Ca-free Medium Research Articles

The Influence of Glutamate on Axonal Compound Action Potential In Vitro.

Background Our previous experiments demonstrated modulation of the amplitude of the axonal compound action potential (CAP) by electrical stimulation. To verify assumption that glutamate released from axons could be involved in this phenomenon, the modification of the axonal CAP induced by glutamate was investigated. Objectives The major objective of this research is to verify the hypothesis that axonal activity would trigger the release of glutamate, which in turn would interact with specific axonal receptors modifying the amplitude of the action potential. Methods Segments of the sciatic nerve were exposed to exogenous glutamate in vitro, and CAP was recorded before and after glutamate application. In some experiments, the release of radioactive glutamate analog from the sciatic nerve exposed to exogenous glutamate was also evaluated. Results The glutamate-induced increase in CAP was blocked by different glutamate receptor antagonists. The effect of glutamate was not observed in Ca-free medium, and was blocked by antagonists of calcium channels. Exogenous glutamate, applied to the segments of sciatic nerve, induced the release of radioactive glutamate analog, demonstrating glutamate-induced glutamate release. Immunohistochemical examination revealed that axolemma contains components necessary for glutamatergic neurotransmission. Conclusion The proteins of the axonal membrane can under the influence of electrical stimulation or exogenous glutamate change membrane permeability and ionic conductance, leading to a change in the amplitude of CAP. We suggest that increased axonal activity leads to the release of glutamate that results in changes in the amplitude of CAPs.

Hake fish bone as a calcium source for efficient bone mineralization

Calcium is recognized as an essential nutritional factor for bone health. An adequate intake is important to achieve or maintain optimal bone mass in particular during growth and old age. The aim of the present study was to evaluate the efficiency of hake fish bone (HBF) as a calcium source for bone mineralization: in vitro on osteosarcoma SaOS-2 cells, cultured in Ca-free osteogenic medium (OM) and in vivo on young growing rats fed a low-calcium diet. Lithotame (L), a Ca supplement derived from Lithothamnium calcareum, was used as control. In vitro experiments showed that HBF supplementation provided bone mineralization similar to standard OM, whereas L supplementation showed lower activity. In vivo low-Ca HBF-added and L-added diet similarly affected bone deposition. Physico-chemical parameters concerning bone mineralization, such as femur breaking force, tibia density and calcium/phosphorus mineral content, had beneficial effects from both Ca supplementations, in the absence of any evident adverse effect. We conclude HBF derived from by-product from the fish industry is a good calcium supplier with comparable efficacy to L.

Role of calcium on the initiation of sperm motility in the European eel

Sperm from European eel males treated with hCGrec was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCl+EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca2+]i fluorescence (and in [Na+]i in some cases) were evaluated by flow cytometry.After sperm motility was activated in a medium containing Ca2+ (seawater, SW) the intracellular fluorescence emitted by Ca2+ increased 4–6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCl+EDTA) resulted in a percentage of motility similar to seawater, the [Ca2+]i levels did not increase at all. This result strongly suggests that increasing [Ca2+]i is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca2+ has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca2+]i concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm.

Effects of sulphonylureas on spontaneous motility and induced contractions in rat isolated uterus.

To clarify the action of sulphonylureas on calcium, the effect of tolbutamide and glibenclamide has been investigated on a Ca-dependent process, the contractile activity of uterine smooth muscle. Both sulphonylureas antagonized the contractions evoked by CaCl2 in a non-competitive manner when the uterus was maintained in depolarizing solution and did not affect the spontaneous contractions of rat uterus. The capacity of tolbutamide and glibenclamide to relax vanadate-induced contraction of rat uterus in Ca-free medium suggests that sulphonylureas may have an intracellular site of action related to cytosolic free Ca levels, or effect a reduction in Ca action.

Role of cell wall bound calcium in Neurospora crassa

Cell wall bound calcium constitutes a significant fraction (25%) of total mycelia calcium in Neurospora crassa. Wall bound calcium increases as a function of growth and calcium concentration, while cell wall bound calcium decreases in Ca-free medium. Removal of wall bound calcium causes its rapid replacement from intracellular pool, inhibited by verapamil, nifedipine, concanamycin A, and wortmanin in a vacuolar mutant (Vma-5), but is unaffected by trifluoropyrazine, and calmidizoluim in a calcineurin mutant (Cnb-1) of N. crassa. Ca 2+ removal from surface with EGTA resulted in leakage of periplasmic enzymes invertase and alkaline phosphatase. Scanning and transmission electron microscopy revealed gross abnormalities represented by giant vacuoles. Toxic metal ions bound to wall fraction by displacing calcium. Our data underline the physiological importance of wall bound calcium in N. crassa.

