Activation of the NFAT-Calcium Signaling Pathway in Human Lamina Cribrosa Cells in Glaucoma.

Purpose: Optic nerve cupping in glaucoma is characterized by remodeling of the extracellular matrix (ECM) and fibrosis in the lamina cribrosa (LC). We have previously shown that glaucoma LC cells express raised levels of ECM genes and have elevated intracellular calcium ([Ca2+]i). Raised [Ca2+]i is known to promote proliferation, activation, and contractility in fibroblasts via the calcineurin-NFAT (nuclear factor of activated T-cells) signaling pathway. In this study, we examine NFAT expression in normal and glaucoma LC cells, and investigate the effect of cyclosporin A (CsA, a known inhibitor of NFAT activity) on [Ca2+]i and ECM gene expression in normal and glaucoma LC cells. Methods: [Ca2+]i was measured with dual-wavelength Ca2+ imaging and confocal microscopy using Fura-2-AM and Fluo-4 under physiological isotonic and hypotonic cell stretch treatment. Human donor LC cells were cultured under normal physiological conditions or using a glaucoma-related stimulus, oxidative stress (H2O2, 100 µM), for 6 hours with or without CsA. NFATc3 protein levels were examined using Western blot analysis. Profibrotic ECM gene transcription (including transforming growth factor-ß1 [TGFß1], collagen 1A1 [Col1A1], and periostin) was analyzed using quantitative real time RT-PCR. Results: Basal and hypotonic cell membrane stretch-induced [Ca2+]i were significantly (P < 0.05) elevated in glaucoma LC cells compared to normal controls. There was a significant delay in [Ca2+]i reuptake into internal stores in the glaucoma LC cells. NFATc3 protein levels were increased in glaucoma LC cells. CsA (10 µM) significantly inhibited the H2O2-induced expression of NFATc3 in normal and glaucoma LC cells. CsA also reduced the H2O2-induced NFATc3 dephosphorylation (and nuclear translocation), and also suppressed the H2O2-induced elevation in profibrotic ECM genes (TGFß1, Col1A1, and periostin), both in normal and in glaucoma LC cells. Conclusions: Intracellular Ca2+ and NFATc3 expression were significantly increased in glaucoma LC cells. CsA reduced the H2O2-induced enhancement in NFATc3 protein expression and nuclear translocation and the profibrotic gene expression both in normal and in glaucoma LC cells. Therefore, targeting the calcineurin-NFATc3 signaling pathway may represent a potential avenue for treating glaucoma-associated LC fibrosis.

Antidepressant-like effect of hyperoside isolated from Apocynum venetum leaves: possible cellular mechanisms.

In the present work, we studied the possible cellular mechanisms of hyperoside isolated from Apocynum venetum leaves in corticosterone-induced neurotoxicity, using PC12 cells as a suitable in vitro model of depression. Cell viability was quantitated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The release amount of lactic dehydrogenase (LDH) and intracellular Ca(2+) concentration were measured using kit and transcript abundances of brain-derived neurotrophic factor (BDNF) and cAMP response element binding protein (CREB) were determined by real-time RT-PCR. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase (LDH) assays showed that 2.5, 5 and 10 µg/ml hyperoside or 10 µM fluoxetine (FLU) protected PC12 cells from the lesion induced by a 48 h treatment with 10 µM corticosterone. Fura-2/AM (acetoxymethyl ester) assays showed that 2.5, 5 and 10 µg/ml hyperoside or 10 µM FLU attenuated the intracellular Ca(2+) overloading in PC12 cells induced by corticosterone. The transcript abundance of BDNF and CREB in PC12 cells was elevated upon hyperoside treatment. These results suggest that the possible cellular mechanisms of hyperoside antidepressant-like effect is a cytoprotective action related to elevation the expression of BDNF and CREB through the signal pathway AC-cAMP-CREB.

TRPV1 activation is required for hypertonicity-stimulated inflammatory cytokine release in human corneal epithelial cells.

