Intracellular Calcium Ion Dynamics Research Articles

A microfluidic system for precisely reproducing physiological blood pressure and wall shear stress to endothelial cells.

To reproduce hemodynamic stress microenvironments of endothelial cells in vitro is of vital significance, by which one could exploit the quantitative impact of hemodynamic stresses on endothelial function and seek innovative approaches to prevent circulatory system diseases. Although microfluidic technology has been regarded as an effective method to create physiological microenvironments, a microfluidic system to precisely reproduce physiological arterial hemodynamic stress microenvironments has not been reported yet. In this paper, a novel microfluidic chip consisting of a cell culture chamber with on-chip afterload components designed by the principle of input impedance to mimic the global hemodynamic behaviors is proposed. An external feedback control system is developed to accurately generate the input pressure waveform. A lumped parameter hemodynamic model (LPHM) is built to represent the input impedance to mimic the on-chip global hemodynamic behaviors. Sensitivity analysis of the model parameters is also elaborated. The performance of reproducing physiological blood pressure and wall shear stress is validated by both numerical characterization and flow experiment. Investigation of intracellular calcium ion dynamics in human umbilical vein endothelial cells is finally conducted to demonstrate the biological applicability of the proposed microfluidic system.

A microfluidic platform enabling real-time control of dynamic biochemical stimuli to biological cells

In the present study, we report a microfluidic platform that enables real-time control of biochemical stimuli to biological cells. The microfluidic platform is designed by integrating a ‘Christmas tree’ inlet for pre-generating spatially linear concentration gradient, with a Y-shaped channel to modulate dynamic signals with an external programmable air pump. The proposed design is simple and straightforward, has negligible response time compared to the traditional Y-shaped channel, and for the first time, provides the capability of generating versatile waveforms of dynamic stimuli and implementing multiple dynamic stimulating signals with different amplitudes synchronously. The feasibility of the microfluidic platform is proved by both computational fluid dynamics simulation and fluorescein experiment. The applicability of the proposed platform is demonstrated by characterizing the intracellular calcium ion dynamics in human umbilical vein endothelial cells in response to multiple dynamic stimuli within a single run. The proposed platform provides a simple approach to rapidly control dynamic stimuli with versatile waveforms to biological cells and shows the potential for the quantitative study of biological cellular dynamic responses.

Different effect of serotonin on intracellular calcium ion dynamics in the smooth muscle cells between rat posterior ciliary artery and vorticose vein.

5-hydroxytriptamine (5-HT: serotonin) is an important transmitter that causes vessel constriction, although few studies have examined the effect of 5-HT on venous smooth muscles. The intracellular Ca(2+) concentration ([Ca(2+)]i) plays an essential role in stimulus-response coupling in numerous tissue/cells including vascular smooth muscle cells. The present study was performed to examine whether differences between arteries and veins in the response to 5-HT can be detected under confocal microscope with respect to [Ca(2+)]i dynamics. In posterior ciliary arteries of rats, 5-HT induced a [Ca(2+)]i increase. The 5-HT-induced responses were caused by both Ca(2+) influx and mobilization. Agonist and antagonist experiments revealed that arterial smooth muscles possess 5-HT1a, 1b, 2 (Gprotein-coupled type) and 5-HT3 (ion channel type) receptors, and that 5-HT2 in particular plays a major role in these responses. For vorticose veins, the 5-HT-induced responses were also caused by both Ca(2+) influx and mobilization. However, the cAMP dependent pathway (5-HT4-7) was found to be significant in vasocontraction with respect to 5-HT in these vessels. Thus, Ca(2+) mobilization was induced by 5-HT2 and 5-HT4-7 in a vessel-dependent manner, whereas Ca(2+) influx universally was induced by 5-HT3. These results indicate that the posterior ciliary arteries and vorticose veins in the same tissue might differ greatly in their responses to stimulus.