Blood Pressure-lowering and Vascular Modulator Effects of Acorus calamus Extract Are Mediated Through Multiple Pathways

This investigation was aimed to provide a pharmacologic basis to the medicinal use of Acorus calamus in cardiovascular disorders. In normotensive anesthetized rats, crude extract of Acorus calamus and its ethylacetate and nHexane fractions caused a fall in mean arterial pressure. In rabbit aorta rings, crude extract was more potent against high K (80 mM), ethylacetate against phenylephrine (1 microM), whereas nHexane fraction was equipotent against both precontractions. Crude extract exhibited a vasoconstrictor effect on baseline. Pretreatment of aortic rings with crude extract and its fractions shifted Ca concentration-response curves to the right, similar to verapamil. Crude extract and ethylacetate fraction suppressed phenylephrine peak formation in Ca-free medium. In rat aorta preparations, crude extract exhibited endothelium-independent relaxation with a vasodilatory effect against high K. nHexane fraction caused an endothelium-dependent Nomega-nitro-l-arginine methyl ester-sensitive vasorelaxant along with ryanodine-sensitive vasoconstrictor effect on baseline tension and partially inhibited high K, although ethylacetate fraction caused an endothelium-independent relaxant and endothelium-dependent vasoconstrictor effect. These data indicate that crude extract possesses a combination of effects, relaxant effects mediated possibly through Ca antagonism in addition to a nitric oxide pathway, although the associated vasoconstrictor effects may be meant by nature to offset excessive vasodilatation, thus providing a pharmacologic rationale to its cardiovascular medi-cinal uses.

OOCYTE ACTIVATION BY STRONTIUM IN THE PRESENCE OF CALCIUM SUPPORTS FULL TERM DEVELOPMENT OF SOMATIC CELL CLONED EMBRYOS

Without using sperm, artificial oocyte activation is essential for the current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in mouse, by which efficient oocyte activation requires calcium-free medium but not culture media since they normally contain calcium. In this study, we considered whether strontium with chelated calcium in culture media is enough to efficiently activate oocytes. Ethylene glycol-bis (beta-aminoethyl ether) - N, N, N', N'-tetraacetic acid (EGTA) were added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because they contain EGTA which preferentially binds calcium ion rather than strontium ion. After optimizing EGTA at 2–5 mM along with 5mM strontium in either CZB, M16 or KSOM, more than 80% of available oocytes were parthenogenetically activated, which was as efficient as standard conditions of strontium in a Ca-free medium. Further, it was demonstrated that this activation method can support full-term development of cloned embryos using trichostatin A with comparable success rates. Thus, addition of EGTA into culture media can easily make activation media without additional preparation of Ca-free medium. (poster)

ICP/OES Application for Assessing Cadmium Uptake (or Toxicity) in Glomerular Cells: Influence of Extracellular Calcium

The risks of metals for health are highlighted by their chemical stability and their persistence in the environment. Chronic exposure to low cadmium (Cd) concentrations results in renal dysfunction mainly. Cd has been regarded primarily as a renal tubular toxicant, but glomerular structures may also be affected. Since the cellular environment may influence metal toxicity, differences concerning Cd uptake and toxicity were evaluated according to calcium (Ca) medium concentrations. An optimized inductively coupled plasma emission spectrometry method (ICP/OES) was developed under defined conditions, as a selective analytical tool to determine cadmium uptake in glomerular mesangial cells. The performance characteristics of the analytical system were evaluated for both Cd and Ca by calibration (50 to 250 μg/L and 1 to 5 mg/L), linearity (r 2 .9968 and .9943), limits of detection (1 μg/L and 0.1 mg/L) and quantitation (3 μg/L and 0.3 mg/L), accuracy with spiking, and repeatability (1.2 and 2.9%) with matrix matched standards. Total intracellular Cd content was significantly threefold lower in 0.175 mM Ca medium (Ca-free Eagle's minumum essential medium [EMEM] medium with 5% fetal bovine serum [FBS]) than in EMEM medium (1.8 mM Ca) with respectively 0.16 and 0.37 μg/mg proteins after 24 h of Cd (1 μM) exposure. Similar differences were obtained in cytotoxicity studies with a fourfold reduction in the mortality index (IC50). Complementary assays using Ca-spiked medium reinforced that Cd cytotoxicity and uptake were significantly dependent on the concentration of extracellular Ca. These findings suggest direct link between Cd uptake and toxicity, underlining the relevance of the analytical method.

