUND: Cellular calcium is an important second messenger for growth regulation. We sought to identify potentially important receptors on prostate tumor cells by screening over 20 agonists for their ability to increase intracellular free calcium ([Ca2+]i) in several human prostate tumor cell lines. METHODS: Intracellular calcium mobilization was detected using fura-2. RESULTS: We found bombesin, GRP, ATP/UTP, lysophosphatidic acid, thrombin, endothelin, histamine, and bradykinin increased [Ca2+]i in the advanced tumor cell lines DU-145, PC3, and PPC-1. Bombesin failed to elevate [Ca2+]i in an immortalized human prostate cell line. Rank-order of potency studies suggested the presence of P2U nucleotide receptors for ATP/UTP on prostate epithelial cells. Potency studies also revealed GRP > > bombesin > > neuromedin B at elevating [Ca2+]i in responding tumor cells. CONCLUSIONS: These findings indicate that androgen independent prostate tumor cell lines express multiple receptors capable of elevating intracellular calcium, and suggest that GRP receptors may be selectively expressed and/or coupled to calcium signaling during prostate tumor progression. Calcium sensitive cellular events may therefore contribute to the progression of prostate cancer.

Inhibition of head and neck squamous cell carcinoma growth and invasion by the calcium influx inhibitor carboxyamido-triazole.

Local invasion and lymph node metastasis are correlated with a decreased overall survival in head and neck cancer patients and warrant new strategies to intervene in the metastatic cascade. One approach is to focus on the intracellular signaling pathways underlying the metastatic process. A common regulatory point in several signal transduction pathways is intracellular calcium homeostasis. We assessed the effect of a novel calcium influx inhibitor, carboxyamido-triazole (CAI), on the growth and invasive phenotype of cell lines derived from head and neck squamous cell carcinoma (HNSCC). CAI inhibited the growth of FaDu and EVSCC17M cells in a dose-dependent (IC50, 13-15 microM) and reversible manner. CAI also caused a generalized attenuation of receptor-mediated calcium elevation to several calcium mobilization agonists, including epidermal growth factor and bradykinin. The effects of CAI on the invasive phenotype of HNSCC cell lines were assessed by a chemo-invasion assay. HNSCC cell lines exhibited a range of invasive potential as measured by the capacity of tumor cells to penetrate a reconstituted basement membrane of Matrigel. HNSCCs were classified as highly invasive (EVSCC14M and EVSCC17M) or weakly invasive (EVSCC18, EVSCC19M, UMSCC10A, and FaDu). Treatment of HNSCC cell lines with 10 microM CAI for 24 h reduced invasion 2-14-fold in a dose-dependent manner. HNSCCs also exhibited different motilities as measured by a chemotaxis assay. EVSCC14M and EVSCC17M were highly motile, whereas EVSCC18, EVSCC19M, UMSCC10A, and FaDu were less motile. CAI reduced the migration of all cell lines. Conditioned medium from HNSCC cell lines was analyzed by zymography for production of Mr 72,000 type IV collagenase [matrix metalloproteinase (MMP)-2)] and Mr 92,000 type IV collagenase (MMP-9). All HNSCC cell lines secreted MMP-2 and/or MMP-9 into conditioned medium. Treatment of cells with 10 microM CAI for 24 h resulted in a reduction of both MMP-2 and MMP-9 production. The results demonstrate that CAI blocks cellular proliferation, migration, chemoinvasion, and MMP production by HNSCC in vitro and identify calcium-dependent signaling as a new target for inhibition of the malignant phenotype of HNSCC.

Effects of the calcium influx inhibitor carboxyamido-triazole on the proliferation and invasiveness of human prostate tumor cell lines.

Aberrant cellular signaling is a central feature of malignant cells and a potential target for anti-cancer therapy. Carboxyamido-triazole (CAI) is a calcium influx inhibitor that alters calcium-sensitive signal transduction pathways and suppresses the proliferative and metastatic potential of malignant cells. We have examined the effects of CAI on several tumor-associated parameters in human prostate cancer cell lines to evaluate the potential of CAI as a signal-transduction therapy agent for advanced-stage prostate cancer. Measuring anchorage-dependent cell growth, continuous application of CAI inhibited the growth of DU-145, PPC-1, PC3 and LNCaP tumor cells with 50% inhibitory concentrations ranging 10-30 microM. Direct cell enumeration assays revealed that the growth-suppressing activity of CAI toward DU-145 cells was reversible, indicating a cytostatic effect of the drug on tumor cells. The drug also inhibited the proliferation of several immortalized human prostatic epithelial cell lines. The proliferation of HaCaT- and RHEK-1-immortalized keratinocyte cell lines was relatively insensitive to CAI. Additionally, invasion by DU-145, PC3 and PPC-1 cells through Matrigel in vitro was reduced approximately 60-70% by 10 microM CAI. Other cellular effects of CAI included an attenuation of the elevation of intracellular free calcium in response to bombesin and carbachol in PC3 cells and a marked dose-dependent inhibition of prostate-specific antigen secretion in LNCaP cell cultures.

