Imaging of Ca2+ Transients in Endothelial Cells of Single Perfused Capillaries: Correlation of Peak [Ca2+]i with Sites of Macromolecule Leakage

Objective: To investigate the mechanisms responsible for variation in the macromolecular leakage (formation of localized leaky sites) in venular microvessels with increased permeability, we examined the hypothesis that cytoplasmic calcium concentration [Ca²⁺]_i, does not increase uniformly within microvessel endothelial cells. Methods: We loaded the endothelial cells forming the walls of venular microvessels in frog mesentery with fura-2, and imaged [Ca²⁺]_i using a cooled CCD camera. Results: Control [Ca²⁺]_i was close to 60 nM in all regions. Control permeability was uniformly low in all microvessels. Exposure to ionomycin (5 mM) increased [Ca²⁺]_i in a biphasic manner, but not uniformly. There was variation in both time to peak (bimodal distribution) and peak [Ca²⁺]_i (274 ± 13 nM; mean variation above or below the peak value was 110 nM). Raising extracellular calcium from 1.1 to 5 mM increased the mean variation of [Ca²⁺]_i about peak values. Extravascular leakage of fluorescently labeled albumin or low-density lipoproteins was most prominent at sites where increases in [Ca²⁺]_i were largest. Conclusions: These data indicate that variation in [Ca²⁺]_i within individual endothelial cells or groups of cells could account, at least in part, for the distribution of localized leakage sites for macromolecules in venular microvessels in the high-permeability state.     

Calcium elevation in mouse pancreatic beta cells evoked by extracellular human islet amyloid polypeptide involves activation of the mechanosensitive ion channel TRPV4

Aims/hypothesis  To investigate the mechanism by which human islet amyloid polypeptide (hIAPP) fibril formation results in calcium influx across the plasma membrane of pancreatic beta cells, and its association with apoptosis. Methods  Cytoplasmic intracellular calcium concentrations ([Ca²⁺]_i) were monitored for 2 h as the 340/380 nm fluorescence ratio in fura-2 loaded cells of the MIN6 mouse pancreatic beta cell line. Cell morphology was evaluated by transmission electron microscopy, and viability by FACS. Results  hIAPP (10 μmol/l) increased [Ca²⁺]_i in 21% of MIN6 cells in standard buffer, and in 8% of cells in Na⁺-free buffer. Transient receptor potential (TRP) channel inhibitors (gadolinium and ruthenium red) prevented the [Ca²⁺]_i rise under both conditions, whilst nifedipine was only effective in the presence of Na⁺. hIAPP increased apoptosis in both insulinoma cells and islets in primary culture, and cell viability was partially rescued by ruthenium red (p < 0.001). By RT-PCR, we detected expression of the mechanosensitive TRP cation channel subfamily V member 4 (Trpv4) in MIN6 cells and mouse pancreas. Small interference RNA against Trpv4 prevented hIAPP-induced [Ca²⁺]_i rises, decreased hIAPP-triggered expression of the endoplasmic reticulum (ER) stress response, and reduced hIAPP-triggered cell death by 50% (p < 0.05). Conclusions/interpretation  Alterations in [Ca²⁺]_i play a key role in hIAPP-induced beta cell cytotoxicity. By electron microscopy, we detected extracellular hIAPP aggregates adjacent to irregular invaginated regions of the plasma membrane. We propose that TRPV4 channels may sense physical changes in the plasma membrane induced by hIAPP aggregation, enabling Ca²⁺ entry, membrane depolarisation and activation of L-type Ca²⁺ channels. Decreasing the activity of TRPV4 prevented hIAPP-induced [Ca²⁺]_i changes, reduced hIAPP-triggered ER stress and improved cell viability.     