Potentiation of ATP-induced Ca 2+ mobilisation in human retinal pigment epithelial cells

Interaction of signalling pathways directs the functional output of many cells. This study investigated the consequences of activating adenosine and adrenergic receptors on ATP-induced Ca 2+ responses in human retinal pigment epithelial (RPE) cells. Intracellular Ca 2+ concentration ([Ca 2+]i) of human RPE cells in primary culture was monitored using Fura-2. Cyclic adenosine monophosphate (cAMP) concentration was measured using an enzyme-linked immunosorbent assay. Both ATP and UTP (10 μ m) increased [Ca 2+]i in human RPE cells. Adenosine (10 n m–10 μ m) had no effect on resting [Ca 2+]i, but potentiated a sub-threshold response to ATP (100 n m) when ATP was added in the presence of adenosine. The potentiation occurred with other G-protein receptor agonists such as acetylcholine. Potentiation persisted in Ca-free medium, but was blocked by prior application of thapsigargin. The A 1 and A 2 adenosine receptor antagonists, DPCPX and MRS1706 (100 n m) respectively, inhibited potentiation in 76±7 and 23±12% of cells, respectively, but the A 3 antagonist MRS1191 had no effect. Conversely, agents that activate the cAMP pathway, including isoproterenol (10 μ m), forskolin (10 μ m), and the protein kinase A (PKA) activator Sp-cBIMPS (1 μ m), potentiated the ATP-induced response in the RPE cells. Agents that are known to inhibit the production of cAMP in other systems also caused potentiation, including clonidine (10 μ m) and the Gi-activator mastoparan (10 μ m). Under resting conditions, cAMP concentration in RPE cells was 7·1±0·5 pmol mg −1 protein. Isoproterenol (10 μ m) and forskolin (10 μ m) increased levels to 104·6±5·2 and 113·7±4·2 pmol mg −1 protein, respectively, while adenosine, clonidine, and mastoparan (all 10 μ m) had no significant effect on cAMP levels. These data indicate that whilst activation of A 1 and A 2 adenosine receptors and α 2 and β adrenergic receptors does not influence basal Ca 2+ levels, stimulation of these receptors can potentiate Ca 2+ signalling by cAMP dependent and independent mechanisms in human RPE cells.

Mitochondrial Na+ overload is caused by oxidative stress and leads to activation of the caspase 3- dependent apoptotic machinery.

Oxidative stress is one of the major causes of cell death. Using time-lapse confocal recording of live cardiomyocytes, we showed that H2O2 (OH*) caused a marked increase in Na+ and Ca2+ levels in both the cytosol ([Na]cyt, [Ca]cyt) and mitochondria ([Na]m, [Ca]m). The H2O2-induced intracellular Na+ ([Na]i) overload contributed to the H2O2-induced [Ca]cyt/[Ca]m overload via activation of the reverse mode of the Na-Ca exchanger. When myocytes were treated for 40 min with 100 microM H2O2 in normal medium, then returned to H2O2-free medium, the percentage of apoptotic cells increased from 4% at 0 h to 55 and 85% at 4.5 and 16 h, respectively. H2O2-induced apoptosis was completely prevented by using Na-free, but not Ca-free, medium. When a Na+ ionophore cocktail in Ca-free medium was used instead of H2O2 to increase the [Na]i by more than 30 mM without any change in the [Ca]i, cytochrome c release and caspase 3-dependent apoptosis occurred, showing that [Na]i overload per se induced apoptosis. We also showed that the increase in the mitochondrial, but not the cytosolic, Na+ levels resulted in the opening of the permeation transition pore, followed by cytochrome c release. Our findings therefore suggest that H2O2-induced [Na]m overload is an important upstream signal for the apoptotic machinery, and the prevention of [Na]m overload thus represents a particularly attractive target for strategies aimed at preventing oxidative stress-induced cell death.

Effect of the aqueous extract of Chrysanthellum indicum on calcium mobilization and activation of rat portal vein

The effects of the aqueous extract of Chrysanthellum indicum (CI) on calcium activation and mobilization were studied using the rat portal vein. The extract caused a concentration-dependent contraction of the portal vein. KCl (80 mM), norepinephrine (NA, 10 −6 M) and CI (4 mg/ml) evoked sustained contraction of the portal vein. In Ca-free medium (with EGTA) the contractions evoked by these agents were reduced significantly. The times-to-peak of KCl, NA and CI were similar -in normal PSS, but in Ca-free medium the times-to-peak for KCl and CI were greatly increased. The contractions induced by CI were not inhibited by chlorpropamide and prazosin, but were blocked by verapamil. The data obtained suggest that the aqueous extract of CI utilizes extracellular calcium pools to bring about contractile response and this effect might be mediated through the activation of potential-sensitive channels.