PURPOSE: To determine whether hypertonic stress promotes increases in inflammatory cytokine release through transient receptor potential vanilloid channel type 1 (TRPV1) signaling pathway activation in human corneal epithelial cells (HCECs). METHODS: Hyperosmotic medium was prepared by supplementing isotonic Ringers solution with sucrose. Ca2+ signaling was measured in fura2-AM-loaded HCECs using a single-cell fluorescence imaging system. Western blot analysis evaluated the phosphorylation status of EGFR, ERK, p38 MAPK, and nuclear factor (NF)-κB. ELISA assessed the effect of TRPV1 activation on the release of IL-6 and IL-8. RESULTS: A 450 mOsm hypertonic stress elicited 2-fold Ca2+ transients that were suppressed by the TRPV1-selective antagonists capsazepine and JYL 1421. Such transients were enhanced by PGE2. Hypertonicity-induced EGF receptor (EGFR) transactivation was suppressed by preincubating HCECs with capsazepine, matrix metalloproteinase 1 (MMP1) inhibitor TIMP-1, broad-spectrum MMP inhibitor GM 6001, heparin-bound (HB)-EGF inhibitor CRM 197, or EGFR inhibitor AG 1478. ERK and p38 MAPK and NF-κB activation after EGFR transactivation occurred in tonicity and in a time-dependent manner. Hypertonicity-induced increases in IL-6 and IL-8 releases were suppressed by exposure to capsazepine, AG 1478, ERK inhibitor PD 98059, p38 inhibitor SB 203580, or NF-κB inhibitor PDTC. CONCLUSIONS: Hypertonic stress-elicited TRPV1 channel stimulation mediates increases in a proinflammatory cytokine IL-6 and a chemoattractant IL-8 by eliciting EGFR transactivation, MAPK, and NF-κB activation. Selective drug modulation of either TRPV1 activity or its signaling mediators may yield a novel approach to suppressing inflammatory responses occurring in dry eye syndrome.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

Phyto- and endogenous estrogens differently activate intracellular calcium ion mobilization in bovine endometrial cells.

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca(2+)](i) in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F(2alpha) production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca(2+)](i) measurement. We also wanted to confirm whether visualization of calcium ion [Ca(2+)](i) in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca(2+)](i) indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-beta (E(2); endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF(2alpha) secretion and [Ca(2+)](i) mobilization in the cells. Equol and para-ethyl-phenol in doses of 10(-8)-10(-6) M increased PGF(2alpha) concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E(2) and PMA induced intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E(2), did not cause intracellular [Ca(2+)](i) mobilization in endometrial cells. The obtained results proved that the [Ca(2+)](i) visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.

A visual thalamocortical slice.

We describe a thalamocortical slice preparation in which connectivity between the mouse lateral geniculate nucleus (LGN) and primary visual cortex (V1) is preserved. Through DiI injections in fixed brains we traced and created a three-dimensional model of the mouse visual pathways. From this computer model we designed a slice preparation that contains a projection from LGN to V1. We prepared brain slices with these predicted coordinates and demonstrated anatomical LGN-V1 connectivity in these slices after LGN tracer injections. We also revealed functional LGN-V1 connectivity by stimulating LGN electrically and detecting responses in layer 4 of V1 using calcium imaging, field potential recordings and whole-cell recordings. We also identified layer-4 neurons that receive direct thalamocortical input. Finally, we compared cortical activity after LGN stimulation with spontaneous cortical activity and found significant overlap of the spatiotemporal dynamics generated by both types of events.

The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins.

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Protective effects of sarpogrelate, a 5-HT2A antagonist, against postischemic myocardial dysfunction in guinea-pig hearts.