Endothelin-Induced Renal Vasoconstriction and Increase in Cytosolic Calcium in Renal Vascular Smooth Muscle Cells

This study was designed to investigate the effects of the potent vasoconstrictor, endothelin, on renal hemodynamics in rats in vivo, and in addition, to measure intracellular calcium ion ([Ca²⁺].) in monolayers of renal vascular smooth muscle cells in culture using the fura-2 method. Endothelin (1 nmol) dramatically decreased renal blood flow from 7.0±0.5 ml/min to 2±6±1±0 ml/min, whereas it increased mean arterial pressure from 100±2 mmHg to 113±7 mmHg. These alterations persisted over 20 minutes in conscious and almost unrestrained rats. Endothelin (10^?8 -10^?7 mol/l) immediately increased [Ca²⁺]_i, although the increase by endothelin (10^?9 mol/l) was relatively slow. The increase persisted in the presence of 1 mmol/l extracellular calcium. In the absence of extracellular calcium, only a small, transient increase of [Ca²⁺]_i was observed. These results indicate that endothelin produces renal vasoconstriction and increases the [Ca²⁺]_i in cultured renal vascular smooth muscle cells. The latter effect is dependent mainly on extracellular calcium.     

Glutamate is an essential mediator in glutamine-amplified insulin secretion

Aims/IntroductionGlutamine is the most abundant amino acid in the circulation. In this study, we investigated cell signaling in the amplification of insulin secretion by glutamine.Materials and MethodsClonal pancreatic β‐cells MIN6‐K8, wild‐type B6 mouse islets, glutamate dehydrogenase (GDH) knockout clonal β‐cells (Glud1KOβCL), and glutamate‐oxaloacetate transaminase 1 (GOT1) knockout clonal β‐cells (Got1KOβCL) were studied. Insulin secretion from these cells and islets was examined under various conditions, and intracellular glutamine metabolism was assessed by metabolic flux analysis. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also measured.ResultsGlutamine dose‐dependently amplified insulin secretion in the presence of high glucose in both MIN6‐K8 cells and Glud1KOβCL. Inhibition of glutaminases, the enzymes that convert glutamine to glutamate, dramatically reduced the glutamine‐amplifying effect on insulin secretion. A substantial amount of glutamate was produced from glutamine through direct conversion by glutaminases. Glutamine also increased [Ca²⁺]_i at high glucose, which was abolished by inhibition of glutaminases. Glutamic acid dimethylester (dm‐Glu), a membrane permeable glutamate precursor that is converted to glutamate in cells, increased [Ca²⁺]_i as well as induced insulin secretion at high glucose. These effects of glutamine and dm‐Glu were dependent on calcium influx. Glutamine also induced insulin secretion in clonal β‐cells MIN6‐m14, which otherwise exhibit no insulin secretory response to glucose.ConclusionsGlutamate converted from glutamine is an essential mediator that enhances calcium signaling in the glutamine‐amplifying effect on insulin secretion. Our data also suggest that glutamine exerts a permissive effect on glucose‐induced insulin secretion.     

The role of Phospholipase A2 in Calcium-Ionophore-Mediated Injury to Rat Gastric Mucosal Cells

