Cytoplasmic Free Calcium Levels Research Articles

ACTIVATION OF DOMESTIC CAT OOCYTES USING THE SULFHYDRYL REAGENT THIMEROSAL

Domestic cats are useful as animal models in comparative medicine programs as they provide insight into disease mechanism and facilitate investigation of new therapeutic options. The application of cloning in these animals is also beneficial in terms of conserving the precious animal models. Although successful cat cloning by nuclear transfer has recently been reported, the technology is still extremely ineffective. One potential reason for the low efficiency is suboptimal embryo development after oocyte activation. During nuclear transfer, the calcium signals that are normally triggered by the fertilizing sperm must be reproduced artificially to initiate the development of the reconstructed embryos. Thimerosal is a chemical which is able to oxidize critical sulfhydryl groups on intracellular calcium release proteins and thus induce the release of calcium from the intracellular stores and trigger oocyte activation. The effects of thimerosal have previously been reported in a number of species; the objective of this study was to evaluate whether thimerosal can induce calcium release and initiate parthenogenetic embryo development in the oocyte of the domestic cat. Cat ovaries were obtained from a local veterinary clinic. Immature oocytes collected from the ovaries were matured in vitro in Feline Optimized Culture Medium (FOCM) supplemented with 0.6 mM cysteine, 0.1 mM cysteamine, 1.0 IU/ml eCG, 2.0 IU/ml hCG, 25 ng/ml EGF, for 24 hours. For intracellular calcium measurements, cat oocytes were incubated in the presence of 2 μM fura-2 AM, a calcium indicator dye and 0.02% pluronic F-127, for 40 minutes. Individual oocytes were then transferred into TL-Hepes medium containing 200 μM thimerosal. Changes in the cytoplasmic free calcium levels were monitored using the InCyt Im2 fluorescence imaging system. Preimplantation embryonic development was also evaluated; for this purpose the oocytes were incubated in TL-Hepes in the presence of 200 μM thimerosal for 30 minutes followed by a 20-min incubation in TL-Hepes with 8 mM dithiothreitol, a sulfhydryl reducing agent. Control oocytes were cultured in TL-Hepes for 50 minutes. After the treatments, the oocytes were held in 7.5 μg/ml cytochalasin B for 4 hours to stimulate the formation of diploid embryos then cultured in FOCM for 6 days. The total number of nuclei in the developing embryos was recorded by staining with Hoechst 33342. After the addition of thimerosal, a series of transient rises in the intracellular free calcium concentration were observed in 81.8% (9 out of 11) of the oocytes during the 30-min incubation period. Furthermore, thimerosal induced cleavage in 21.8% (14/64) of the activated oocytes; 4.7% (3/64) of the oocytes developed to morula or blastocyst stage. The average cell number of those morulae/blastocysts was 19.7±3.2. In the control group, no cleavage was observed (0/43). The results support the hypothesis that thimerosal can induce an oscillation in the intracytoplasmic free calcium levels in oocytes by oxidizing sulfhydryl groups on intracellular calcium release proteins. The series of calcium increases was sufficient to trigger the signal transduction pathway in cat oocytes that resulted in embryonic development up to the blastocyst stage. The results indicate the potential application of thimerosal for oocyte activation during cat nuclear transfer. Additional studies are needed for the optimization of the activation parameters. (poster)

Regulatory roles for mitochondria in the peri-implantation mouse blastocyst: possible origins and developmental significance of differential ΔΨm