The protective effects of sarpogrelate (SG), a 5-HT2A antagonist, were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca2+ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored using an nitric oxide (NO) electrode, fluorometry and 31P-NMR. The recovery of LVDP from ischemia by reperfusion was 30.1% in the control, while the treatment with SG (5 x 10(-7) M) in pre- and post-ischemia hearts produced a gradual increase to 73.1 and 53.6%, respectively. At the final stage of ischemia, the intracellular concentration of Ca2+ ([Ca2+]i) and release of NO increased with no twitching and remained at a high steady level. The addition of SG increased the transient NO signal (TNO) level at the end of ischemia compared with the control, but [Ca2+]i during ischemia decreased. Meanwhile, mitochondrial Ca2+ uptake on acidification or Ca2+ content changes of the perfusate was suppressed by pre-treatment with SG or the KATP channel opener diazoxide, but not the KATP channel blocker 5-HD. The myocardial NO elevated with 5-HT in normal Langendorff hearts was suppressed by the treatment with SG. Therefore, the existence of the 5HT2A receptor in a Langendorff heart was anticipated. By in vitro EPR, SG was found to directly quench the hydroxy radical. Thus, these findings suggested that the 5-HT2A receptor induced in ischemia-reperfusion plays an important role in the mitochondrial KATP channel of hearts in close relation with NO and active oxygen radicals.

Prolongation and enhancement of gamma-aminobutyric acid receptor mediated excitation by chronic treatment with estradiol in developing rat hippocampal neurons.

GABA(A) receptor activation during brain development is a critical source of excitation. This is due to the positive equilibrium potential for chloride relative to resting membrane potential, resulting in membrane depolarization sufficient to open voltage sensitive calcium channels. The gonadal steroid estradiol has pronounced trophic effects on the developing hippocampus, promoting cell survival and synaptogenesis. In the current study, we investigated the effect of estradiol on GABA(A) receptor-mediated calcium transients in cultured neonatal hippocampal neurons, from Sprague-Dawley rats, using the calcium sensitive dye, Fura-2-AM. Treatment of hippocampal neurons with physiological levels of estradiol significantly increased the peak amplitude of calcium transients, increased the number of cells responding to the GABA(A) agonist muscimol with membrane depolarization, and delayed the rate of clearance of free intracellular calcium. These effects were significantly attenuated by pretreatment with the oestrogen receptor antagonist ICI-182,780. This suggests that estradiol, via its action on the oestrogen receptor, prolongs the developmental duration of depolarizing GABA. Estradiol likely maintains GABA-mediated excitation by promoting increased protein levels of the active/phosphorylated form of the chloride cotransporter Na+K+2CL- and L-type voltage sensitive calcium channels containing the alpha1C subunit. We propose that a component of the trophic effects of estradiol on hippocampal development results from enhanced calcium influx subsequent to GABA(A) receptor activation.

Agrin signaling in cortical neurons is mediated by a tyrosine kinase-dependent increase in intracellular Ca2+ that engages both CaMKII and MAPK signal pathways.

Agrin has been implicated in multiple aspects of central nervous system (CNS) neuron differentiation and function including neurite formation, synaptogenesis, and synaptic transmission. However, little is known about the signaling mechanisms whereby agrin exerts its effects. We have recently identified a neuronal receptor for agrin, whose activation induces expression of c-fos, and provided evidence that agrin binding to this receptor is associated with a rise in intracellular Ca2+, a ubiquitous second messenger capable of mediating a wide range of effects. To gain further insight into agrin's role in brain, we used Ca2+ imaging to explore agrin signal transduction in cultured cortical neurons. Bath application of either z+ or z-agrin isoforms resulted in marked changes in intracellular Ca2+ concentration specifically in neurons. Propagation of the Ca2+ response was a two-step process characterized by an initial increase in intracellular Ca2+ mediated by ryanodine receptor (RyR) release from intracellular stores, supplemented by influx through voltage-gated calcium channels (VGCCs). Agrin-induced increases in intracellular Ca2+ were blocked by genistein and herbimycin, suggesting that the agrin receptor is a tyrosine kinase. Ca2+ release from intracellular stores activates both calcium/calmodulin-dependent kinase II (CaMKII) and mitogen activated protein kinase (MAPK). Activation of CaMKII is required for propagation of the Ca2+ wave itself, whereas both MAPK and CaMKII play a role in mediating long latency responses such as induction of c-fos. These results suggest that an agrin-dependent tyrosine kinase could play a critical role in modulating levels of intracellular Ca2+ and activity of MAPK and CaMKII in CNS neurons.