Hydroxyflutamide alters the characteristics of live boar spermatozoa

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 μg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the “C-Ruch” computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P < 0.05) in sperm mitochondrial membrane potential and a concomitant increase (P < 0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 μg OH-Flu compared with the respective controls and other doses used (P < 0.05). The adverse effects of OH-Flu become strengthened over time (P < 0.05). Notably, 50 and 100 μg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P < 0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P < 0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P < 0.05) only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility.

Monitoring Intracellular Calcium Ion Dynamics in Hair Cell Populations with Fluo-4 AM

We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca2+ signals in populations of hair cells. The bundle Ca2+ signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca2+ entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca2+ chelators or blocking Ca2+ entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca2+ decrease, which could serve to stimulate the hair cell’s tip-link regeneration process. Measurement of bundle Ca2+ with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca2+ signaling in the hair cell.

Intracellular calcium ion dynamics involved in long-term potentiation in hippocampal CA1 neurons in mice lacking the IP 3 type 1 receptor

In the present study, mice lacking the type 1 inositol-1,4,5-trisphosphate receptor (IP 3R) were used to study the role of type 1 IP 3Rs in the induction of long-term potentiation (LTP) in hippocampal CA1 neurons. The magnitude of the LTP induced by high frequency stimulation (HFS) consisting of 20 pulses at 30 Hz in mice lacking type 1 IP 3Rs was significantly larger than that in wild-type mice in terms of the field excitatory postsynaptic potential and population spike. By measuring changes in the intracellular Ca 2+ concentration ([Ca 2+] i) in CA1 pyramidal neurons using fluorometry, we found that the decay time of the transient increase in the [Ca 2+] i evoked by the HFS in mutant mice was significantly longer than that in wild-type mice, whereas the [Ca 2+] i at rest and the magnitude of the [Ca 2+] i increases caused by the HFS were no different from those in wild-type mice. In slices from the mutant mice, paired-pulse stimulation (PPS) delivered at an interval of 10 ms resulted in significantly weaker paired-pulse inhibition (PPI) than in wild-type mice, suggesting that lack of type 1 IP 3Rs reduces the PPI induced by PPS in the CA1 region. These results indicate that a lack of type 1 IP 3Rs causes a slower decay of the transient [Ca 2+] i in CA1 pyramidal neurons and attenuates the activity of inhibitory interneurons, resulting in enhancement of LTP induction.

The role of protease-activated receptors on the intracellular calcium ion dynamics of vascular smooth muscles, with special reference to cerebral arterioles

Protease-activated receptors (PARs) mediate cellular responses to various proteases in numerous cell types, including smooth muscles and the endothelium of blood vessels. To clarify whether the stimulation of PARs induces responses in smooth muscle cells of cerebral arterioles, intracellular Ca2+([Ca2+]i) dynamics and nitric oxide (NO) production during PARs stimulation were investigated in the rat cerebral arterioles by real-time confocal microscopy, since [Ca2+]i and NO are both key factors in the maintenance of strain in blood vessels. Testicular arterioles were also investigated for comparison. In smooth muscle cells of small cerebral arterioles (< 50 microm in diameter), thrombin and PAR1-activating peptide (AP) induced an increase in [Ca2+]i and contraction. The response to PAR1 activation was caused by Ca2+ mobilization from intracellular Ca2+ stores. Trypsin and PAR2-AP induced a decrease in [Ca2+]i in the cells which was considered to be mediated by endothelium-derived NO and/or by promoting a Ca2+ sequestration mechanism. PAR3- and 4-AP had little effect. In contrast to small cerebral arterioles, [Ca2+]i dynamics in smooth muscle cells of large cerebral arterioles (< 150 microm in diameter) or testicular arterioles remained unchanged during PARs activation. The effects of PARs activation on the [Ca2+]i dynamics and the contraction/relaxation of cerebral arterioles are also discussed in relation to the role of proteases in the regional tissue circulation of the brain.