Studies of mitochondria in mouse and human oocytes and preimplantation stage embryos have focused primarily on their metabolic capacity to generate ATP. However, it is becoming increasingly apparent that mitochondria are also regulatory agents in other processes involved in the establishment of developmental competence, including calcium homeostasis and apoptosis. The magnitude of the inner mitochondrial membrane potential, or its polarity (DeltaPsim), is a physiochemical property of mitochondria related to levels of organelle activity, and differences in the magnitude and spatial distribution of high- and low-polarized mitochondria have been suggested to influence oocyte and early embryo competence. Here, we investigated mitochondrial polarity in normal and diapausing peri-implantation-stage mouse blastocysts, and their corresponding outgrowths, for indications of cell-type-specific regulatory functions or activities in which these organelles may be engaged. The results demonstrate that cell-type- and location-specific domains of differential DeltaPsim exist in the peri-implantation blastocyst and remain unchanged during blastocyst outgrowth and during delayed implantation, which for the latter, is accompanied by the suppression of mitochondrial oxidative phosphorylation. Our findings demonstrate that cell-type-specific DeltaPsim in the peri-implantation blastocyst is not an intrinsic property of the corresponding mitochondria but one that can be mediated by the dynamics of intercellular contact. Cells with high- or low-polarized mitochondria are differentially affected by photosensitization, with developmental consequences related to embryo behavior and outgrowth performance. Differences in polarity are discussed with respect to the participation of mitochondria in regulatory and morphogenetic processes in the normal peri-implantation embryo. The persistence of high DeltaPsim in the diapausing embryo is suggested to be associated with the regulation of levels of cytoplasmic free calcium and the ability of the embryo to reactivate development when delayed implantation terminates.

Systematic significance of cell inclusions in Haemodoraceae and allied families: silica bodies and tapetal raphides.

This paper presents the first record of silica deposits in tissues of Haemodoraceae and adds new records of tapetal raphides in this family. Within the order Commelinales, silica is present in leaves of three families (Hanguanacaeae, Haemodoraceae and Commelinaceae), but entirely absent from the other two (Pontederiaceae and Philydraceae). Presence or absence of characteristic cell inclusions may have systematic potential in commelinid monocotyledons, although the existing topology indicates de novo gains and losses in individual families. Silica sand was observed in leaves of five out of nine genera examined of Haemodoraceae, predominantly in vascular bundle sheath cells and epidermal cells. Within Haemodoraceae, silica is limited to subfamily Conostylidoideae. The occurrence of silica in Phlebocarya supports an earlier transfer of this genus from Haemodoroideae to Conostylidoideae. The presence of raphides (calcium oxalate crystals) in the anther tapetum represents a rare character, only reported in a few monocot families of the order Commelinales, and possibly representing a mechanism for regulation of cytoplasmic free calcium levels. Tapetal raphides were observed here in Anigozanthus and Conostylis (both Haemodoraceae), and Tradescantia (Commelinaceae), thus supplementing two earlier records in Haemodoraceae, Philydraceae and Commelinaceae.

Making sense of oxygen sensing.

Making sense of oxygen sensing.

Deficiency of presenilin-1 increases calcium-dependent vulnerability of neurons to oxidative stress in vitro.

We examined the function of presenilin-1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1-deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild-type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L-type voltage-dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2-induced death, while an N-type voltage-dependent calcium channel blocker, omega-conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild-type and PS1-deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1-deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium-dependent manners.

Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury

Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca 2+] n), perinuclear ([Ca 2+] pn), and cytoplasmic free calcium ([Ca 2+] i) levels are regulated by the concentration of extracellular free magnesium ions ([Mg 2+] o). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca 2+ responses to stepwise reduction in [Mg 2+] o (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg 2+] o-containing incubation media, basal mean [Ca 2+] i was 89.6±15 nM. Lowering [Mg 2+] o to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca 2+] i going to 213±43, 368±67, 471±77, and 642±98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg 2+] o, the higher the [Ca 2+] i. Restoration of [Mg 2+] o to normal caused decreases in [Ca 2+] i to 215.9±42.3 nM, but only complete removal of [Ca 2+] o returned [Ca 2+] i to basal levels. Results show that basal [Ca 2+] pn (282±92 nM) exceeds basal cytoplasmic Ca 2+ (61±27.8 nM) and [Ca 2+] n (20±7.6 nM). However, reduction of normal [Mg 2+] o to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca 2+] pn (1503±102 nM)>cytoplasmic Ca 2+ (688±49 nM)≡[Ca 2+] n (674±12 nM). Nuclear Ca 2+ rose dramatically (e.g., 35–40 times basal levels). Both verapamil (1 μM) and Ni 2+ (5 mM) prevented, completely, the rises in Ca 2+ in all compartments, suggesting that Mg 2+-dependent Ca 2+ accumulation may be dependent on nuclear, endoplasmic reticulum–Golgi, and cytoplasmic L-type voltage membrane-regulated Ca 2+ channels. The normally low [Ca 2+] n suggests that Ca 2+ does not transport passively across the nuclear membrane in cerebral vascular smooth muscle cells. These results may help to explain much of the impact of hypomagnesemic states on cerebral–central nervous system pathobiology, and, particularly, alcohol-induced strokes.