Increased calcium influx through acetylcholine receptors in dunce neurons.

Calcium homeostasis was studied in dunce, a Drosophila mutant that is defective in learning and memory. Fura 2-AM fluorescence photometry was used to measure the intracellular calcium concentration in wild type and dunce cleavage-arrested neurons under resting conditions and in response to neurotransmitters. After acetylcholine application, the peak [Ca2+]i was greater in dunce (693 +/- 125 nM) than in wild type neurons (464 +/- 154 nM), but half decay time was shorter in dunce (66 +/- 15 s) than in wild type neurons (104 +/- 40 s). In contrast, the application of glutamate, NMDA, dopamine, and serotonin had no effect on [Ca2+]i. These results indicate that calcium influx through acetylcholine receptors is increased in dunce, compared to wild type neurons. The results also suggest that calcium extrusion to the outside and/or calcium buffering are enhanced in dunce, compared to wild type neurons. This disturbance in the homeostasis of cytosolic calcium concentration in dunce may be implicated in defective associative learning in Drosophila, and may play a role in acute and chronic neurodegenerative disorders in the mammalian brain.

Cardiomyocyte dysfunction in sucrose-fed rats is associated with insulin resistance.

Diabetes is associated with impaired cardiac dysfunction in both humans and animals. Specific phenotypic changes-prolonged action potentials, slowed cytosolic Ca2+ clearing, and slowed relaxation-that contribute to this whole heart dysfunction occur in isolated ventricular myocytes. The present study was designed to determine whether cardiomyocyte abnormalities occur early in the development of type 2 diabetes (in this case, insulin resistance) and whether an insulin-sensitizing drug (metformin) is cardioprotective. In the study, high-sucrose feeding was used to induce whole-body insulin resistance. Wistar rats were maintained for 7-10 weeks on a starch (ST) diet, sucrose (SU) diet, or diet supplemented with metformin (SU + MET). Whole-body insulin resistance was measured in SU and SU + MET rats by performing euglycemic-hyperinsulinemic clamps. Mechanical properties of isolated ventricular myocytes were measured by high-speed video edge detection, and [Ca2+]i transients were evaluated with Fura-2 AM. Untreated SU rats were insulin-resistant (glucose infusion rate [GIR] = 14.5 +/- 1.1 mg.kg(-1).min(-1)); metformin treatment in SU + MET rats prevented this metabolic abnormality (GIR = 20.0 +/- 2.2 mg.kg(-1).min(-1)). Indexes of myocyte shortening and relengthening were significantly longer in SU rats (area under the relaxation phase [AR/peak] = 103 +/- 3 msec) when compared to ST and SU + MET rats (AR/peak = 73 +/- 2 and 80 +/- 1 msec, respectively). The rate of intracellular Ca2+ decay and the integral of the Ca2+ transient through the entire contractile cycle were significantly longer in myocytes from SU than from ST rats (Ca2+ signal normalized to peak amplitude = 152 +/- 8 vs. 135 +/- 5 msec, respectively). Collectively, our data showed the presence of cardiomyocyte abnormalities in an insulin-resistant stage that precedes frank type 2 diabetes. Furthermore, metformin prevented the development of sucrose-induced insulin resistance and the consequent cardiomyocyte dysfunction.

Transport of magnesium by two isoforms of the Na+-Ca2+ exchanger expressed in CCL39 fibroblasts.

