Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat

In few mammalian species including rat, post-ovulatory aging induces abortive spontaneous egg activation (SEA), which is morphologically characterized by exit from metaphase-II (M-II) arrest. A possibility exists that the RyR channel-mediated insufficient increase of cytosolic free Ca²⁺ level could be one of the causes for post-ovulatory aging-induced abortive SEA. To test this possibility, eggs collected after 17 h post-hCG surge were cultured with or without various concentrations of nifedipine (NF), ruthenium red (RR), and KN-93 for 3 h in vitro. Morphological changes characteristic of abortive SEA, cytosolic free Ca²⁺ level, cyclin B1 level, and meiotic status were analyzed. Data of the present study indicate that NF and RR inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. Further, RR protected against RyR channel as well as caffeine-mediated increase of cytosolic free Ca²⁺ level. In addition, KN-93 inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. An increase of cytosolic free Ca²⁺ level was associated with a reduction of cyclin B1 level during post-ovulatory aging-induced abortive SEA. These data indirectly suggest the involvement of RyR channels in the increase of cytosolic free Ca²⁺ level. The increased cytosolic free Ca²⁺ level triggers cyclin B1 degradation possibly through CaMK-II activity during post-ovulatory aging-induced abortive SEA in rat eggs cultured in vitro.     

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.     

Serotonin-induced cytosolic free calcium transients in cultured vascular smooth muscle cells

Serotonin induced a transient elevation in the levels of cytosolic calcium in cultured rat vascular smooth muscle cells. Ketanserin, a selective antagonist of serotonin 2 receptors, dose-dependently inhibited the elevation of cytosolic calcium induced by serotonin, and ultimately unmasked a serotonin-induced decrease in the levels of cytosolic calcium. These observations show that serotonin has direct and dual effects, that is, it increases and decreases cytosolic free calcium concentrations in vascular smooth muscle cells, in culture. Knowledge of such events is important because serotonergic inhibitors may prove to be useful drugs for treating clinical hypertension and vasospastic disorders.     

Positive correlation between cytosolic free calcium and surfactant secretion in cultured rat alveolar type II cells

To determine whether increases in the cytosolic free Ca2+ concentration ([Ca2+]i) accompany agonist-stimulated surfactant secretion by cultured alveolar type II cells, we measured the [Ca2+]i of quin2-loaded cells isolated from adult rats before and after cells were stimulated with ionomycin, terbutaline or tetradecanoylphorbol acetate (TPA). To determine whether increases in [Ca2+]i are necessary for stimulated surfactant secretion to occur, we measured secretion in cells after [Ca2+]i had been reduced by loading cells with quin2 in medium containing low [Ca2+]. Ionomycin increased [Ca2+]i and stimulated surfactant secretion in a dose-dependent manner. Reductions in [Ca2+]i correlated with reductions in secretion stimulated by ionomycin, terbutaline or TPA. Ionomycin-stimulated secretion was most sensitive to reductions in [Ca2+]i; terbutaline-stimulated secretion was more sensitive than TPA-stimulated secretion. When [Ca2+]i was less than 65 nM, all stimulated secretion was blocked. Restoration of [Ca2+]i to greater than 100 nM restored ionomycin-stimulated secretion. We conclude that ionomycin increases [Ca2+]i and stimulates surfactant secretion in cultured alveolar type II cells, and that increased [Ca2+]i appears to be necessary for ionomycin-stimulated secretion to occur. Terbutaline-stimulated surfactant secretion seems to be more easily inhibited by a reduction in [Ca2+]i than does TPA-stimulated secretion.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

A wave of free cytosolic calcium traverses zebrafish eggs on activation.

The activation process in a variety of deuterostome and protostome eggs is accompanied by cytosolic calcium transients that usually take the form of either a single or multiple propagating waves. Here we report that the eggs of zebrafish (Danio rerio) are no exception in that they generate a single activation wave that traverses the egg at a velocity of around 9 microm/s. There appears, however, to be no difference between the calcium-mediated activation response of eggs with regard to the presence or absence of sperm in the spawning medium. This leads us to suggest that these eggs are normally activated when they come in contact with their spawning medium and are then subsequently fertilized. The aspermic wave is initiated at the animal pole in the region of the micropyle, appears to propagate mainly through the yolk-free egg cortex, and then terminates at the vegetal pole. As neither sperm nor external calcium is required for the initiation (or propagation) of the activation wave, this suggests that an alternative wave trigger must be involved.     

Glucose raises cytosolic free calcium in the rat pancreatic islets

Cytosolic free calcium [( Ca2+]i) was measured using fura 2 in the whole pancreatic islets obtained from male Wistar rats by collagenase dispersion. The pattern of change of [Ca2+]i in response to high glucose, potassium (K+) depolarization or the removal of extracellular calcium was compared with the temporal profile of insulin secretion. Twenty-nine mM glucose produced a gradual increase in [Ca2+]i with approximately 1.5 min of latency period. It remained elevated until the end of observation period (25 min) during which period the first phase of insulin secretion ceased and the second phase of secretion gradually increased. Depolarizing concentration of KCl also produced an elevation of [Ca2+]i, without detectable latency period, which lasted at a sustained level for the entire observation period (30 min). KCl caused a rapid increase of insulin secretion followed by a gradually decreasing level of secretion. Elevated [Ca2+]i and insulin secretion in response to high glucose returned to the basal level when external calcium was removed by the addition of EGTA. We conclude that high glucose and K+ depolarization raise [Ca2+]i in the pancreatic islet. However, the elevation of [Ca2+]i and insulin secretion are not always correlated in the later period of stimulation.     