Mechanism of glutamate stimulation of CO production in cerebral microvessels.

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.

Regulation of CO production in cerebral microvessels of newborn pigs.

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.

Contractile responses of the human umbilical artery to KCl and serotonin in Ca-free medium and the effects of levcromakalim

It is known that K ATP channel openers inhibit the release and refilling of Ca 2+ from intracellular stores. The present study was designed to test the effects of levcromakalim in human umbilical artery (HUA) rings stimulated by serotonin (5-HT) and KCl in Ca-free medium. Umbilical cords were obtained at vaginal or cesarean deliveries from healthy, term pregnancies. After the isolation, HUA rings were placed in organ baths in solution with indomethacin (10 −5 M) and N G-nitro-L-arginine methyl ester (L-NAME) (10 −3 M) at 37 °C and aerated with 95% O 2 and 5% CO 2 for the measurement of isometric force. In Ca-free solution with Ethylene glycol-bis (ß-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (2 mM) the contractions produced by 5-HT (10 −6 M) and KCl (40 mM) decreased significantly. Afterwards, HUA rings were treated with 5-HT and KCl in repeated manner in Ca-free medium. In contrast to KCl, 5-HT induced contractions reduced in each application, progressively. Levcromakalim (10 −4 M) abolished the contractions elicited by 5-HT. On the other hand, levcromakalim had a little but significant inhibitory effect on KCl induced contraction in Ca-free medium. These results suggest that Ca 2+ is not the only transduction pathway in KCl produced contractions of HUA smooth muscle cells.

Recycle use of Sphingomonas sp. CDH-7 cells for continuous degradation of carbazole in the presence of MgCl2.

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 m M K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 m M MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 m M EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 m M MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 m M) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 m M) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

A Calcium Aspect of the Effect of Noradrenaline on the Resting Membrane Potential in Somatic Muscle Cells of the Earthworm

A hyperpolarizing effect of noradrenaline (NA) on muscle cells of the earthworm caused by activation of the membrane ion pumps is eliminated in a Ca-free medium, in the case of replacement of Na+ by Mn2+, or when verapamil or chlorpromazine have been added to the bath solution. A decrease or an increase in the Ca2+ concentration in the solution, as well as caffeine application, do not influence the resting membrane potential (RMP) of muscle cells. It is supposed that signal transmission from the membrane adrenoreceptors to the ion pump of earthworm muscle cells by NA is provided via entry of extracellular Ca2+ ions into the cell with subsequent involving of Ca2+ acceptor proteins similar to calmodulin in vertebrate animals.

Bradykinin induced contraction of human gallbladder muscle in vitro

Aim To examine the mechanisms of Bradykinin (BK)-induced contraction on human gallbladder muscle and to investigate possible receptors that may mediate this action. Methods A full-thickness segment from the anterior aspect of healthy and stone-hearing gallbladders was removed and placed in cold Krebs solution in an organ bath. Concentration-response curves (CRCs) were obtained for BK (0.1-100/LM). The BK response curves were repeated in the presence of a number of antagonists and channel blockers BI & B2 receptor antagonists, SR 27897 (0.5nM,a CCK-A receptor antagonist), mepyramine (l/LM, a histamine receptor antagonist), atropine (l/LM, a muscarinic receptor antagonist), prazosin (O.l/LM,a adrenoceptor blocker), tetrodotoxin (O.03/LM, a Na channel blocker), quinidine (l/LM, a K channel blocker) and indomethacin (3/LM, a cyclo-oxygenase inhibitor). Extracellular Ca involvement was examined by use of Ca free medium. Ryanodine (lO/LM) was used to examine intracellular involvement. Ethics committee approval for tissue sampling was obtained. Statistical significance was performed using t-test, P < .05 or less was considered as indicative of significance. Results Healthy (n=6) and stone-bearing (n=9) gallbladders showed similar and repeatable CRCs to BK (EDso=4.95 & 5.5/LM). The BK response was significantly reduced only in healthy strips by BI receptor blockade (EDso=31.5/LM) and in both tissue types by B2 receptor blockade (EDso=37.2 & 29./LM). Neither antagonist affected the maximum BK response. No significant rightward shift of the response to BK was observed in the presence of mepyramine (n=4), atropine (n=), prazosin (n=6), tetrodotoxin (n=), quinidine (n=7) or SR27897 (n=5). A significant rightward shift of the curve was noted in the presence of indomethacin (n=20,EDso=55/LM). BK-induced contraction was not modified in Ca-free medium (n=7), however the presence of ryanodine caused a significant rightward shift of the BK dose-response curve (n=7,EDso=46/LM. Conclusion Gallbladder strips contract to BK in a dose-dependent manner. A difference in receptor population seems to exist between normal and stone-bearing gallbladders. BK appears to have a direct myogenic effect on the human gallbladder and this may be mediated by a third BK receptor or a BK-independent mechanism. The contraction occurs independently of extracellular Ca but intracellular Ca is vital. In addition, part of this BK-induced contraction is probably mediated by the release of a cyclo-oxygenasc-dcrived product from the arachidonic acid pathway.

