SK&F 96365, a novel inhibitor of receptor-mediated calcium entry.

A novel inhibitor of receptor-mediated calcium entry (RMCE) is described. SK&F 96365 (1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride) is structurally distinct from the known 'calcium antagonists' and shows selectivity in blocking RMCE compared with receptor-mediated internal Ca2+ release. Human platelets, neutrophils and endothelial cells were loaded with the fluorescent Ca2(+)-indicator dyes quin2 or fura-2, in order to measure Ca2+ or Mn2+ entry through RMCE as well as Ca2+ release from internal stores. The IC50 (concn. producing 50% inhibition) for inhibition of RMCE by SK&F 96365 in platelets stimulated with ADP or thrombin was 8.5 microM or 11.7 microM respectively; these concentrations of SK&F 96365 did not affect internal Ca2+ release. Similar effects of SK&F 96365 were observed in suspensions of neutrophils and in single endothelial cells. SK&F 96365 also inhibited agonist-stimulated Mn2+ entry in platelets and neutrophils. The effects of SK&F 96365 were independent of cell type and of agonist, as would be expected for a compound that modulates post-receptor events. Voltage-gated Ca2+ entry in fura-2-loaded GH3 (pituitary) cells and rabbit ear-artery smooth-muscle cells held under voltage-clamp was also inhibited by SK&F 96365; however, the ATP-gated Ca2(+)-permeable channel of rabbit ear-artery smooth-muscle cells was unaffected by SK&F 96365. Thus SK&F 96365 (unlike the 'organic Ca2+ antagonists') shows no selectivity between voltage-gated Ca2+ entry and RMCE, although the lack of effect on ATP-gated channels indicates that it discriminates between different types of RMCE. The effects of SK&F 96365 on functional responses of cells thought to be dependent on Ca2+ entry via RMCE were also studied. Under conditions where platelet aggregation is dependent on stimulated Ca2+ entry via RMCE, the response was blocked by SK&F 96365 with an IC50 of 15.9 microM, which is similar to the IC50 of 8-12 microM observed for inhibition of RMCE. Adhesion and chemotaxis of neutrophils were also inhibited by SK&F 96365. SK&F 96365 is a useful tool to distinguish RMCE from internal Ca2+ release, and to probe the role of RMCE in mediating functional responses of cells. However, SK&F 96365 is not as potent (IC50 around 10 microM) or selective (also inhibits voltage-gated Ca2+ entry) as would be desirable, so caution must be exercised when using this compound.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Ligands to the platelet fibrinogen receptor glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca2+ or cyclic AMP.

Previous studies have suggested that the platelet glycoprotein complex GPIIb-IIIa, which is the putative fibrinogen receptor, regulates Ca2+ influx into platelets, possibly operating as a Ca2+ channel. We have used RGD-peptides (peptides containing the sequence Arg-Gly-Asp; disintegrins), isolated from snake venoms, that have a high affinity and specificity for the fibrinogen-binding site of GPIIb-IIIa to address the question of whether blocking this site inhibits Ca2+ movement from the extracellular medium to the cytosol. Using fura-2-loaded human platelets, we found that neither disintegrins nor a monoclonal antibody (M148) to the GPIIb-IIIa complex altered the level of cytosolic Ca2+ obtained when the cells were stimulated with various agonists in the presence of either nominal or 1 mM extracellular Ca2+. In the presence of Mn2+, an ion that quenches fura-2 fluorescence, fura-2-loaded platelets were stimulated with thrombin or ADP. Neither disintegrins nor the monoclonal antibody altered the kinetics or the amount of quenching of fura-2 fluorescence by Mn2+. These data indicate that the binding of ligands to the fibrinogen receptor is not associated with an inhibition of Ca2+ movement through a receptor-operated channel. Furthermore, the disintegrins have no effect on platelet cyclic AMP metabolism in either the presence or the absence of phosphodiesterase inhibitors.     

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.     