Identification of Membrane Calcium Channels Essential for Cytoplasmic and Nuclear Calcium Elevations Induced by Vascular Endothelial Growth Factor in Human Endothelial Cells

Vascular endothelial growth factor (VEGF) is mitogenic for endothelial cells and has been shown to induce angiogenesis and endothelial cell migration through stimulation of endothelial tyrosine-kinase receptors. Here, using confocal microscopy and the patch-clamp technique on endothelial cells, membrane permeability to calcium as well as cytoplasmic and nuclear free calcium levels have been investigated in the fmt stages of tyrosine-kinase receptor activation by VEGF. VEGF (0.5nM) as well as inositol trisphosphate (IP3) induced an activation of membrane calcium-permeable channels exhibiting a similar low conductance in the range of 10 pS. The VEGF-triggered activation of these calcium channels, mediated by IP3 and involving the intracellular calcium stores, results in an increase in both cytoplasmic and nuclear calcium levels in endothelial cells, potentially modulating gene expression. Finally, the effect of Ni2+, a calcium channel blocker, on endothelial cell proliferation has been studied. The results show that inhibition of extracellular calcium influx significantly inhibits VEGF-induced cell proliferation. In the process of cell stimulation by VEGF, and possibly by other growth factors, activation of calcium channels could then be a key step in calcium-regulated gene expression and cell activation. These results suggest that the use of calcium channel blockers could be a novel way of prevention or reversion of VEGF-induced tumoral angiogenesis.

Small molecular weight heat shock-related protein, HSP20, exhibits an anti-plate activity by inhibiting receptor-mediated calcium influx

We have shown that Hsp20, one of small molecular weight heat shock protein, which is present at a high concentration both in vascular smooth muscle cells and in circulating blood in patient with vascular disease, strongly inhibits platelet aggregation in vitro and ex vivo. To clarify the mechanism, we investigated the effect of Hsp20 on free calcium concentration in human platelet cytoplasm using fura 2. Hsp20 inhibited thrombin-induced calcium influx without affecting calcium release from intracellular calcium stores. The degree of inhibition is well-correlated with that of suppression of thrombin-induced platelet aggregation by this substance. Hsp20 also inhibited the elevation of cytoplasmic free calcium level triggered by collagen, but not that by A-23187. In contrast, Hsp28, another type of small molecular weight Hsp, failed to affect the cytoplasmic free calcium level. These findings suggest that Hsp20 inhibits the receptor-mediated calcium influx of platelets without affecting calcium release from intracellular calcium stores, leading to its anti-platelet activity.

Inhibitory Effects of Sasa senanensis Rehder Extract (SE) on Calcium-Ionophore A23187-Induced Histamine Release From Rat Peritoneal Exudate Cells

The effects of Sasa senanensis Rehder extract (SE) by alkaline hydrolysis on histamine release from rat peritoneal exudate cells (PECs) were examined. Preincubation with SE for 5 min suppressed calcium-ionophore A23187-induced histamine release in a concentration-dependent manner. A23187 evoked a quick increase in cytoplasmic free calcium ([Ca2+]i) levels in the presence of extracellular calcium. Preincubation with SE also had an inhibitory effect on calcium influx increases induced by A23187. These results indicate that SE prevents degranulation from rat PECs by inhibiting [Ca2+]i level elevation.

Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells.

The extracellular matrix protein "elastin" is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium- and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.

