Oxysterol activation of arachidonic acid release and prostaglandin E2 biosynthesis in NRK 49F cells is partially dependent on protein kinase C activity [corrected]

We previously demonstrated that the oxysterol potentiation of arachidonic acid release and prostaglandin biosynthesis induced by foetal calf serum activation of normal rat kidney (NRK) cells (fibroblastic clone 49F) was not related to a direct effect of oxysterols on cell free Ca²⁺ level. Since both Ca²⁺ variations and protein C are involved in arachidonic acid release in some models, we looked for a possible modulation by protein C in the oxysterol effect on arachidonic acid release. We show that when the phorbol ester 12-O-tetradecanoyl-phorbol-13acetate (TPA), a protein kinase C activator, was added to the culture medium, the oxyterol effect on arachidonic acid release and prostaglandin synthesis clearly increased. Moreover, the effect of TPA was dose-dependent and TPA EC₅₀ (4 × 10⁻⁹ M) was unchanged in the presence of the oxysterol. Preincubation of cells with TPA for 24 h prevented the arachidonic acid release induced by TPA alone, whereas the oxysterol effect was decreased but not abolished. In the absence of serum, TPA and ionomycin added together induced the same noticeable (arachidonic acid) release and PGE₂ synthesis as serum alone. Nevertheless, the potentiating effect of cholest-5-ene-3β,25-diol was much higher when serum itself was used to activate NRK cells than it was in the present serum-mimicking experimental conditions. Thus, the presence of growth factors is probably required to obtain a full oxysterol effect. We conclude that the oxysterol effect was synergistic with, but not fully dependent on, protein kinase C and Ca²⁺ ion fluxes, therefore oxysterols could affed earlier events triggered by serum growth factor binding to their cell membrane receptors.     

5-hydroxytryptamine-evoked [Ca]i oscillations in rat C6 glioma cells

We have investigated the modulation of the intracellular calcium concentration ([Ca²⁺]_i) in rat C6 glioma cells following their activation by the agonists 5-hydroxytryptamine·HCl (5-HT) and bradykinin, using single cell imaging of [Ca²⁺]_i with the calcium-sensitive dye Fura-2. The majority of the signals observed involved release of calcium from intracellular stores, and after prolonged application of 5-HT, but not bradykinin, the cells exhibited oscillations in [Ca²⁺]_i levels. These calcium oscillations were dependent on the presence of extracellular calcium, and were unaffected by the calcium channel antagonists nifedipine and verapamil. Caffeine, which in other cell types is able to release calcium from inositol trisphosphate-insentive stores, had very little effect on [Ca²⁺]_i levels in C6 cells. On the other hand, bradykinin, although able to elevate [Ca²⁺]_i probably by acting via the B₂-receptor subtype, was unable to induce any calcium oscillations in these cells.     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Ca(2+) as second messenger in PTTH-stimulated prothoracic glands of the silkworm,Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

2, 5-Di(Tert-Butyl)-1, 4-Benzohydroquinone —A Novel Mobilizer Of The Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Pool