Although transient increases in intracellularCa²⁺ ([Ca²⁺]_i) underliea number of important physiological processes, sustainedelevations in [Ca²⁺]_i mediatedamage to a number of tissues and cell types including gastric mucosal cells. Increases in[Ca²⁺]_i can activate phospholipidhydrolysis via increases in phospholipase A₂(PLA₂) activity and subsequent cell injury.In the present study we have examined whether [Ca²⁺]_i-inducedgastric cellular injury is mediated by PLA₂activation. Gastric mucosal cells were harvested fromrat stomachs after pronase digestion. Cell integrity wasassessed using trypan blue dye exclusion and release of lysozomal enzymes.PLA₂ activity was estimated colorimetricallyby determination of thiol release from the substrate,arachidonyl thio-PC. In these studies calcium ionophore A23187 (3-25 M) resulted in an increase incell injury. The damage produced by A23187 (12.5 M)was inhibited by preincubation of cells with thePLA₂ inhibitor, quinacrine (1-100 M).Quinacrine did not reduce ethanol (10% w/v) mediated-celldamage. Similarly Ca²⁺ ionophore A23187treatment resulted in a concentration-dependent increasein PLA₂ activity in gastric cells. Theincrease in PLA₂ activity was attenuated if cells were incubated inCa²⁺-depleted medium containing EGTA (4 mM).Furthermore lysophospholipids generated byPLA₂ (lysophosphatidylethanolamine andlysophosphatidylcholine; 100 M) also increased the degree of cell injury.Pretreatment of cells with the PAF antagonist WEB 2086(10^-6 and 10^-5 M), the leukotrienesynthase inhibitor 5,6-dehydroarachidonic acid (10M), or the thromboxane synthase inhibitor furegrelate (1 M)decrease A23187-mediated cell injury. These data suggestthat Ca²⁺ ionophore-mediated increases in[Ca²⁺]_i result in gastric cellinjury and this effect is mediated in part by PLA₂ activation andsubsequent release of free fatty acids andlysophosphatides.     

Insulin-secreting INS-1E cells express functional TRPV1 channels

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for β-cells, and in primary β-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca²⁺]_i in a concentration-dependent manner, and the [Ca²⁺]_i increase was dependent on extracellular Ca²⁺. AM404 also increased [Ca²⁺]_i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca²⁺]_i increases. Capsaicin did not increase [Ca²⁺]_i in the primary β-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary β-cells, express functional TRPV1 channels.     

A link between polymorphonuclear leukocyte intracellular calcium,plasma insulin,and essential hypertension

BackgroundIntracellular ionized calcium ([Ca²⁺]_i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca²⁺]_i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca²⁺]_i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP).MethodsThe PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca²⁺]_i was measured by flow cytometry in isolated cells stained with Fluo-3.ResultsThe EH PMNs showed significantly increased [Ca²⁺]_i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca²⁺]_i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca²⁺]_i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04).ConclusionsThis study adds PMN to previously described cells exhibiting elevated [Ca²⁺]_i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca²⁺]_i and plasma insulin levels, together with the fact that elevated [Ca²⁺]_i mediates PMN priming, suggest that elevated [Ca²⁺]_i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.     

Nisoldipine blocks the increase of intracellular free calcium-ion concentration associated with elevated sodium–lithium countertransport activity in erythrocytes in patients with NIDDM

To understand the mechanism by which elevated sodium–lithium countertransport activity (SLC) associates with increased intracellular free calcium-ion concentration ([Ca²⁺]_i), we investigated the relationship between SLC and the effects of the extracellular Ca²⁺ concentration ([Ca²⁺]_o) and a Ca²⁺-channel blocker, nisoldipine, on [Ca²⁺]_i in erythrocytes from 48 patients with non-insulin-dependent (Type 2) diabetes mellitus (NIDDM). There was a significant correlation between SLC and [Ca²⁺]_i. Nisoldipine in the incubation medium significantly decreased [Ca²⁺]_i, and there was a significant positive correlation between SLC and the degree of [Ca²⁺]_i decrease. When the [Ca²⁺]_o was elevated, [Ca²⁺]_i was significantly increased, but nisoldipine almost completely suppressed this increase of [Ca²⁺]_i. There was a significant positive correlation between SLC and the degree of the suppression. These data suggest that elevated SLC correlates with increased [Ca²⁺]_i, and that the increased [Ca²⁺]_i might be due to the increased Ca²⁺ influx through a dihydropyridine-sensitive Ca²⁺ pathway. © 1997 by John Wiley & Sons, Ltd.     