Regulatory processes on the cytoplasmic surface of the Na(+)/Ca(2+) exchanger from lobster exoskeletal muscle.

A partially purified preparation of the lobster muscle Na(+)/Ca(2+) exchanger was reconstituted with, presumably, random orientation in liposomes. Ca(2+) efflux from (45)Ca-loaded vesicles was studied in exchanger molecules in which the transporter cytoplasmic surface was exposed to the extravesicular (ev) medium. Extravesicular Na(+) (Na(ev))-dependent Ca(2+) efflux depended directly upon the extravesicular Ca(2+) concentration ([Ca(2+)](ev)) with a half-maximal activation at [Ca(2+)](ev) = 0.6 microm. This suggests that the lobster muscle exchanger is catalytically upregulated by cytoplasmic Ca(2+), as in most other species. In contrast, at low [Na(+)](ev), the Ca(ev)-binding site (i.e., on the cytoplasmic surface) for Ca(2+) transported via Ca(2+)/Ca(2+) exchange was half-maximally activated by about 7.5 microm Ca(2+). Mild proteolysis of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin also upregulated the Na(ev)-dependent Ca(2+) efflux. Following proteolytic digestion in Ca-free medium, the exchanger was no longer regulated by nontransported ev Ca(2+). Proteolytic digestion in the presence of 1.9 microm free ev Ca(2+), however, induced only a 1. 6-fold augmentation of Ca(2+) efflux, whereas, after digestion in nominally Ca-free medium, a 2.3-fold augmentation was observed; Ca(2+) also inhibited proteolytic degradation of the Na(+)/Ca(2+) exchanger measured by immunoblotting. These data suggest that Ca(2+), bound to a high affinity binding site, protects against the activation of the Na(+)/Ca(2+) exchanger by alpha-chymotrypsin. Additionally, we observed a 6-fold increase in the Na(+)/Ca(2+) exchange rate, on average, when the intra- and extravesicular salt concentrations were increased from 160 to 450 mm, suggesting that the lobster muscle exchanger is optimized for transport at the high salt concentration present in lobster body fluids.

Mechanisms of calcium transport in human endothelial cells subjected to hyperthermia

We have probed the mechanisms by which severe heat alters cytosolic calcium ion concentrations (Cai) in individual cultured human endothelial cells (ATCC ♯1998). Cells adhering to glass coverslips were heated to as high as 50°C and Cai determined by means of a fluorescence laser imaging system using the calcium-sensitive dye, indo-1, in the presence of thapsigargin, and in Na-free and Ca-free media. Baseline Cai varied between 175–225 nM. When cells were heated to 50°C in a complete Ca-containing medium, there was first a transient fall in Cai, then a rapid rise of 50–100 nM in Cai, followed by a slower, secondary rise of 50–75 nM. Depleting the intracellular calcium stores with thapsigargin blocked both the transient fall and the secondary rise in Cai. Placement of the cells into a Ca-free medium blocked both the transient fall and the initial rapid rise, while use of a Na-free buffer prevented the initial rapid rise only. These data suggest that in human endothelial cells, extreme heat accelerates the CaATPase pumps of the intracellular Ca stores causing the transient fall in Cai which is soon followed by activation of the reverse mode of the Na/Ca exchanger to cause the initial rapid rise in Cai. The Ca-release channels of the intracellular stores become activated by heat to cause the secondary, slow rise in Cai. This preliminary work indicates that the application of heat to cultured cells can be a useful probe to examine the kinetics and unmask mechanisms of intracellular Ca fluctuations.

Are salivary glands cell lines in culture a good model for purinergic receptors in salivary glands?

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic, mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in MY cells shows single Michaelis-Menten kinetics with an apparent K a of 0.8 μM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca 2+ ] i rise. Both ATP (10 μM) and carbachol (100 μM) increased IP 3 production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix. We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP 3 Production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP 3 production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca 2+ ] i and IP 3 means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.