Unusual steroid specificity of the cell surface progesterone receptor on human sperm.

The steroid specificity of the cell surface progesterone receptor in human sperm was examined with the use of progesterone, testosterone, and androstane analogues. Many compounds were shown to be more effective than progesterone at increasing intracellular free calcium concentration, e.g., 2 alpha-methyl-17beta-methoxy-5 alpha-androstan-3-one. Several testosterone analogues were demonstrated to be antagonists of progesterone, e.g., 9(11)-dehydro-2 alpha,17alpha-dimethyltestosterone. The synthetic potent progestigens, norethynodrel, cyproterone acetate, norethindrone, and megestrol acetate, were found to be only weak stimulators of the sperm cell surface receptor. Furthermore, these compounds were shown to antagonize the effect of progesterone to elevate intracellular free calcium concentration in sperm. It is known that progesterone and some of its analogues bind to the intracellular progesterone nuclear receptor via the alpha-face of the steroid molecule. In stark contrast, it was concluded from the analysis of the steroid analogues examined on human sperm in this study that intimate contact exists between the effective progesterone analogues and the sperm cell surface progesterone receptor across the beta-face of the steroid C/D-ring "upper" edge (C11, C12, and C17). Positioning of the C21 methyl group is also critical for efficacy, and recognition of the steroid A-ring seems not to be involved.

Steroid specificity of the human sperm membrane progesterone receptor.

The aim of this study was to evaluate the effect of several abeopregnane, steroidal heterocycles (A/B-transandrostano [2,3-d]isoxazole, and 17-spiroandrostano[2,3-c]furazan), and 6 alpha, 11 beta, 16 alpha-trisubstituted 19-norpregnadienedione on the influx of extracellular Ca2+ in human sperm. These steroidal compounds had minimal genomic progestational, androgenic, or estrogenic activity with the exception of 16 alpha-ethyl-6 alpha-methyl-11 beta-(4-N,N-dimethylaminophenyl)-19- norpregna-4,9-diene-3,20-dione which was four times more progestational than progesterone. Some of the steroidal compounds, e.g., 16 alpha-ethyl-6 alpha-methyl-11 beta-(4-N,N-dimethylaminophenyl)-19-nor- pregna-4,9-diene-3,20-dione and 2',3',4',5'-tetrahydrospiro[furan-2' beta, 17-androstano] [2,3-c]furazan produced an influx of Ca2+ into human spermatozoa. These studies indicate that high (10 microM) concentrations of certain steroidal compounds are selective for the sperm membrane progesterone receptor, since most of them have minimal genomic activity. The steroidal compounds that elicited an influx of Ca2+ caused an initial high influx but were not as potent as progesterone, since no effects were observed below 1 microM, whereas progesterone at 1 microM produced a maximum effect. Progesterone as well as the steroidal compounds caused a modest increase in the number of acrosome-reacted spermatozoa. Molecular modeling revealed that 5 alpha-dihydro-2,3-fused and 4,4-dimethyl-5-ene-2,3-fused steroidal heterocycles possessing different conformations compared to that of progesterone are responsible for elevation of Ca2+. In conclusion, a unique non-genomic progesterone receptor is present on human spermatozoa and several steroidal compounds that do not have progestational effects may activate this sperm membrane receptor, resulting in Ca2+ influx.

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells.

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A2 (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219-225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced as significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA2 and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA2 activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMPL was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMPL-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA2 rather than secretory PLA2. Inhibition of PLA2 with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA2-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.

The role of endothelin-1 in regulating human granulosa cell proliferation and steroidogenesis in vitro.