Metformin Attenuates Agonist-Stimulated Calcium Transients in Vascular Smooth Muscle Cells

Metformin, an antidiabetic agent that increases insulin sensitivity, has been shown to lower blood pressure. However, the mechanism of action of metformin in vascular smooth muscle (VSM) cell is not fully understood. We have tested the hypothesis that metformin produces vascular changes by direct interaction with VSM cells by investigating its effect on platelet-derived growth factor (PDGF)- and angiotensin II (ANG II)-stimulated intracellular calcium concentration ([Ca²⁺]_i) and VSM cell proliferation in response to PDGF in cultured cells. VSM cells were cultured from rat thoracic aorta and [Ca²⁺]_i was estimated in single cells by image analysis. Treatment of VSM cells with 1 or 2 μg/ml metformin significantly decreased (p < 0.05) PDGF- or ANG II-stimulated [Ca²⁺]_i. Treatment of VSM cells with 1, 2, 5, or 10 μg/ml metformin had no significant effect on PDGF-stimulated [³H]-thymidine incorporation. However, metformin at pharmacological doses of 20 and 50 μg/ml significantly reduced (p< 0.05) PDGF-stimulated thymidine incorporation. We conclude that metformin mediates its vascular effects by attenuating agonist-stimulated [Ca²⁺]i.     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.     

Carbachol-Stimulated Ca2+ Increase in Single Neuroblastoma SH-SY5Y Cells: Effects of Ethanol

The effect of ethanol on the characteristics of carbachol-stimulated release of Ca²⁺ from intracellular Ca²⁺ stores was studied in single SH-SY5Y cells. Stimulation with carbachol, in the absence of extracellular Ca²⁺, elicited a rapid Ca²⁺ increase in SH-SY5Y cells peaking within seconds after addition of maximal agonist concentration. The Ca²⁺ response pattern in single cells resembled the population response, and there was no evidence of oscillatory changes in cytosolic [Ca²⁺] ([Ca²⁺]_i). However, cell-to-cell variability could be detected in the magnitude and the latency time of the response, and in the rate of [Ca²⁺]_i increase. In a carbachol dose-response analysis, the EC₅₀ for the number of responsive cells and for the peak [Ca²⁺]_i response was lower than that for carbachol-induced inositol 1,4,5-trisphosphate formation by a factor of 5 to 50. Ethanol (100 mM) caused a significant suppression of the number of responsive cells, but only when cells were stimulated with nonsaturating carbachol concentrations (1 and 10 μM). The suppression by ethanol was evident primarily in those cells that gave a Ca²⁺ response after several seconds of stimulation, whereas cells that responded within the initial seconds of receptor stimulation remained relatively unaffected. In responding cells stimulated with 10 μM carbachol, ethanol exposure also suppressed the maximal Ca²⁺ increase primarily in those cells that responded late. We suggest that ethanol suppression of muscarinic receptor-mediated signal transduction through the phospholipase C pathway may depend on the potentiation of feedback inhibition that requires receptor stimulation.     

Crosstalk between membrane potential and cytosolic Ca2+ concentration in beta cells from Sur1 −/− mice

Aims/hypothesis Islets or beta cells from Sur1^–/– mice were used to determine whether changes in plasma membrane potential (V_m) remain coupled to changes in cytosolic Ca²⁺ ([Ca²⁺]_i) in the absence of K_ATP channels and thus provide a triggering signal for insulin secretion. The study also sought to elucidate whether [Ca²⁺]_i influences oscillations in V_m in sur1^–/– beta cells.Methods Plasma membrane potential and ion currents were measured with microelectrodes and the patch–clamp technique. [Ca²⁺]_i was monitored with the fluorescent dye fura-2. Insulin secretion from isolated islets was determined by static incubations.Results Membrane depolarisation of Sur1^–/– islets by arginine or increased extracellular K⁺, elevated [Ca²⁺]_i and augmented insulin secretion. Oligomycin completely abolished glucose-stimulated insulin release from Sur1^–/– islets. Oscillations in V_m were influenced by [Ca²⁺]_i as follows: (1) elevation of extracellular Ca²⁺ lengthened phases of membrane hyperpolarisation; (2) simulating a burst of action potentials induced a Ca²⁺-dependent outward current that was augmented by increased Ca²⁺ influx through L-type Ca²⁺ channels; (3) Ca²⁺ depletion of intracellular stores by cyclopiazonic acid increased the burst frequency in Sur1^–/– islets, elevating [Ca²⁺]_i and insulin secretion; (4) store depletion activated a Ca²⁺ influx that was not inhibitable by the L-type Ca²⁺ channel blocker D600.Conclusions/interpretation Although V_m is largely uncoupled from glucose metabolism in the absence of K_ATP channels, increased electrical activity leads to elevations of [Ca²⁺]_i that are sufficient to stimulate insulin secretion. In Sur1^–/– beta cells, [Ca²⁺]_i exerts feedback mechanisms on V_m by activating a hyperpolarising outward current and by depolarising V_m via store-operated ion channels.     