Cytoplasmic concentrations of Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were measured with fluorescent indicators in CCL39 cells, a cell line established from Chinese hamster lung fibroblasts, transfected with complementary deoxyribonucleic acid (cDNA) of the Na+-Ca2+ exchanger isolated either from canine heart (NCX1) or from rat brain (NCX3). Raising extracellular [Mg2+] to 10 mM increased Mg2+ influx and the resultant change in [Mg2+]i (delta[Mg2+]i) was monitored with furaptra under Ca2+-free conditions. In control (vector-transfected) cells, delta[Mg2+]i at 45 min was similar with or without extracellular Na+ (130 mM or 0 mM) and when [Na+]i was raised by 1 mM ouabain treatment. delta[Mg2+]i in NCX1-transfected cells was attenuated significantly in the presence of 130 mM Na+, but became comparable to (or slightly larger than) that in control cells on either removal of extracellular Na+ or treatment with 1 mM ouabain. Cells expressing NCX3 showed an intermediate dependence of delta[Mg2+]i on Na+, probably reflecting a lower degree of expression of the exchanger protein. Extracellular Na+-dependent changes in [Ca2+]i (measured with fura-2 in the presence of extracellular Ca2+ and 10 microM ionomycin, a Ca2+ ionophore) were minimal in control cells, marked in the NCX1-transfected cells and intermediate in the NCX3-transfected cells. These results suggest that the Na+-Ca2+ exchanger (either NCX1 or NCX3) can transport Mg2+ and may play a role in the extrusion of magnesium from cells.

Coordinated transitions in neurotransmitter systems for the initiation and propagation of spontaneous retinal waves.

Spontaneous waves of excitation in the developing mammalian retina are mediated, to a large extent, by neurotransmission. However, it is unclear how the underlying neurotransmitter systems interact with each other to play specific roles in the formation of retinal waves at various developmental stages. In particular, it is puzzling why the waves maintain a similar propagation pattern even after underlying neurotransmitter systems have undergone drastic developmental changes. Using Ca(2+) imaging and patch clamp in a whole-mount preparation of the developing rabbit retina, we discovered two dramatic and coordinated transitions in the excitatory drive for retinal waves: one from a nicotinic to a muscarinic system, and the other from a fast cholinergic to a fast glutamatergic input. Retinal waves before the age of postnatal day 1 (P1) were blocked by nicotinic antagonists, but not by muscarinic or glutamatergic antagonists. After P3, however, the spontaneous wave, whose basic spatiotemporal pattern remained similar, was completely inhibited by muscarinic or glutamate antagonists, but not by nicotinic antagonists. We also found that the muscarinic drive, mediated primarily by M1 and M3 receptors, was particularly important for wave propagation, whereas the glutamatergic drive seemed more important for local excitation. Our results suggest (1) a novel mechanism by which a neurotransmitter system changes its functional role via a switch between two completely different classes of receptors for the same transmitter, (2) the cholinergic system plays a critical role in not only early but also late spontaneous waves, and (3) the continued participation of the cholinergic system may provide a network basis for the consistency in the overall propagation pattern of spontaneous retinal waves.

Fluorescence methods to assess multidrug resistance in individual cells.

PURPOSE: Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were developed. METHODS: Multidrug-resistant (MDR) and parental lines of hamster CHO and pituitary GH3 cells were incubated with the acetoxymethylester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by intracellular esterases and then trapped in cells, and the AM forms of the dyes are excellent substrates for P-glycoprotein (Pgp). RESULTS: The fluorescent free acid forms of fura2, indol and fluo3 did not accumulate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)verapamil, quinidine, or progesterone was included during loading to prevent the cells from extruding the AM forms of the dyes before they could be hydrolyzed. Cyclosporin A increased the fluorescence due to intracellularly trapped fura2 free acid from 8- to 20-fold and was maximally effective at < 5 microM. Fluorescence microscopy was employed to measure fura2 free acid accumulation by parental and MDR cell lines using excitation at the Ca2+-insensitive wavelength. When MDR cells were incubated with rhodamine 123 and fura2/AM, no fluorescence was detectable. Cellular fluorescence was dramatically increased by inclusion of cyclosporin A, quinidine, progesterone, or R(+)verapamil. There was no measurable decline in the fura2 free acid fluorescence in 1 h while the fluorescence due to rhodamine 123 diminished rapidly in cells overexpressing Pgp. CONCLUSIONS: These fluorescence methods detect drug-extruding activity in individual cells and therefore have the potential to provide complementary information to studies quantifying protein or mRNA levels of Pgp or other efflux pumps. In addition, they provide a rapid and quantifiable method for screening multidrug resistance reversing agents.

