Calcium Fluorescent Dye Research Articles

Utilization of the genetically encoded calcium indicator Salsa6F in cardiac applications

Calcium signaling is a critical process required for cellular mechanisms such as cardiomyocyte contraction. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

MO059CONTRIBUTION OF OPIOID RECEPTOR SIGNALING IN PODOCYTES TOWARDS THE DEVELOPMENT OF SALT-SENSITIVE HYPERTENSION AND KIDNEY INJURY

Abstract Background and Aims The global increase in opioid use requires practitioners to be aware of the side effects and contraindications of these medications in order to avoid complications, especially in patients with organ dysfunction such as cardiovascular disease and chronic kidney disease. It has been reported that opioid use is strongly associated with predictors of poor cardiovascular outcomes such as increased albuminuria and reduced GFR. Opioids work by binding to kappa-, mu- and delta-opioid receptors (ORs). We hypothesize that stimulation of ORs modulates calcium flux in podocytes ultimately leading to cell apoptosis, development of albuminuria, glomeruli damage and progression of hypertension. Method To test this hypothesis, we applied specific OR agonists to: 1) in vitro - immortalized cultured human podocytes (hPod), 2) ex vivo - freshly isolated human and rat glomeruli; and 3) in vivo - Dahl salt-sensitive (SS) rats. Calcium response in the podocytes was analyzed via ratiometric confocal fluorescent microscopy using calcium fluorescent dyes. For chronic studies Dahl SS rats were fed a high salt (HS; 8%) NaCl diet for 14 days with or without a daily i.v. bolus infusion of BRL52537, a potent and selective kappa-OR agonist. Results The hPods displayed a fast increase in fluorescence following an addition of BRL52537 (EC50 = 162 ± 2 µM) in a 2 mM calcium solution. A pre-application of kappa-OR antagonist (NorBNI) disrupted the effect of BRL52537 on calcium flux. The effect of BRL52537 on calcium in podocytes was completely abolished when we used the calcium-free solution or a pre-application of SAR7334 (a TRPC6 channel inhibitor), which revealed that calcium flux is mediated by activation of TRPC6 channels rather than other plasma membrane channels or depletion of intracellular calcium stores. Immunofluorescent analysis of human kidney validated kappa-OR protein expression in podocytes. Podocytes of the freshly isolated human glomeruli also revealed robust increases in fluorescent intensity following a single application of BRL52537, which was abolished by a pre-application of SAR7334. Effect of kappa-OR agonist was specific since application of Damgo (mu-OR agonist) or DPDPE (delta-OR agonist) had either no or minimal effect in normotensive rats. Chronic studies in Dahl SS rats showed that in BRL-treated group animals had elevation of microalbuminuria (13 ± 3 vs. 3 ± 1 Alb/Cre at day 7 of HS), higher mean arterial pressures (179 ± 15 mmHg vs. 151 ± 11 mmHg), and nephrinuria (50,232 ± 22,254 vs. 15,230 ± 4,647 a.u./Cre) compared to control rats. Glomeruli isolated from the hypertensive opioid treated rats had a significantly higher level of basal calcium (245 ± 23 vs. 139 ± 9 nM) in podocytes compared to non-treated group. Additionally, acute application of BRL52537 to podocytes of hypertensive animals resulted in a sustained increase in total [Ca2+]i influx. Conclusion Our data establish that stimulation of kappa-ORs are specifically involved in increasing of intracellular calcium in podocytes through the TRPC6 pathway, which causes calcium overload and cellular damage. Observed increase in the calcium flux in podocytes most likely further contribute to the kidney injury and progression of salt-induced hypertension.

Estimation of presynaptic calcium currents and endogenous calcium buffers at the frog neuromuscular junction with two different calcium fluorescent dyes.