Modulation of intercellular adhesion molecule 1 (ICAM-1) expression on the human mast-cell line (HMC)-1 by inflammatory mediators

The effect of inflammatory mediators on the expression of several surface adhesion molecules on the human mast-cell line (HMC)-1 was studied. By flow cytometry, it could be shown that among several surface adhesion molecules (ICAM-1/CD54, VLA-4/CD49d, Mac-1/CD11b, LFA-1/CD11a, LFA-2/CD2, LFA-3/CD58, VCAM-1), only the constitutively expressed immunoglobulin family member intercellular adhesion molecule-1 (ICAM-1) is modulated by proinflammatory cytokines on HMC-1 mast cells. Stimulation with tumor necrosis factor-a (TNF-alpha) and interferon-gamma (IFN-gamma) resulted, in addition to interleukin-(IL-)4, in selective upregulation of ICAM-1 expression. Costimulation of either IL-4 or IFN-gamma with TNF-alpha further increased the ICAM-1 expression as compared to the stimuli alone. In contrast, stem-cell factor (SCF), granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-10, IL-8, monocyte chemotactic and activating factor (MCAF), and the complement split product C5a failed to modulate the expression of any adhesion molecule examined. The levels of cytoplasmic free calcium in HMC-1 mast cells were not altered by cross-linking surface ICAM-1, suggesting linkage of other intracellular signaling pathways. This cytokine-induced upregulation of ICAM-1 expression might reveal a putative regulatory mechanism of mast-cell interaction with effector cells bearing the counterparts of ICAM-1 (CD54), the molecules Mac-1 (CD11b/CD18) and leukosialin (CD43), and the principal ligand LFA-1 (CD11a/CD18).

Ca 2+ homeostasis and Ca 2+ signalling in sulphur mustard-exposed normal human epidermal keratinocytes

To investigate the role of increased cytoplasmic free calcium levels ([Ca] i) in sulphur mustard-induced cellular toxicity, the [Ca] i of human epidermal keratinocytes exposed to sulphur mustard concentrations between 50 and 800 μM were measured. No early increase in [Ca] i was observed during the first 20 min after application of the agent. Furthermore, at 3, 6 and 24 h after exposure to sulphur mustard, the [Ca] i of sulphur mustard-exposed cells differed only slightly from that of control cells. However, sulphur-mustard exposed cells responded with a weaker rise of [Ca] i upon stimulation with histamine and ATP than control cells. These results suggest that sulphur mustard can induce small increases in [Ca] i, which reflect abnormal cellular physiology rather than acute toxicity. In addition, it has been shown that sulphur mustard disturbs cellular Ca 2+ signalling in human epidermal keratinocytes.

Complementary vascular-protective actions of magnesium and taurine: A rationale for magnesium taurate

By a variety of mechanisms, magnesium functions both intracellularly and extracellularly to minimize the cytoplasmic free calcium level, [Ca 2+] i. This may be the chief reason why correction of magnesium deficiency, or induction of hypermagnesemia by parenteral infusion, exerts anti hypertensive, anti-atherosclerotic, anti-arrhythmic and antithrombotic effects. Although the amino acid taurine can increase systolic calcium transients in cardiac cells (and thus has positive inotropic activity), it has other actions which tend to reduce [Ca 2+] i. Indeed, in animal or clinical studies, taurine lowers elevated blood pressure, retards cholesterol-induced atherogenesis, prevents arrhythmias and stabilizes platelets — effects parallel to those of magnesium. The complex magnesium taurate may thus have considerable potential as a vascular-protective nutritional supplement, and might also be administered parenterally, as an alternative to magnesium sulfate, in the treatment of acute myocardial infarction as well as of pre-eclampsia. The effects of magnesium taurate in diabetes deserve particular attention, since both magnesium and taurine may improve insulin sensitivity, and also may lessen risk for the micro- and macrovascular complications of diabetes.

Cytoplasmic calcium level and membrane fluidity of platelets contacting poly(acrylamide-co-methacrylic acid) particles with different surface properties.