Role of intracellular calcium concentration on tumor cell death from hyperthermia.

The intracellular calcium ion concentration of SCK cells was measured after the cells were heated to determine the relationship between thermotolerance and intracellular calcium concentration. SCK cells, which are breast cancer cells that spontaneously arise in A/J mice, were used. Exponentially growing cells were trypsinized and incubated for 60 min with fura-2/AM at a concentration of 10 M. The incubation was carried out at a density of 1x106 cells/ml in serum-free RPMI medium. Thermotolerance was induced by incubating cells for 5 h at 37yC after heating them at 43yC for 30 min. When heated at 44yC in a calcium containing buffer, the intracellular calcium concentration of non-thermotolerant and thermotolerant cells increased continuously as the cells were heated for longer periods of time. The same increase in intracellular calcium concentration was seen in these two cell lines when heated at 43yC in a calcium-free buffer. Thus, our data indicated that intracellular calcium ion increased during heating due to an influx into the cytoplasm from both internal stores and the extracellular medium.

Ca(2+)-induced Ca2+ oscillations in parathyroid cells.

Parathyroid cells from patients with hyperparathyroidism were first cultured in a growth cocktail supplemented version of Dulbecco's MEM/F12 medium and then used for measurements of the cytoplasmic Ca2+ concentration ([Ca2+]i). Elevation of extracellular Ca2+ from 0.5 to 1.5-3 mM often triggered rhythmic oscillations of [Ca2+]i. Whereas the amplitude of the oscillations remained constant their frequency (0.4-2.6 /min) increased with the Ca2+ concentration and there was sometimes transformation into sustained increase of [Ca2+]i at the highest extracellular Ca2+ concentration. The oscillations were abolished by the inorganic Ca2+ channel blocker Ce3+ and by K+ depolarisation, which lowered [Ca2+]i, as well as by thapsigargin, an inhibitor of the intracellular Ca(2+)-pumping ATPase. The data indicate that extracellular Ca2+ can trigger [Ca2+]i oscillations in parathyroid cells, dependent on mobilisation of the ion from intracellular Ca2+ stores and on influx through non-voltage dependent channels.

Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa.

Progesterone induced a rapid, long-lasting, dose-dependent increase of intracellular free calcium concentration ([Ca2+]i) in human sperm capacitated overnight. This effect was not counteracted by the cytosolic progesterone receptor antagonist RU486 (1 mumol/L) nor by the GABA-A receptor antagonists bicuculline (10 mumol/L) and picrotoxin (50 mumol/L). Also, the rank order of potency of several progestative steroids on [Ca2+]i differed from that previously reported for uterine intracellular progesterone receptor or for P-GABA interaction in the central nervous system, indicating a different pathway for progesterone stimulation of human sperm. Modifications of basal and progesterone-stimulated [Ca2+]i during sperm capacitation were also studied. A progressive, parallel increase of basal and progesterone-stimulated [Ca2+]i in capacitating spermatozoa was found. In particular, progesterone-stimulated [Ca2+]i increased from a basal concentration of 147% +/- 17% at 10 minutes to 327% +/- 65% after 120 minutes of incubation in capacitating medium. This increase was well correlated with basal [Ca2+]i (r = 0.93). In contrast, basal and progesterone-stimulated [Ca2+]i concentrations were constantly low in spermatozoa incubated in noncapacitating medium. In capacitated spermatozoa, initial responsiveness to progesterone and basal [Ca2+]i was higher than in capacitating and noncapacitated samples, and remained constant throughout the duration of the experiment. The progressive, parallel increase of [Ca2+]i and response to progesterone observed during in vitro capacitation of human spermatozoa might be physiologically relevant in vivo during capacitation of sperm in the female genital tract.

Myoplasmic calcium transients in intact frog skeletal muscle fibers monitored with the fluorescent indicator furaptra.

Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura-2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a frog skeletal muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half-width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.

Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2.

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.