At the frog neuromuscular junction, under physiological conditions, the direct measurement of calcium currents and of the concentration of intracellular calcium buffers—which determine the kinetics of calcium concentration and neurotransmitter release from the nerve terminal—has hitherto been technically impossible. With the aim of quantifying both Ca2+ currents and the intracellular calcium buffers, we measured fluorescence signals from nerve terminals loaded with the low-affinity calcium dye Magnesium Green or the high-affinity dye Oregon Green BAPTA-1, simultaneously with microelectrode recordings of nerve-action potentials and end-plate currents. The action-potential-induced fluorescence signals in the nerve terminals developed much more slowly than the postsynaptic response. To clarify the reasons for this observation and to define a spatiotemporal profile of intracellular calcium and of the concentration of mobile and fixed calcium buffers, mathematical modeling was employed. The best approximations of the experimental calcium transients for both calcium dyes were obtained when the calcium current had an amplitude of 1.6 ± 0.08 pA and a half-decay time of 1.2 ± 0.06 ms, and when the concentrations of mobile and fixed calcium buffers were 250 ± 13 μM and 8 ± 0.4 mM, respectively. High concentrations of endogenous buffers define the time course of calcium transients after an action potential in the axoplasm, and may modify synaptic plasticity.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca “depleted”. Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca2+-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na+(1H+)/1 Ca2+ exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans.

Prediction of Twitch and High Frequency Local Calcium Dynamics in Mouse EDL Fibers at 15-35°C

Sites of calcium release, uptake and action are highly organized and densely packed in skeletal muscle cells. This organization suggests an important role for the spatial distribution of organelles and calcium sensitive proteins in muscle function. The current measurement techniques are not able to measure cytosolic [Ca2+] with both high temporal and high spatial resolution. In addition, it is known that all processes are highly temperature dependent. Therefore, the model of Groenendaal et al. [1], that describes local calcium dynamics at physiological frequencies at 35°C, was extended with Q10 correction factors and calcium-fluorescent dye binding kinetics, to predict local calcium dynamics at 15-35°C.For model validation, simultaneously calcium fluorescent dye and force kinetic measurements were performed. Hereto, murine EDL muscles were isolated and mounted in the set up. Muscle length was adjusted to optimal resting length and stimulation voltage was adjusted to generate maximal force. Rhod-2 AM was loaded in whole muscle at 37°C for 30 minutes or until force was decreased with >25%. Muscles were stimulated at 15-35°C at frequencies between twitch and tetanus.A significant relation was found between the temperature and the fluorescence characteristics, e.g. decrease in decay time with increasing temperature (one-way ANOVA, p-value<0.05). Model simulations predicted a comparable range. In addition, simulations showed an approximately five-fold difference in calcium gradient throughout the sarcomere between 15 and 35°C.The validated model is now able 1. to explain calcium fluorescent dye measurements, 2. to translate data at unphysiological to physiological temperature and 3. to predict local calcium dynamics at low and high frequency stimulations at a wide temperature range.[1] Groenendaal et al. IET Systems Biology, in press.

Extracellular Ca2+ requirement for serotonin-induced release and meiosis reinitiation from prophase in oocytes of the scallop Argopecten purpuratus

Summary The requirement for Ca2+in the external medium for the release and reinitiation of meiosis in the scallop Argopecten purpuratus was examined. In vitro experiments demonstrated that the serotonin-induced release of oocytes is inhibited in a calcium-free medium. Caffeine (10 and 20 mM), a known activator of ryanodine channels, increased oocyte release by gonadal pieces and 50 μM ryanodine inhibited this release and blocked the serotonin-induced effect. The number of oocytes released under control conditions was not affected when ryanodine was used in concentrations in the nanomolar range. Germinal vesicle breakdown, indicative of meiosis reinitiation, was inhibited in gonadal pieces incubated in calcium-free normal or artificial seawater and serotonin does not overcome this inhibition in the same conditions. An increase in fluorescence on adding serotonin (10−5 M, final concentration) was detected under the confocal microscope when oocytes charged with the calcium fluorescent dye Fluo-3 AM were studied. We postulate that there is a rise in cellular calcium in the serotonin-induced spawning process and that the source for the increase is influx from the external medium and release from internal stores. We suggest that this calcium rise plays a role in germinal vesicle breakdown and in the contraction of gonadal smooth muscle.