The effects of endothelin-1 (ET-1) on luteinized human granulosa cells (L-HGCs) have not been examined. It is well known that there are differences of actions of several autocrine/paracrine regulators between L-HGCs and GCs of other species, and therefore the present study was designed to examine the effects of ET-1 1) on intracellular Ca2+ concentrations ([Ca2+]i) using the Ca(2+)-responsive fluorescent indicator Fura-2, 2) on cell proliferation by the nonradioactive method using bromodeoxyuridine, and 3) on basal and gonadotropin-stimulated steroidogenesis, and to examine the expression of ET receptor messenger RNA (mRNA) using freshly isolated and cultured L-HGCs obtained from patients undergoing in vitro fertilization. ET-1 increased [Ca2+]i in L-HGCs in a dose-dependent manner between 1 and 1000 nmol/L. High concentrations (100-1000 nmol/L) of ET-1 produced a more rapid and transient increase in [Ca2+]i than that observed with low concentrations (1-10 nmol/L) of ET-1. The increase in [Ca2+]i elicited by ET-3 (1000 nmol/L) and IRL-1620 (1000 nmol/L), a selective ETB receptor agonist, was 16% and 3% (vs. ET-1, 100%), respectively. BQ-123 (1000 nmol/L), an ETA receptor antagonist, inhibited the increase in [Ca2+]i elicited by ET-1 (by 50% at 1000 nmol/L ET-1 and by > 90% at < 500 nmol/L ET-1). mRNAs for the two known receptor subtypes (ETA and ETB) were also present in L-HGCs; however, the expression of ETA receptor mRNA was much greater than that of ETB receptors. ET-1 stimulated cell proliferation in L-HGCs in a dose-dependent manner (1000 nmol/L, 210.5 +/- 13.1%; 100 nmol/L, 198 +/- 11%; 10 nmol/L, 146 +/- 18%; and 1 nmol/L, 103 +/- 9%; vs. control, 100%). These stimulatory effects were completely blocked by BQ-123 (1000 nmol/L). ET-3 and IRL-1620 had no effects on cell proliferation in L-HGCs. Significant stimulatory effects on cell proliferation by the calcium ionophore, ionomycin (10-1000 nmol/L), were observed. ET-1, ET-3, and IRL-1620 attenuated basal progesterone secretion in L-HGCs. These results suggest that ETA receptor predominantly exist in L-HGCs and that ET-1 may stimulate cell proliferation of L-HGCs by increasing [Ca2+]i via ETA receptors.

Cellular regulation of arachidonate mobilization and metabolism.

Synthesis of eicosanoids is initiated by signal transduction cascades which result in the hydrolysis of free arachidonic acid from membrane phospholipids. Both a cytosolic 85 kDa and a nonpancreatic 14 kDa PLA2 may contribute to cellular arachidonate mobilization. In many cells, agonist-stimulated fatty acid release is dependent upon increases in intracellular free calcium and is enhanced by pretreatment with agents such as phorbol esters and soluble diglycerides. The response is specific for arachidonate and structurally similar polyunsaturated fatty acids containing a cis 5, 6 double bond. DMSO-differentiation of U937 cells markedly enhances the A23187-stimulated release of [3H]arachidonate, which appears to be coupled to differentiation-induced enhancement of capacitance calcium entry. Although both phorbol esters and soluble diglycerides enhance subsequent fMLP or A23187-stimulated arachidonate release in human neutrophils, several lines of evidence indicate that the effects of oleoylacetylglycerol and 1,2-dioctanoylglycerol are protein kinase C-independent. Soluble diglycerides, but not phorbol esters, enhance the coupling of arachidonate mobilization to subsequent leukotriene B4 synthesis. Further studies will be required to elucidate the mechanisms which regulate activation of cellular phospholipases and subsequent synthesis.

The cell surface progesterone receptor which stimulates calcium influx in human sperm is unlike the A ring reduced steroid site on the GABAA receptor/chloride channel.

Progesterone elicits a rapid, transient calcium influx in sperm that is a prerequisite for the progesterone-induced acrosome reaction. The possibility that the GABAA receptor/chloride channel was the receptor that mediated the progesterone-induced calcium influx in human sperm was examined. A-ring reduced 3 alpha-hydroxy pregnane steroids (e.g. alfaxalone, allopregnanolone, pregnanolone), which are active on the GABAA receptor/chloride channel, were found to be much weaker than progesterone at stimulating Ca2+ influx in sperm. The effects of a variety of progesterone metabolites and analogs and other steroids were compared for their ability to (i) stimulate GABA-induced 36Cl- uptake in synaptoneurosomes, (ii) stimulate GABA-induced Cl- currents in HEK-293 cells transfected with alpha 1, beta 2, and gamma 2 subunits of the GABAA receptor/chloride complex, and (iii) elicit a rapid Ca2+ influx in sperm. No correlation was observed between the ability of a given steroid to stimulate Ca2+ influx and efficacy in eliciting either 36Cl- uptake or chloride currents. Importantly, the action of progesterone to stimulate Ca2+ influx was not modified by GABA, diazepam, picrotoxin and pentobarbitol (known regulators of the GABAA receptor/chloride channel). It is concluded from these studies that the cell surface progesterone binding site on human sperm that mediates progesterone-induced changes in [Ca2+]i is unlike the steroid binding site on the GABAA receptor/chloride channel.(ABSTRACT TRUNCATED AT 250 WORDS)