Activation of protein kinase C in human neutrophils attenuates agonist-stimulated rises in cytosolic free Ca2+ concentration by inhibiting bivalent-cation influx and intracellular Ca2+ release in addition to stimulating Ca2+ efflux.

Stimulation of fura-2-loaded human neutrophils with formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin elevated the cytosolic free Ca2+ concentration, [Ca2+], to a maintained elevated level. Activation of protein kinase C (C-kinase) with phorbol 12-myristate 13-acetate, 4 beta-phorbol 12,13-didecanoate or dioctanoylglycerol caused decreases in [Ca2+]i from this level. 4 alpha-Phorbol didecanoate, which does not activate C-kinase, had no effect. These results confirm previous reports that C-kinase activation decreases neutrophil [Ca2+]i by stimulating removal of Ca2+ from the cytosol. Further experiments showed that activation of C-kinase attenuated the component of the FMLP-stimulated [Ca2+]i rise that was dependent on external Ca2+. C-kinase activation also inhibited FMLP-stimulated entry of the quenching cation, Mn2+, used as an indicator of bivalent-cation entry. In contrast, C-kinase activation caused only a partial inhibition of FMLP-stimulated release of Ca2+ from intracellular stores. 4 alpha-Phorbol didecanoate was ineffective in inhibiting Ca2+ entry, Mn2+ entry and intracellular Ca2+ release. Addition of FMLP also stimulated a decrease in the ionomycin-elevated [Ca2+]i, and this effect was blocked by staurosporine, a protein kinase inhibitor. These results show that, in addition to stimulating Ca2+ efflux, C-kinase activation in neutrophils inhibits FMLP-stimulated entry of bivalent cations, and partially inhibits intracellular release of Ca2+. Further, FMLP itself can modulate [Ca2+]i by activation of C-kinase.     

Mechanisms of miconazole-induced rise in cytoplasmic calcium concentrations in Madin Darby canine kidney (MDCK) cells

The effect of miconazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ indicator. Miconazole increased [Ca2+]i dose-dependently at concentrations of 5-100 microM. The [Ca2+]i transient consisted of an initial rise, a gradual decay and an elevated plateau (220 s after addition of the drug). Removal of extracellular Ca2+ partly reduced the miconazole response. Mn2+ quench of fura-2 fluorescence confirmed that miconazole induced Ca2+ influx. The miconazole-sensitive intracellular Ca2+ store overlapped with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 20 microM miconazole depleted the thapsigargin (1 microM)-sensitive store, and conversely, thapsigargin abolished miconazole-induced internal Ca2+ release. Miconazole (20-50 microM) partly inhibited the capacitative Ca2+ entry induced by 1 microM thapsigargin, measured by depleting intracellular Ca2+ store in Ca(2+)-free medium followed by addition of 10 mM CaCl2. Miconazole induced capacitative Ca2+ entry on its own. Pretreatment with 0.1 mM La3+ partly inhibited 20 microM miconazole-induced Mn2+ quench of fura-2 fluorescence and [Ca2+]i rise, suggesting that miconazole induced Ca2+ influx via two pathways separable by 0.1 mM La3+. Miconazole-induced internal Ca2+ release was not altered when the cytosolic level of inositol 1,4,5-trisphosphate (IP3) was substantially inhibited by the phospholipase C inhibitor U73122.     

Refill status of the agonist-sensitive Ca2+ pool regulates Mn2+ influx into parotid acini

We have utilized fura-2 and a Ca2+ surrogate, Mn2+, to assess the mechanism of Ca2+ entry involved in the refill of the internal agonist-sensitive Ca2+ pool in parotid acini. Both the muscarinic agonist, carbachol, and the alpha-adrenergic agonist, epinephrine, stimulate Mn2+ entry into dispersed parotid acini, which is detected as an augmentation in fura-2 fluorescence quench rate. The rate of Mn2+ entry into parotid acini, depleted of internal agonist-sensitive Ca2+ pools by prolonged carbachol stimulation in a nominally Ca2(+)-free medium, is not significantly changed by the addition of the muscarinic antagonist, atropine, but is significantly attenuated when these internal pools are allowed to either partially or totally reload with Ca2+. Also, we provide evidence which suggests that under conditions which promote refill, Mn2+ appears to directly enter the cytosol from the extracellular medium and is not accumulated into an internal Ca2+ pool either directly from the medium or via a cytosolic route. Thus, we suggest that during refill, Ca2+ enters into the cytosol prior to its recruitment into the internal agonist-sensitive Ca2+ pool and in turn, the magnitude of this entry is modulated by the refill status of this pool.     