Endothelin receptors in cultured and native human radial artery smooth muscle.

In human vascular smooth muscle cells endothelin-1, acting at both endothelin A and endothelin B receptors, has been demonstrated to be both a potent vasoconstrictor and mitogen. Our aim was to study the functional expression of endothelin receptors in human radial artery smooth muscle using both native tissue and cultured cells (RASMCs). Radial artery smooth muscle cells were cultured from arterial explants and loaded with the calcium fluorescent dye fura-2. Cells responded to endothelin-1 and a variety of other vasoconstrictors with rises in cytoplasmic calcium ([Ca2+]c). Arterial rings responded to endothelin-1 with an increase in tension. The response of both cells and arterial rings to endothelin-1 was characterized using the selective endothelin A receptor antagonist BQ123 and the endothelin B receptor antagonist BQ788. The RASMCs were found to express [Ca2+]c responses consistent with the expression of only the endothelin A receptor. Endothelin-1-mediated vasoconstriction in radial artery rings was unaffected by BQ788 but was completely blocked by BQ123. Using the selective radioligands [125I]-PD151242 and [125I]-BQ3020 and a combination of in vitro receptor autoradiography and isolated cell preparations, endothelin A receptors were confirmed to be present on RASMCs and on arterial sections, whereas endothelin B binding was barely detectable on native smooth muscle and on RASMCs.

Protein Kinase C Activation in the Heart: Effects on Calcium and Contractile Proteins

Background Cardiac contractile function is dependent on the energetic state of the heart, intracellular calcium levels, and the interaction of the contractile proteins with both adenosine triphosphate and calcium. Protein kinase C (PKC) is a ubiquitous intracellular mediator that has been found in the heart and has been shown to phosphorylate proteins that regulate calcium homeostasis (calcium channels) and the contractile proteins themselves (troponin I and troponin T). Methods To determine the role of PKC activation on cardiac contractile function, direct activation of PKC was achieved by the infusion of phorbol 12-myristate 13-acetate, an activating phorbol ester. The effects of PKC activation were evaluated in Langendorff-perfused rabbit hearts. Contractile function, high-energy phosphate content (phosphorous-31 nuclear magnetic resonance spectroscopy), oxygen consumption, and intracellular calcium levels (calcium fluorescent dye Rhod-2) were determined. Results Activation of PKC in the heart by phorbol 12-myristate 13-acetate resulted in a significant decrease in both systolic and diastolic function while oxygen consumption and adenosine triphosphate production remained unchanged. Both baseline and peak intracellular calcium levels decreased, which may contribute to the impaired systolic function. Conclusions Activation of PKC in the heart leads to significant loss of contractile function without affecting energetics. The effect is most likely due to alteration in cytosolic calcium regulation and altered contractile sensitivity to calcium. Cardiac contractile function is dependent on the energetic state of the heart, intracellular calcium levels, and the interaction of the contractile proteins with both adenosine triphosphate and calcium. Protein kinase C (PKC) is a ubiquitous intracellular mediator that has been found in the heart and has been shown to phosphorylate proteins that regulate calcium homeostasis (calcium channels) and the contractile proteins themselves (troponin I and troponin T). To determine the role of PKC activation on cardiac contractile function, direct activation of PKC was achieved by the infusion of phorbol 12-myristate 13-acetate, an activating phorbol ester. The effects of PKC activation were evaluated in Langendorff-perfused rabbit hearts. Contractile function, high-energy phosphate content (phosphorous-31 nuclear magnetic resonance spectroscopy), oxygen consumption, and intracellular calcium levels (calcium fluorescent dye Rhod-2) were determined. Activation of PKC in the heart by phorbol 12-myristate 13-acetate resulted in a significant decrease in both systolic and diastolic function while oxygen consumption and adenosine triphosphate production remained unchanged. Both baseline and peak intracellular calcium levels decreased, which may contribute to the impaired systolic function. Activation of PKC in the heart leads to significant loss of contractile function without affecting energetics. The effect is most likely due to alteration in cytosolic calcium regulation and altered contractile sensitivity to calcium.