Isolated hepatocytes and the isolated perfused rat liver have been used to study the alterations of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) produced by 2,5-di(tert-butyl)-l.4-benzohydroquinone (tBuBHQ), a potent inhibitor of hepatic microsomal Ca²⁺ sequestration (Moore. G.A., McConkey. D.J., Kass, G.E.N., OBrien, P.J. and Orrenius, S. FEBS Lett.,224, 331-336). (1987). Addition of tBuBHQ to isolated hepatocytes caused a rapid increase in [Ca²⁺]_i which was similar in magnitude to the [Ca²⁺]_i elevation induced by the Ca²⁺ mobilizing hormone, vasopressin. In contrast with vasopressin which caused a Ca²⁺ transient, tBuBHQ elevated [Ca²⁺]_i to a new steady state that was maintained for up to 15-20min. When vasopressin was administered during the tBuBHQ-induced period of elevated [Ca²⁺]_i. [Ca²⁺]_i, rapidly returned to basal levels. Similarly, if vasopressin was administered just prior to tBuBHQ, the resultant tBuBHQ-dependent change in [Ca²⁺]_i was transient. and not sustained. The hydroquinone mobilized the same intracellular Ca²⁺ pool as inositol 1,4,5-trisphosphate. but tBuBHQ did not produce any detectable inositol polyphosphate accumulation. IBuBHQ stimulated glucose release from perifused hepatocytes. mimicking the effect of vasopressin. In the perfused liver, tBuBHQ infusion produced a single, slow and prolonged release of Ca²⁺ into the perfusate and inhibition of subsequent vasopressin-induced Ca²⁺ effluxes. Inhibition of the response to vasopressin was reversed over time, and closely correlated with the extent of inhibition of both Ca²⁺ sequestration and (Ca²⁺-Mg²⁺)-ATPase activity in microsomes isolated from the isolated perfused liver. The present results are consistent with tBuBHQ inhibiting ATP-dependent Ca²⁺ sequestration by a direct effect on the endoplasmic reticular Ca²⁺ pump, which results in net Ca²⁺ release and elevation of [Ca²⁺]_i. Furthermore. vasopressin appears to stimulate active removal of increased [Ca²⁺] from the hepatocyte cytosol by a mechanism which does not depend on reuptake of Ca²⁺ into the endoplasmic reticulum

2,5-Di(tert-butyl) -l,4-benmhydroquinone. calcium. hepatocytes. perfused liver, endoplasmic reticulum     

Insulin secretion and intracellular Ca rises in monolayer cultures of neonatal rat β-cells

Glucose-induced insuline release, glucose-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i), and voltage-dependent Ca²⁺ channel activity were assessed in monolayer cultures of β-vells 3–5 day-old rats. The glucose-stimulated insulin secretory responses and [Ca²⁺]_i rises were like those in adult rat β-cells rather than fetal rat β-cells. Voltage-dependent Ca²⁺ channel antagonists decreased glucose-induced insulin secretion, aborted the [Ca²⁺]₂ rise and, like deprivation of extracellular Ca²⁺, prevented the glucose-induced rise in [Ca²⁺]_i when added before the glucose challenge. The presence of nifedipine-sensitive, voltage-dependent Ca²⁺ channels was demonstrated directly by measuring Ca²⁺ currents using the whole-cell configuration of the patch-clamp technique and indirectly by measuring [Ca²⁺]₁ after membrane depolarization by 45 mMm K⁺ or 200 μM tolbutamide. Thus, in cultured β-cells of 3–5 day-old rats the coupling of glucose stimulation to Ca²⁺ influx is essentially mature, in contrast to what has been reported for fetal or very early neonatal cells.     

Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs

In cultured pituatary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca²⁺]_i) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca²⁺]_i and LH secretion. The spike phase of the [Ca²⁺]_i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca²⁺. In contrast, the prolonged phase of the Ca²⁺ signal depends exclusively on Ca²⁺ entry from the extracellular pool. The influx of Ca²⁺ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca²⁺]_i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca²⁺ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca²⁺ signaling and LH exocytosis. In contrast to [Ca²⁺]_i measurements in cell suspension, single cell Ca²⁺ measurements revealed the existence of a more complicated pattern of Ca²⁺ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca²⁺ spiking. In addition, analysis of the magnitudes of the magnitudes of the [Ca²⁺]_i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca²⁺, and to K⁺ and phorbol ester stimulation, showed non-linearity between these parameters with amplification of [Ca²⁺]_i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplication, especially during the plateau phase of the secretory response to GnRH.     

Lysophosphatidic Acid Sensitises Ca Influx through Mechanosensitive Ion Channels in Cultured Lens Epithelial Cells

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca²⁺]_i in the presence of LPA and this increase was concentration-dependent (1–10 μM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca²⁺]_i. The mechanical stress-induced increase in [Ca²⁺]_i in the presence of LPA was inhibited by removing extracellular Ca²⁺ or by addition of Gd³⁺, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca²⁺ influx through cation-selective mechanosensitive channels, but does not sensitise Ca²⁺ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca²⁺ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca²⁺ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca²⁺]_i induced by mechanical stress.     

Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.     

Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca concentration but not by an increase of cAMP in rat hepatocytes

A wasp venom, mastoparan, rapidly increased the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca²⁺]_i even in the absence of extracellular Ca²⁺, but a larger increase was observed in the presence of extracellular Ca²⁺. Thus, mastoparan mobilized Ca²⁺ from intracellular and extracellular Ca²⁺ stores. It also activated inositol triphosphate (IP₃) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP₃, which causes an increase of [Ca²⁺]_i but not that mediated by cAMP.     

V-1, a catecholamine biosynthesis regulatory protein,positively controls catecholamine secretion in PC12D cells

Stably transfected PC12D cell lines overexpressing a catecholamine biosynthesis regulatory protein, V-1, were used to examine the functional role of V-1 in catecholamine secretion. High K⁺-induced dopamine secretion in V-1 overexpressing clones was shown to be markedly potentiated compared with control clones carried with a vector alone. As assayed intracellular calcium concentration ([Ca²⁺]_i) using fura-PE3, V-1 overexpression was observed to enhance high K⁺-elicited [Ca²⁺]_i elevation. Electron microscopic analysis revealed an increase in dense-cored vesicle formation by V-1 overexpression. These results suggest that the enhancement of high K⁺-induced dopamine secretion by V-1 overexpression results from the potentiation of high K⁺-induced [Ca²⁺]_i elevation and the increase in the number of dense-cored vesicles.     

Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle?

Stretch of the myocardium influences the shape and amplitude of the intracellular Ca²⁺([Ca²⁺]_i) transient. Under isometric conditions stretch immediately increases myofilament Ca²⁺ sensitivity, increasing force production and abbreviating the time course of the [Ca²⁺]_i transient (the rapid response). Conversely, muscle shortening can prolong the Ca²⁺ transient by decreasing myofilament Ca²⁺ sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca²⁺ sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca²⁺ sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca²⁺ transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca²⁺]_i and the underlying mechanisms.

Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca²⁺]_i and in this review we discuss how stretch-induced changes in [Ca²⁺]_i can influence electrical activity via the modulation of these Ca²⁺-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca²⁺]_i with BAPTA, suggesting that both SACs and [Ca²⁺]_i are important for the full effects of axial stretch on electrical activity to develop.     

Release of azurophilic granule contents in fMLP-stimulated neutrophils requires two activation signals, one of which is a rise in cytosolic free Ca

We have used a continuous spectrofluorimetric method to analyse the role of cytosolic free Ca²⁺ ([Ca²⁺]_i) in the lysosomal enzyme release from the azurophilic granules in human neutrophils stimulated with f-Met-Leu-Phe (fMLP) in the presence of cytochalasin B. Measurements were performed with the β-glucuronidase substrate 4-methylumbelliferyl-β- -glucuronide. We found that the transient rise in [Ca²⁺]_i induced by fMLP is a necessary signal to obtain to obtain maximal degranulation. When this Ca²⁺ transient is prevented by the Ca²⁺ chelator BAPTA, degranulation can still be induced by a stimulated Ca²⁺ influx, albeit to a lower extent. We also studied the degranulation process in the neutrophils of a patient with a generalized chemotactic defect. Release of β-glucuronidase from the patient's neutrophils could not be induced despite the occurrence of a normal Ca²⁺ response and normal degranulation of specific granules. We conclude that, besides an increase in [Ca²⁺]_i], an additional signal is required for the fusion of azurophilic granules with the plasma membrane in human neutrophils.     

Effects of cholinergic agonists on diacylglycerol and intracellular calcium levels in pancreatic β-cells

We have studied the effects of cholinegic agonists on the rates of insulin release and the concentrations of diacylglycerol (DAG) and intracellular free Ca²⁺ ([Ca²⁺]_i) in the β-cell line MIN6. Insulin secretion was stimulated by glucose, by glibenclamide and by bombesin. In the presence of glucose, both acetylcholine (ACh) and carbachol (CCh) produced a sustained increase in the rate of insulin release which was blocked by EGTA or verapamil. The DAG content of MIN6 β-cells was not affected by glucose. Both CCh and ACh evoked an increase in DAG which was maximal after 5 min and returned to basal after 30 min; EGTA abolished the cholinergic-induced increased in DAG. ACh caused a transient rise in [Ca²⁺]_i which was abolished by omission of Ca²⁺ or by addition of devapamil. Thus, cholinergic stimulation of β-cell insulin release is associated with changes in both [Ca²⁺]_i and DAG. The latter change persists longer than the former and activation of protein kinase C and sensitization of the secretory process to Ca²⁺ may underlie the prolonged effects of cholinergic agonists on insulin release. However, a secretory response to CCh was still evident after both [Ca²⁺]_i and DAG had returned to control values suggesting that additional mechanisms may be involved.     