Imaging analysis of effect of ATP on intracellular calcium ion dynamics of dorsal root ganglia in immature and mature rats

Imaging analysis of effect of ATP on intracellular calcium ion dynamics of dorsal root ganglia in immature and mature rats

Expression of group I metabotropic glutamate receptors in rat hippocampal cells in culture and their characterization by intracellular calcium ion dynamics.

The distribution of group I metabotropic glutamate receptors in rat hippocampal cells in culture was examined by calcium imaging and immunocytochemistry. To distinguish different cell types in the culture, the effects of t-ACPD ((1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid) and of NMDA (N-methyl-D-aspartate) were examined. About 40% of the cultured cells showed either a transient increase or a sustained or oscillatory increase in the intracellular calcium concentration ([Ca(2+)](i)) during t-ACPD administration, while about 60% of the cells showed a sustained [Ca(2+)](i) increase in response to NMDA. Cells that showed an oscillatory [Ca(2+)](i) change during t-ACPD administration did not respond to NMDA administration, while cells that showed a sustained [Ca(2+)](i) increase during NMDA administration did not show any oscillatory response to t-ACPD. After pharmacological examination using those two agonists, the cultured cells were subjected to immunocytochemistry using anti-GFAP and ant-MAP-2 antibodies to distinguish, respectively, astrocytes and neurons. All cells responding to NMDA with a sustained [Ca(2+)](i) increase were MAP-2-positive, whereas all cells showing either oscillatory or sustained [Ca(2+)](i) increase in response to t-ACPD were GFAP-positive. The present results show that, in these cultures, group I metabotropic glutamate receptors are mainly expressed on glial cells and contribute to dynamic [Ca(2+)](i) changes in astrocytes.

Expression of Group I Metabotropic Glutamate Receptors in Rat Hippocampal Cells in Culture and Their Characterization by Intracellular Calcium Ion Dynamics

Expression of Group I Metabotropic Glutamate Receptors in Rat Hippocampal Cells in Culture and Their Characterization by Intracellular Calcium Ion Dynamics

Comparison of the effect of ATP on intracellular calcium ion dynamics between rat testicular and cerebral arteriole smooth muscle cells

Adenosine 5′-triphosphate (ATP), when released from neuronal and non-neuronal tissues, interacts with cell surface receptors produces a broad range of physiological responses. The goal of the present study was to examine the issue of whether vascular smooth muscle cells respond to ATP. To this end, the dynamics of the intracellular concentration of calcium ions ([Ca 2+] i) in smooth muscle cells in testicular and cerebral arterioles was examined by laser scanning confocal microscopy. ATP produced an increase in [Ca 2+] i in arteriole smooth muscle cells. While P1 purinoceptor agonists had no effect on this process, P2 purinoceptor agonists induced a [Ca 2+] i increase and a P2 purinoceptor antagonist, suramin, completely inhibited ATP-induced [Ca 2+] i dynamics in both arteriole smooth muscle cells. In testicular arterioles, Ca 2+ channel blockers and the removal of extracellular Ca 2+, but not thapsigargin pretreatment, abolished the ATP-induced [Ca 2+] i dynamics. In contrast, Ca 2+ channel blockers and the removal of extracellular Ca 2+ did not completely inhibit ATP-induced [Ca 2+] i dynamics in cerebral arterioles. Uridine 5′-triphosphate caused an increase in [Ca 2+] i only in cerebral arterioles and α,β-methylene ATP caused an increase in [Ca 2+] i in both testicular and cerebral arterioles. We conclude that testicular arteriole smooth muscle cells respond to extracellular ATP via P2X purinoceptors and that cerebral arteriole smooth muscle cells respond via P2X and P2Y purinoceptors.

Application of real-time confocal microscopy to intracellular calcium ion dynamics in rat arterioles.