Acute hypoxia activates store-operated Ca2+ entry and increases intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle cells

Rationale

Exposure to acute hypoxia causes vasoconstriction in both pulmonary arteries (PA) and pulmonary veins (PV). The mechanisms on the arterial side have been studied extensively. However, bare attention has been paid to the venous side.

Objectives

To investigate if acute hypoxia caused the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i), and Ca²⁺ influx through store-operated calcium channels (SOCC) in pulmonary venous smooth muscle cells (PVSMCs).

Methods

Fluorescent microscopy and fura-2 were used to measure effects of 4% O₂ on [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) in isolated rat distal PVSMCs.

Measurements and main results

In PVSMCs perfused with Ca²⁺-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca²⁺ stores in the sarcoplasmic reticulum (SR) and nifedipine to prevent Ca²⁺ entry through L-type voltage-depended Ca²⁺ channels (VDCC), hypoxia markedly enhanced both the increase in [Ca²⁺]_i caused by restoration of extracellular [Ca²⁺] and the rate at which extracellular Mn²⁺ quenched fura-2 fluorescence. Moreover, the increased [Ca²⁺]_i in PVSMCs perfused with normal salt solution was completely blocked by SOCC antagonists SKF-96365 and NiCl₂ at concentrations that SOCE >85% was inhibited but [Ca²⁺]_i responses to 60 mM KCl were not altered. On the contrary, L-type VDCC antagonist nifedipine inhibited increase in [Ca²⁺]_i to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca²⁺]_i caused by hypoxia was completely abolished by perfusion with Ca²⁺-free KRBS.

Conclusions

These results suggest that acute hypoxia enhances SOCE via activating SOCCs, leading to increased [Ca²⁺]_i in distal PVSMCs.KEYWORDS : Calcium signaling, pulmonary venous smooth muscle (PVSM), store-operated Ca²⁺ entry (SOCE), intracellular Ca²⁺ concentration ([Ca²⁺]_i)     

Fluorescent Indicators Give Biased Estimates of Intracellular Free Calcium Change in Aggregating Platelets: Implication for Studies with Human von Willebrand Factor