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells

Using a variety of synthetic analogs of porcine endothelin (pET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(1-15) and (16-21), substitution of disulfide bond, Cys(3-11) or Cys(1-15), by Cys (Acm), all resulted in a complete loss of receptor binding activity and [Ca2+]i response, while N-terminal elongation of Lys-Arg residues, but not oxidation of Met7 residue, decreased receptor binding activity and [Ca2+]i response. [Cys1-15,Cys3-11]pET was far more potent than [Cys1-11,Cys3-15]pET in receptor binding and [Ca2+]i response. These data indicate that the C-terminal Trp21 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the pET molecule are essential for receptor binding and subsequent [Ca2+]i increase in rat VSMC.     

Activin A increases cytosolic free calcium concentration in rat pituitary somatotropes.

The effect of activin A on the cytosolic free calcium concentration ([Ca2+]i) in normal rat pituitary cells was examined using a calcium sensitive fluorescent dye, indo 1 AM, and a digital imaging fluorescent microscope system. The cells showing an increase in [Ca2+]i in response to activin A were then characterized by comparison with cells responding to growth hormone releasing hormone (GRH), thyrotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), and gonadotropin releasing hormone (GnRH) in monolayer cultures of normal rat pituitary cells. Activin A increased [Ca2+]i in some cells in a mixed population of normal rat pituitary cells. The cells that responded to activin A also responded to GRH. Most of these cells were not affected by other tropic hormones (CRH, TRH, and GnRH), but a few cells responded to both GRH and TRH. None of the activin A-responding cells responded to CRH or GnRH, and none of the CRH- or GnRH-responding cells responded to activin A. In a preparation of somatotropes purified 80-90% by fluorescence-activated cell sorting, activin A increased [Ca2+]i in 30% of the cells that shows a [Ca2+]i-response to GRH. These findings suggest direct involvement of somatotropes in activin A-induced biological events in the rat pituitary gland.     

Histamine activates H1-receptors to induce cytosolic free calcium transients in cultured vascular smooth muscle cells from rat aorta

Using an intracellularly trapped dye, quin 2, the effects of histamine on cytosolic free calcium concentrations in rat aortic vascular smooth muscle cells in primary culture were recorded, microfluorometrically. When the cells were exposed to histamine, both in the presence and the absence of extracellular Ca2+, there was a rapid, transient and dose-dependent elevation of cytosolic Ca2+ concentrations, with a similar time course. This elevation of cytosolic Ca2+ was dose-dependently inhibited by mepyramine, but not by cimetidine. Thus, histamine activates H1- but not H2- receptors to mediate a release of Ca2+ from the store sites, and there is a rapid and transient elevation of cytosolic Ca2+.     

Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway

Activated Akt kinase has been proposed as a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins and/or caspase-9. To study the mechanism underlying the anti-apoptotic effect of taurine, the interaction between taurine and Akt/caspase-9 pathway was examined using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Taurine (20mM) treatment attenuated simulated ischemia-induced decline in the activity of Akt. Although taurine treatment had no effect on the expression of Bcl-2 in mitochondria and the level of cytosolic cytochrome c, it inhibited ischemia-induced cleavage of caspases 9 and 3. Moreover, adenovirus transfer of the dominant negative form of Akt objected taurine-mediated anti-apoptotic effects, cancelling the suppression of caspase-9 and caspase-3 activities by taurine. These findings provide the first evidence that taurine inhibits ischemia-induced apoptosis in cardiac myocytes with the increase in Akt activities, by inactivating caspase-9.     

Fc-gamma- and complement receptor mediated elevation in the cytosolic calcium level in human neutrophils.

The effects of differently opsonized zymosan particles, acting solely at Fc-gamma or at complement receptors or at both, on the level of intracellular calcium ([Ca2+]i) in human neutrophils were studied. A biphasic, long-lasting increase in [Ca2+]i was seen in response to IgG-, C3- and fresh serum-opsonized zymosan particles in the presence of extracellular Ca2+. Unopsonized zymosan, acting mainly at CR3 failed to elevate [Ca2+]i. Addition of 1.4 mM EGTA reduced but did not abolish the rise in [Ca2+]i triggered by opsonized zymosan, indicating that Ca2+ is released from intracellular stores. EGTA changed also the kinetic patterns of Ca(2+)-responses possibly by indirectly affecting the extrusion of Ca2+ in neutrophils.     

Transient increase of cytosolic free calcium in cultured human vascular endothelial cells by platelet-activating factor

The effect of platelet-activating factor (PAF-acether) on cytosolic free calcium, [Ca2+]i, in adherent human vascular endothelial cells in culture was directly determined using a new fluorescent calcium indicator, fura-2. It was found that PAF-acether but not lyso PAF-acether induced a rapid and transient increase in [Ca2+]i in endothelial cells. Restimulation with PAF-acether after the first challenge did not cause further response, while the cells were able to respond to thrombin. In the absence of extracellular calcium, PAF-acether evoked a similar transient increase, suggesting that PAF-acether raises [Ca2+]i mainly by discharging calcium from intracellular pools. PAF-acether-induced rise in [Ca2+]i was completely blocked by a specific antagonist, BN 52021. These results suggest the receptor-mediated increase in [Ca2+]i as an early event in PAF-acether activation of human vascular endothelial cells.