Modelling of calcium handling in airway myocytes

Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca²⁺]_i increase via Ca²⁺ release from the sarcoplasmic reticulum through InsP₃ receptors. Several models have been developed to explain the characteristics of InsP₃-induced [Ca²⁺]_i responses, in particular Ca²⁺ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca²⁺ handling, i.e., InsP₃ receptors, SERCAs, mitochondria and Ca²⁺-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca²⁺ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP₃ receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca²⁺ clearance in the pattern of [Ca²⁺]_i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca²⁺ influx. Stimulation of this model by simulated increase in [Ca²⁺]_i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca²⁺ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.     

Effects of Basal [Ca]i on Calcium Handling in Ca-Overloaded Rat Cultured Heart Muscle Cells

To elucidate the relationship between intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺-signalling by the sarcoplasmic reticulum (SR) in Ca²⁺-overloaded heart muscle cells, the direct effects of “basal” [Ca²⁺]_i on calcium waves were investigated by altering the membrane potential. When basal inter-calcium wave (BCW) [Ca²⁺]_i was maintained at a high level, (i) calcium waves showed more gradual and more rapidly suppressed increase in [Ca²⁺]-profile (P < 0.005), and (ii) calcium waves occurred at a significantly higher frequency and velocity (259% and 137%), than when low BCW [Ca²⁺]_i was maintained. Similar investigations on inhibition of the Na⁺-Ca²⁺ exchanger, however, showed that membrane potential did not elicit direct effects on calcium waves. These results showed that the elevation of BCW [Ca²⁺]_i per se directly influences Ca²⁺-signalling in heart muscle cells through non-equilibrated release-restoration Ca²⁺-handling by the SR.     

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10⁻⁶ M LTD₄, LTB₄ and FMLP with an increase in both InsP formation and [Ca²⁺]_i. FMLP caused a greater rise in InsPs than either LTD₄ or LTB₄, which were equivalent. LTD₄, however, caused a greater increase in [Ca²⁺]_i than LTB₄ (4-fold) or FMLP. The FMLP [Ca²⁺]_i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10⁻⁷ M for 3 min). In contrast, the LTD₄ [Ca²⁺]_i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca²⁺]_i and InsP responses to LTD₄ by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD₄ evoked increases in [Ca²⁺]_i.     

Extracellular Na removal enhances granule secretion in platelets 3-evidence that Na/H exchange is inhibitory to secretion induced by some agonists

The effect of extracellular Na⁺ ([Na⁺]_e) removal on agonist-induced granule secretion in platelets in relation to [ph]_i and [Ca²⁺]_i changes was investigated. Substitution of [Na⁺]_e with choline⁺ of K⁺ resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC₈. Increases in [Ca²⁺]_i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH]_i increases induced by thrombin, U46619, PMA and diC₈ were abolished and a drop in [pH]_i (0.05–0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH]_i and greatly increased secretion with all the agonists, particularly in the absence of [Na⁺]_e, clear evidence that [pH]_i rises due to Na⁺/H⁺ exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH₄Cl, which restored the increase in [pH]_i in the absence of [Na⁺]_e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin-induced secretion was observed when Na⁺ was replaced with Li⁺, which allowed a normal increase in [pH]_i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH]_i rise and potentiated thrombin-induced secretion. With collagen and U46619, the results suggest that removal of [Na⁺]_e per se rather than inhibition of Na⁺/H⁺ exchange results in enhanced secretion. It is concluded that [Na⁺]_e per se and [pH]_i elevations via Na⁺/H⁺ exchange both have important inhibitory roles in the control of platelet granule secretion.