Platelets and parotid acinar cells have different mechanisms for agonist-stimulated divalent cation entry

Stimulation of platelets with thrombin or parotid acinar cells with carbachol results in an increase in [Ca2+]i which is due to both release from internal stores and influx across the plasma membrane. In platelets, thrombin also stimulates Mn2+ entry into the cytosol; this is seen as a stimulated quench of fura-2 fluorescence. In the parotid, however, carbachol does not stimulate Mn2+ entry. This result suggests different mechanisms of stimulated divalent cation entry in the two cell types and could have important implications in the study of receptor-mediated Ca2+ entry mechanisms.     

Functional implications of Ca2+ mobilizing properties for nitric oxide production in aortic endothelium

We have investigated the relationship between Ca2+ mobilization and the cellular production of nitric oxide (NO) by using fura-2 and diaminofluorescein-2 (DAF-2), an NO-sensitive dye, in bovine aortic endothelial cells (BAEC). High concentrations of ATP (100 microM) or thapsigargin (1 micro M) depleted intracellular Ca2+ store sites with a single Ca2+ transient, and induced an increase in DAF-2 fluorescence even in Ca2+-free solution, thereby indicating that store depletion leads to NO production. The same level of increase in DAF-2 fluorescence was elicited by low concentrations of ATP (1 micro M), which induced Ca2+ oscillations but did not deplete store sites, only in the presence of extracellular Ca2+. Furthermore, inhibition of ATP (1 micro M)-induced Ca2+ entry with La3+ suppressed DAF-2 fluorescence. ATP (0.3 micro M), applied in Ca2+-free, Mn2+-containing solution induced Mn2+ entry-coupled fura-2 quenching, repeating shortly after each oscillation peak. These results indicate that NO is produced preferentially by entered Ca2+, and that Ca2+ oscillations, which are induced by low levels of stimulation, play a significant role in NO production by strongly modulating Ca2+ entry.     

A role for Ca2+ in mediating hormone-induced biphasic pepsinogen secretion from the chief cell determined by luminescent and fluorescent probes and X-ray microprobe

In isolated chief cells from the guinea pig, cholecystokinin (10 nM) and a high concentration of ionomycin each caused a biphasic pattern of pepsinogen secretion. The initial fast response to cholecystokinin was not dependent on medium Ca2+ ans was mimicked by low concentration of ionomycin (100 nM). Inositol 1,4,5-trisphosphate caused a similar fast release from permeabilized cells. The slow component of release was dependent on medium Ca2+, however, and was mimicked by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) (100 nM) or the diacylglycerol analogue 1-oleoyl-2-acetylglycerol (OAG) (100 microM). Ionomycin (100 nM) and TPA (and/or OAG), when applied together, reproduced the biphasic pattern of pepsinogen secretion, suggesting that the signalling pathways utilized by both types of agonist contribute to the response evoked by cholecystokinin-hormone stimulation. Both fura-2 and aequorin were used to monitor changes of intracellular Ca2+. Three pathways were found to contribute to the Ca2+ transient. A rapid release of Ca2+ from intracellular store(s), a rapid Ca2+ entry from the extracellular space, and a more sustained Ca2+ entry from the extracellular space. Cholecystokinin induced a rapid increase in cytoplasmic Ca2+ ([Ca2+]i) as estimated with fura-2 and aequorin. This rise was reduced but not abolished upon removal of extracellular Ca2+, suggesting that both Ca2+ entry from the extracellular space and Ca2+ mobilization from the intracellular store(s) contribute to the initial, fast component of the Ca2+ transient. A second, more sustained component of the Ca2+ transient induced by cholecystokinin was abolished by lanthanum. TPA and OAG induced a biphasic Ca2+ transient that could be detected only with aequorin. The late, sustained component of this response was again abolished by lanthanum as well as by removal of extracellular Ca2+. It appears that the late component of the Ca2+ transient is dependent on Ca2+ influx from the extracellular space and is too localized to be detected by fura-2. Prestimulation of cells with TPA or OAG prevented the aequorin transient caused by cholecystokinin and vice versa, suggesting that TPA, OAG and cholecystokinin activate the same pathways of Ca2+ entry into the cytosol from the intracellular store(s) or the extracellular space. The stimulation-sensitive Ca2+ pool was examined with electron probe X-ray microanalysis. It appears to be restricted to an area enriched in secretory granules or peripheral endoplasmic reticulum just beneath the apical plasma membrane and in close association with the microtubular-microfilamentous system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arachidonic acid-induced calcium influx in human platelets. Comparison with the effect of thrombin.