ABSTRACT: The ratiometric fluorescent indicators Fura-2 and Indo-1 are considered optimal probes for monitoring intracellular free calcium concentration ([Ca²⁺]_i). Unique problems arise, however, in studying [Ca²⁺]_ichanges induced in platelets by von Willebrand factor (vWF). Binding of native multimeric vWF causes extensive platelet aggregation, and is reported to evoke a gradual [Ca²⁺]_iincrease. The present investigation examined the reliability of platelet [Ca²⁺]_imeasurements in these circumstances. Ristocetin-mediated binding of vWF to human platelets promoted a slow rise in Fura-2 fluorescence ratio. Fura-2 extrusion contributed substantially to this rise, unless blocked by probenecid. Despite this precaution, the platelets were invariably contaminated slightly with extracellular indicator. As aggregation progressively reduced the number of platelets in the spectrofluorometer beam, through settling of the larger aggregates, such extracellular Fura-2 contributed proportionately more to the observed fluorescence. This extraneous signal accounted completely for the fluorescence ratio increase, and apparent [Ca²⁺]_irise, in response to native multimeric vWF. The same problem arose with Indo-1, whereas the single wavelength indicator Fluo-3 showed the opposite pattern of apparent [Ca²⁺]_ichanges. Thus, none of these indicators provides reliable data on [Ca²⁺]_isignals in aggregating platelets. Use of a dimeric form of vWF eliminated the problem of platelet aggregates settling out of suspension, but also virtually abolished the [Ca²⁺]_iincrease. These observations may explain some of the inconsistencies among previous investigations of vWF-induced calcium signaling. Moreover, similar problems may arise in studies with other adhesive proteins.     

Effects of Erythropoietin on Endothelin-1 Synthesis and the Cellular Calcium Messenger System in Vascular Endothelial Cells

A rise in blood pressure is the main side effect of erythropoietin (EPO) treatment in patients with renal anemia. The mechanisms, however, by which EPO may cause hypertension are still unclear. We therefore investigated the effects of EPO on endothelin (ET) synthesis and cytosolic free calcium concentration ([Ca²⁺]_i) in vascular endothelial cells. Porcine endothelial cells were isolated from thoracic aorta, pulmonary artery, and vena cava. Studies were performed with cells of the first subculture. ET concentrations were measured radioimmunologically. Changes in [Ca²⁺]_i were determined with the fluorescent probe fura-2. Cytotoxicity was assessed by sodium 3′-[1-(phenyl-amino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT) assay. ET synthesis was similar in cells of different vascular origins and was time-dependent, reaching approximately 2 pmol ET/mg protein within 12 h of incubation. EPO (12 to 200 U/mL) stimulated ET release time- and dose-dependently by up to 83.2% (P < .01) within 12 h in the absence of fetal calf serum and heparin. EPO induced an immediate significant rise in [Ca²⁺]_i from 58 ± 12 nmol/L to 495 ± 85 nmol/L (P < .01) with a subsequent slow return to 257 ± 3 nmol/L. During 2 h of incubation, the Ca-ionophore A 23187 (10⁻⁸ mol/L) moderately but significantly stimulated endothelial ET synthesis. However, the Ca-channel blocker verapamil, the intracellular Ca-release blocker TMB-8, and nickel, an unspecific calcium channel blocker, had no consistent effects on [Ca²⁺]_i or ET synthesis. The protein kinase C inhibitor H-7 stimulated basal [Ca²⁺]_i and cellular ET synthesis. The tyrosine kinase inhibitor genistein suppressed the EPO-induced rise in [Ca²⁺]_i and cellular ET synthesis. From these data we conclude that EPO may stimulate ET synthesis in vascular endothelial cells by activation of an EPO-receptor and via intracellular signalling mechanisms that comprise tyrosine kinase activation and a rise in [Ca²⁺]_i. Therefore, the systemic hypertensive effects of EPO may be due at least in part to local stimulation of vascular endothelial ET synthesis via calcium mobilization.     

Effects of external ATP on Ca(2+) signalling in endothelial cells isolated from mouse islets