Changes in cytoplasmic free calcium levels and membrane fluidity of platelets in contact with poly(acrylamide-co-methacrylic acid) (PAAmMAc) particles were examined to analyze the mechanistic aspect of regulating platelet function. Our previous studies demonstrated interesting features of PAAmMAc particles during interaction with platelets: (1) PAAmMAc particles induce no calcium increase but enhance membrane fluidity of platelets: (2) thrombin induces no calcium increase in platelets when the platelets were mixed previously with PAAmMAc particles; and (3) PAAmMAc particles induce a calcium increase in platelets when they were treated previously with sodium azide (NaN3). These results suggest the possibility that PAAmMAc surfaces may regulate the calcium level by influencing platelet metabolism. In this study, non-cross-linked PAAmMAc solution with the same chemical composition as the particles showed a suppressive effect on thrombin-induced calcium increase, but, no influence on membrane fluidity. This result indicates that aggregated macromolecular surface assemblies of PAAmMAc may dominate the increase in membrane fluidity of platelets although the calcium change is induced by discrete molecular level interaction between the PAAmMAc and platelet membranes. It was also revealed that the suppression of thrombin-induced calcium increase and the membrane fluidity increase in platelets by PAAmMAc particles were reduced by albumin-treatment of the particles. This result suggests that such phenomena may be due to a decrease in any physicochemical interaction of PAAmMAc surfaces with albumin, rather than platelet metabolic change. PAAmMAc particle surfaces with higher carboxyl groups exhibited a more suppressive effect on thrombin-induced calcium increase, whereas those with lower carboxyl groups derived a higher calcium increase when the platelets were treated previously with NaN3. These results suggest the importance of electrostatic and any other physicochemical interaction of PAAmMAc chains on regulating cytoplasmic calcium levels.

Chronic overcrowding decreases cytoplasmic free calcium levels in T lymphocytes of aged CBA/CA mice.

Intracellular calcium concentration is a sensitive marker of the homeostasis of living cells, and its increase is an essential step of T lymphocyte activation. Changes in the environment provoke an adaptive stress-response of the organism. In our present work we have investigated the effect of chronic overcrowding on resting and lectin-stimulated cytoplasmic free calcium concentration of splenic T lymphocytes from young and aged CBA/CA mice (50 animals total). The animals were kept under 'normal' (68 cm2/animal) or 'overcrowded' (22 cm2/animal) conditions for 3 months. Young animals showed no change in resting and stimulated calcium after overcrowding. T cells from aged mice, however, displayed significantly smaller levels of both resting and lectin-stimulated intracellular calcium concentration (p < 0.01 each), as compared to those of the non-stressed, aged animals. This inadequate adaptation in the calcium metabolism of T lymphocytes may significantly contribute to the diminished immune response of the aged in stress.

FK 506 and cyclosporin each block antigen-induced T cell receptor signalling that is dependent on CD 4 co-receptor and operates in the absence of detectable cytoplasmic calcium fluxes

The T cell hybridoma "171", which lacks CD4 but expresses T cell receptor (TCR) for hen egg white lysozyme, requires introduction of wild-type CD4 for antigen-mediated induction and secretion of interleukin-2 (IL-2). Mutant CD4, which fails to associate with the tyrosine kinase p56lck does not support IL-2 secretion, suggesting that a role of CD4 is to bring cytoplasmic p56lck into alignment for signal transduction to the IL-2 promotor. Using 171, 171-CD4 (wild-type) and 171-CD4 (mutant), we found that IL-2 secretion was inhibited by FK 506 and cyclosporin but not by rapamycin. However, this inhibition was not associated with calcium fluxes since no change in cytoplasmic free calcium levels ([Ca]i; resting level 80 nM) was detectable during antigen stimulation of the 171 or 171-CD4 cells. Thus, although FK 506 and cyclosporin inhibited calcium-dependent signalling to the IL-2 promoter via inhibition of the protein phosphatase calcineurin, it is possible that IL-2 induction via TCR/CD4 requires an FK 506 (and cyclosporin) sensitive step which is independent of cytoplasmic calcium changes.