The regulation of cytosolic Ca(2+) homeostasis is essential for cells, including vascular smooth muscle cells. Arterial tone, which underlies the maintenance of peripheral resistance in the circulation, is a major contributor to the control of blood pressure. Confocal microscopy was employed to study the alteration in intracellular calcium ion concentration ([Ca(2+)](i)) in arterioles (external diameters <100 microm) with respect to selected modifying reagents. 5-Hydroxytryptamine (1 microM), ATP (10 microM), and endothelin 1-3 (5 nM) elicited an increase in [Ca(2+)](i) in most arteriole smooth muscle cells. The [Ca(2+)](i) increase sometimes propagated in an intercellular manner. When noradrenaline (10 microM) was used as a stimulant, [Ca(2+)](i) increase was observed only in a portion of the smooth muscle cells. It was also noted that the reaction of these cells with respect to ATP is different between testis and brain arterioles; the [Ca(2+)](i) increase in testicular arterioles is dependent on Ca(2+) influx from extracellular space, whereas in cerebral arterioles it plays a role in both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores (i.e., sarco/endoplasmic reticulum). These results indicate that arterioles in different tissues may differ greatly in their responses. Real-time confocal microscopy was found to be a useful tool for investigating the structural and functional changes in living tissues.

Effects of carbachol and catecholamines on ultrastructure and intracellular calcium-ion dynamics of acinar and myoepithelial cells of lacrimal glands.

The current study was carried out to investigate autonomic nervous control of secretory functions in the lacrimal gland. To distinguish the difference between the responses to cholinergic and adrenergic agonists in acinar and myoepithelial cells in the lacrimal gland of guinea pigs, the morphological and functional responses to the agonists were examined by electron microscopy and by digital-imaging analysis of the intracellular concentration of Ca2+ ([Ca2+]i) using fluorescent Ca2+-indicators (Fura-2/AM and Indo-1/AM). In the resting state, exocytosis was rare, and the [Ca2+]i in acinar and myoepithelial cells was low (less than 300 nM). Stimulation with carbachol (CCh) induced a rapid rise in [Ca2+]i reaching a peak level followed by gradual decay and an appearance of many exocytotic figures. Approximately 4-8 s after an initial increase of [Ca2+]i, myoepithelial cells commenced contraction. Noradrenaline or adrenaline induced an increase in [Ca2+]i and exocytosis in acinar cells, but caused no [Ca2+]i increase in myoepithelial cells. In a Ca2+-deficient environment, the responses to CCh in myoepithelial cells and those to noradrenaline in acinar cells were inhibited, whereas the responses to CCh in acinar cells remained unchanged. Isoproterenol caused no effect on [Ca2+]i dynamics, although it occasionally induced exocytosis. Different cellular signaling pathways may be involved in the responses in acinar and in myoepithelial cells to different agonists. Lacrimation mechanisms are redundant.

Calcium antagonists in manic-depressive illness.

Increased free intracellular calcium ion concentration ([Ca2+]i) has been found in lymphocytes and blood platelets of patients with bipolar affective disorders when they are acutely ill, but not after recovery. Because lithium alters intracellular calcium ion dynamics and lowers platelet [Ca2+]i in affectively ill patients but not controls, drugs whose primary action is to modulate [Ca2+]i in hyperactive cells have been used as antimanic agents. The best studied of these is verapamil, a calcium channel blocking agent (CCB) that appears most effective for lithium-responsive patients. Because they interact with different central CCB receptors, second-generation CCBs may have a different spectrum of action. CCBs are usually well tolerated and may be useful for a number of other psychiatric, neurological and medical conditions.

Elevated platelet intracellular calcium concentration in bipolar depression

Baseline and thrombin-stimulated free intracellular calcium concentrations in blood platelets were significantly higher in untreated depressed bipolar patients than in untreated unipolar depressed patients or controls. Platelet intracellular calcium ion concentrations in euthymic-treated bipolar patients were equivalent to control values, suggesting but not proving a state-dependent change in intracellular calcium ion dynamics in bipolar depression. Unipolar and some subsets of bipolar patients appear not to exhibit this change.