External ATP is believed to initiate and propagate Ca²⁺ signals co-ordinating the insulin release pulses within and among the different islets in the pancreas. The possibility that islet endothelial cells participate in this process was evaluated by comparing the effects on [Ca²⁺]_i of purinoceptor activation in these cells with those in β-cells. β-Cell-rich pancreatic islets were isolated from ob/ob mice and dispersed into single cells/aggregates. After culture with or without endothelial cell growth supplement (ECGS), the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was measured with ratiometric fura-2 technique. Presence of ECGS or prolongation of culture (>5 days) resulted in proliferation of endothelial cells and altered their phenotype from rounded to elongated. Endothelial cells, preliminarily identified by attachment of Dynabeads coated with the Bandeiraea simplicifolia 1 lectin (BS-1), responded in a similar way as those stained with CD31 antibodies after measurements of [Ca²⁺]_i. Spontaneous transients and oscillations of [Ca²⁺]_i were seen in β-cells, but not in endothelial cells exposed to 20 mM glucose. Addition of ATP (10 μM) resulted in pronounced and more extended rise of [Ca²⁺]_i in endothelial cells than in β-cells. The endothelial cells differed from the β-cells by also responding with a rise of [Ca²⁺]_i to 10 μM UTP, but not to equimolar ADP and acetylcholine. The results support the idea of mutual interactions between islet endothelium and β-cells based on ATP-induced Ca²⁺ signals. It is suggested that the endothelial cells have a tonic inhibitory action on β-cell P2 purinoceptors resulting in impaired synchronization of the insulin release pulses.     

Spontaneous and agonist-induced calcium oscillations in single human nonfunctioning adenoma cells

The effects of gonadotropin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) on cytosolic free calcium concentration ([Ca²⁺]_i) were investigated in 20 human nonfunctioning pituitary adenomas. We divided these tumors into three classes according to their response pattern to hypothalamic peptides. In type I adenomas (8 out of 20 adenomas), GnRH and GAP mobilized intracellular calcium ions stored in a thapsigargin (TG)-sensitive store. For the same concentration of agonist, two distinct patterns of GnRH-GAP-induced Ca²⁺ mobilization were observed (1) sinusoidal oscillations, and (2) monophasic transient. The latter is followed by a protein kinase C (PKC)-dependent increase in calcium influx through L-type channels. In type II adenomas (7 out of 20 adenomas), GnRH and GAP only stimulate calcium influx through dihydropyridine-sensitive Ca²⁺ channels by a PKC-dependent mechanism. TG (1 μM) did not affect [Ca²⁺]_i in these cells, suggesting that they do not possess TG-sensitive Ca²⁺ pools. All the effects of GnRH and GAP were blocked by an inhibitor of phospholipase C (PLC), suggesting that they were owing to the activation of the phosphoinositide turnover. Type I and type II adenoma cells showed spontaneous Ca²⁺ oscillations that were blocked by dihydropyridines and inhibition of PKC activity. GnRH and GAP had no effect on the [Ca²⁺]_i of type III adenoma cells that were also characterized by a low resting [Ca²⁺]_i and by the absence of spontaneous Ca²⁺ fluctuations. K⁺-induced depolarization provoked a reduced Ca²⁺ influx, whereas TG had no effect on the [Ca²⁺]_i of type III adenoma cells. The variety of [Ca²⁺]_i response patterns makes these cells a good cell model for studying calcium homeostasis in pituitary cells.     

Adenosine 5'-triphosphate (ATP) receptors induce intracellular calcium changes in mouse leydig cells

Cytoplasmic calcium ([Ca²⁺]_i) changes evoked by adenosine 5¹-triphosphate (ATP) were recorded in cultured individual Leydig cells within 10–18 h after cell dispersion. [Ca²⁺]_i was monitored using Fura-2AM loaded cells with a digital ratio imaging system. Five micromolars ATP induced biphasic [Ca²⁺]_i responses in most cells (94%,n=100), characterized by a fast increase from a basal level (126±5 nMSE,n=60 cells) to a peak (5–7 times above basal levels) within seconds, followed by a slow decrease toward a plateau level (2–3 times above basal) within 5 min. The peak phase of the [Ca²⁺]_i response increased with ATP concentrations (1–100 μM ATP) in a dose-dependent manner with an IC₅₀ of 5.9±1.2 μM, and it desensitized in a reversible manner with repeated application of 5 μM ATP at <5-min intervals. The [Ca²⁺]_i peak response was dependent on Ca²⁺ release from an intracellular pool, whereas the plateau phase was dependent on extracellular [Ca²⁺]. ATP did not appear to induce formation of nonspecific membrane pores, since stimulation for 10 min with ATP (10–100 μM) in the presence of extracellular Lucifer yellow (LY) (5 mg/mL) did not result in dye loading of the cells. [Ca²⁺]_i transients were elicited by other adenosine nucleotides with an order of potencies (ATP>Adenosine diphosphate [ADP]>Adenosine> Adenosine monophosphate [AMP]) that was compatible with the expression of P₂ receptors. [Ca²⁺]_i responses were suppressed by the purinergic P₂ receptor antagonist, suramin. These results provide functional evidence for the expression of purinergic P₂ receptors in Leydig cells.     