Contractile failure in early myocardial ischemia: Models and mechanisms

In early myocardial ischemia we find a number of salient electrical and ionic alterations. This article reviews action potential shortening, K accumulation, and contractile failure. Enhanced K efflux during early myocardial ischemia has been attributed to a number of mechanisms, including: the inhibition of active K uptake, osmotic changes, efflux of K ions linked to anion extrusion, cation exchange, altered cellular energy levels, in particular, the opening of ATP-dependent K channels, the involvement of other ion channels, a H/K-ion exchanger, and a catecholamine-dependent pathway. The different mechanisms are discussed. Action potential shortening was described as a salient characteristic of myocardial ischemia in 1954 by Trautwein and Dudel, and was attributed to enhanced outward current. Recently it has been shown by several authors that ATP-dependent potassium channels play a key role in this context. Contractile failure in early myocardial ischemia has been explained by shortening of the action potential duration, reduced cytoplasmic free calcium levels, intracellular acidification, and a rise in inorganic phosphate and Mg. In summary, it is concluded that ATP-dependent K channels may be involved in each of these three phenomena.

The survival of developing neurons: A review of afferent control

Developmental cell death is a major event of neurogenesis, and emphasis has systematically been placed on the roles of either the peripheral targets or central postsynaptic neurons in the control of neuronal survival. In this article, the main types of experimental design used to test the control of neuronal death by the afferent supply are compared with analogous data indicating neurotrophic support by the targets. It is argued that targets and afferents may have equivalent roles and interact in the control of neuron numbers during development of the vertebrate nervous system. Possible mechanisms of anterograde trophic control include contact-mediated cell interactions, activity-dependent processes mediated by neurotransmitters or neuromodulators, modulation of the levels of cytoplasmic free calcium and the involvement of neurotrophic factors.

Synergistic insulin release induced by glucose and carbachol is not associated with an increase in cytoplasmic free calcium levels.

Synergistic insulin release is observed in combination with muscarinic agonist and glucose. In order to define the role of changes in the cytoplasmic free Ca2+ level ([Ca2+]i), the effect of combination with carbachol and glucose on both phosphoinositide breakdown in rat pancreatic islets and [Ca2+]i dynamics in single fura 2-loaded B-cells was investigated. When the islets were prelabeled with myo-[2-3H]inositol, phosphoinositide breakdown was obtained and inositol trisphosphate levels were increased in the presence of carbachol (0.5 mM). Inositol trisphosphate levels induced by carbachol were further increased by co-stimulation with 16.7 mM glucose. [Ca2+]i in single-B-cells was increased by 16.7 mM glucose. A prompt, transient rise in [Ca2+]i was induced by carbachol. However, carbachol failed to augment the [Ca2+]i response in the presence of 16.7 mM glucose. These data suggest that a synergistic release of insulin occurs without an amplification of [Ca2+]i regardless of an increase in inositol trisphosphate.

Prevention of changes in platelet cytoplasmic free calcium levels by interaction with 2-hydroxyethyl methacrylate/styrene block copolymer surfaces.

Changes in platelet cytoplasmic free calcium levels were investigated in contact with cast film surfaces of a block copolymer containing 2-hydroxyethyl methacrylate (HEMA) and styrene (St) (0.5 mole fraction HEMA). These copolymer surfaces demonstrate microdomain alternating lamellae structures composed of hydrophilic HEMA domains (5 nm width) and hydrophobic St domains (20 nm width). The results were compared with those obtained from platelets contacting a random copolymer of HEMA-St (0.5 mole fraction HEMA) and from homopolymers of polystyrene (PSt) and poly(2-hydroxyethyl methacrylate) (PHEMA). Cytoplasmic free calcium levels in platelets contacting the microdomain structured surfaces of the HEMA-St block copolymer remained relatively constant in contrast to the significant increases observed for the radically prepared HEMA-St copolymer, PSt, and PHEMA surfaces. Adhering platelets were stimulated by exogenously introduced thrombin and calcium ionophore A23187 20 min after platelet adherence to the polymer surfaces. Only platelets on the block copolymer surfaces showed active metabolic responses. These results suggest that adhering platelets on the microdomain structured surfaces maintain high sensitivities to external stimulation due to an intrinsic strong inhibition of platelet functional changes induced by surface contact.