Protein Kinase C activation in the heart: Effects on calcium and contractile proteins

Cardiac contractile function is dependent on the energetic state of the heart, intracellular calcium levels, and the interaction of the contractile proteins with both adenosine triphosphate and calcium. Protein kinase C (PKC) is a ubiquitous intracellular mediator that has been found in the heart and has been shown to phosphorylate proteins that regulate calcium homeostasis (calcium channels) and the contractile proteins themselves (troponin I and troponin T). To determine the role of PKC activation on cardiac contractile function, direct activation of PKC was achieved by the infusion of phorbol 12-myristate 13-acetate, an activating phorbol ester. The effects of PKC activation were evaluated in Langendorff-perfused rabbit hearts. Contractile function, high-energy phosphate content (phosphorous-31 nuclear magnetic resonance spectroscopy), oxygen consumption, and intracellular calcium levels (calcium fluorescent dye Rhod-2) were determined. Activation of PKC in the heart by phorbol 12-myristate 13-acetate resulted in a significant decrease in both systolic and diastolic function while oxygen consumption and adenosine triphosphate production remained unchanged. Both baseline and peak intracellular calcium levels decreased, which may contribute to the impaired systolic function. Activation of PKC in the heart leads to significant loss of contractile function without affecting energetics. The effect is most likely due to alteration in cytosolic calcium regulation and altered contractile sensitivity to calcium.

Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb.

The effect of antidiuretic hormone [( Arg]vasopressin, ADH) on intracellular calcium activity [Ca2+]i of isolated perfused rabbit cortical thick ascending limb (cTAL) segments was investigated with the calcium fluorescent dye fura-2. The fluorescence emission ratio at 500-530 nm (R) was monitored as a measure of [Ca2+]i after excitation at 335 nm and 380 nm. In addition the transepithelial potential difference (PDte) and transepithelial resistance (Rte) of the tubule were measured simultaneously. After addition of ADH (1-4 nmol/l) to the basolateral side of the cTAL R increased rapidly, but transiently, from 0.84 +/- 0.05 to 1.36 +/- 0.08 (n = 46). Subsequently, within 7-12 min R fell to control values even in the continued presence of ADH. The increase in R evoked by the ADH application corresponded to a rise of [Ca2+]i from a basal level of 155 +/- 23 nmol/l [Ca2+]i up to 429 +/- 53 nmol/l [Ca2+]i at the peak of the transient, as estimated by intra- or extracellular calibration procedures. The electrical parameters (PDte and Rte) of the tubules were not changed by ADH. The ADH-induced Ca2+ transient was dependent on the presence of Ca2+ on the basolateral side, whereas luminal Ca2+ had no effect. d(CH2)5[Tyr(Me)2]2,Arg8vasopressin, a V1 antagonist (Manning compound, 10 nmol/l), blocked the ADH effect on [Ca2+]i completely (n = 5). The V2 agonist 1-desamino-[D-Arg8]vasopressin (10 nmol/l, n = 4), and the cAMP analogues, dibutyryl-cAMP (400 mumol/l, n = 4), 8-(4-chlorophenylthio)-cAMP (100 mumol/l, n = 1) or 8-bromo-cAMP (200 mumol/l, n = 4) had no influence on [Ca2+]i. The ADH-induced [Ca2+]i increase was not sensitive to the calcium-channel blockers nifedipine and verapamil (100 mumol/l, n = 4). We conclude that ADH acts via V1 receptors to increase cytosolic calcium activity transiently in rabbit cortical thick ascending limb segments, possibly by an initial Ca2+ release from intracellular stores and by further Ca2+ influx through Ca2+ channels in the basolateral membrane. These channels are insensitive to L-type Ca2+ channel blockers, e.g. nifedipine and verapamil.