Effects of new intravascular contrast agents on [Ca2+]i transients and contraction in cultured ventricular myocytes

Summary We examined the effects of four kinds of intravascular contrast agents (amidtrizoic acid, iohexol, iopamidol, and ioxaglic acid) on [Ca²⁺]_i transients (indo-1 fluorescence) and cell contraction (video motion analyzer), using cultured chick embryo ventricular myocytes. Exposure of ventricular myocytes to amidtrizoic acid (a conventional contrast agent) reduced the [Ca²⁺]_i transients and the sensitivity of the contractile elements to [Ca²⁺]_i. Ioxaglic acid (a low osmotic contrast agent) also reduced the [Ca²⁺]_i transients, but did not significantly change the sensitivity of the contractile elements to [Ca²⁺]_i. Neither iohexol nor iopamidol (nonionic contrast agents) reduced the [Ca²⁺]_i transients, but both significantly decreased the sensitivity of the contractile elements to [Ca²⁺]_i. A marked negative inotropic effect of amidtrizoic acid was caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by ioxaglic acid is possibly the result of calcium binding, but is not caused by hypertonicity. The negative inotropism produced by nonionic contrast agents (iohexol and iopamidol) was due to hypertonicity, but not due to alterations in the [Ca²⁺]_i transients.Exposure of ventricular myocytes to nonionic contrast agents (iohexol and iopamidol) slowed decay in the [Ca²⁺]_i transients with increased end-diastolic [Ca²⁺]_i. After washing out the nonionic contrast agents, these parameters returned to control levels. On the other hand, exposure to amidtrizoic acid decreased end-diastolic [Ca²⁺]_i without changing decay time in the [Ca²⁺]_i transients. After washing out amidtrizoic acid, there was a prolongation of half decay time in [Ca²⁺]_i transients with a significant increase in end-diastolic [Ca²⁺]_i and cell position. Diastolic dysfunction just after washout of amidtrizoic acid was possibly caused by an increase in [Na⁺]_i due to sodium influx during exposure to the contrast agent.     

Oral taurine supplementation prevents fructose-induced hypertension in rats

Taurine is known to have antihypertensive and lipid-lowering effects in some experimental models and patients. On the other hand, intracellular free calcium and magnesium play important roles in regulating the tonus of blood vessels and insulin sensitivity. We examined the effect of oral taurine supplementation on blood pressure, serum metabolic parameters, and platelet cytosolic free calcium ([Ca²⁺]_i) and magnesium ([Mg²⁺]_i) concentration in fructose-fed Sprague-Dawley rats. Systolic blood pressure and platelet [Ca²⁺]_i were significantly higher in rats fed a 60% fructose diet. Oral taurine supplementation (1% in drinking water) completely prevented the elevation of blood pressure and an increase in platelet [Ca²⁺]_i, but exacerbated hyperinsulinemia, hypertriglyceridemia, and a decrease in platelet [Mg²⁺]_i. In conclusion, taurine may ameliorate fructose-induced hypertension in rats by preventing an increase in intracellular free calcium concentration. The blood pressure-lowering effect of taurine appeared to be independent from its effect on glucose and lipid metabolism in this model.