The effects of arachidonic acid and thrombin on calcium movements have been studied in fura-2-loaded platelets by a procedure which allows simultaneous monitoring of the uptake of manganese, a calcium surrogate for Ca2+ channels, and the release of Ca2+ from intracellular stores. Arachidonic acid induced both Ca2+ (Mn2+) entry through the plasma membrane and Ca2+ release from the intracellular stores. The release of Ca2+ was prevented by cyclo-oxygenase inhibitors and mimicked by the prostaglandin H2/thromboxane A2 receptor agonist U46619. Ca2+ (Mn2+) entry required higher concentrations of arachidonic acid and was not prevented by either cyclo-oxygenase or lipoxygenase inhibitors. Several polyunsaturated fatty acids reproduced the effect of arachidonic acid on Ca2+ (Mn2+) entry, but higher concentrations were required. The effects of maximal concentrations of arachidonic acid and thrombin on the uptake of Mn2+ were not additive. Both agonists induced the entry of Ca2+, Mn2+, Co2+ and Ba2+, but not Ni2+, which, in addition, blocked the entry of the other divalent cations. However, arachidonic acid, but not thrombin, increased a Ni2(+)-sensitive permeability to Mg2+. The effect of thrombin but not that of arachidonic acid was prevented either by pretreatment with phorbol ester or by an increase in cyclic-AMP levels. Arachidonic acid also accelerated the uptake of Mn2+ by human neutrophils, rat thymocytes and Ehrlich ascites-tumour cells.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Release of Ca2+ from a non-mitochondrial store site in peritoneal macrophages treated with saponin by inositol 1,4,5-trisphosphate.

The effects of inositol 1,4,5-trisphosphate, prepared from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated guinea pig peritoneal macrophages. Micromolar concentrations of inositol 1,4,5-trisphosphate released Ca2+ within 1 min from store sites which had accumulated Ca2+ in the presence of 10 mM-NaN3. In the presence of 10 mM-NaN3, the Ca2+ accumulated in the presence of oxalate was seen in the endoplasmic reticulum of saponin-treated macrophages by electron microscopy, indicating that the site of Ca2+ released by inositol 1,4,5-trisphosphate may be endoplasmic reticulum-like membranes. When the concentrations of free Ca2+ were over 3.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. This inhibition may be due to either the higher concentration of extra-vesicular free Ca2+ or the larger accumulation of Ca2+ into the store site or perhaps both effects. MgCl2 also had an inhibitory effect on the Ca2+ release. Inositol 1,4,5-trisphosphate also released Ca2+ from cardiac sarcoplasmic reticulum, but not from erythrocyte inside-out vesicles.     

Increase in Ca2+ permeability of intracellular Ca2+ store membrane of saponin-treated guinea pig peritoneal macrophages by inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) releases Ca2+ from the non-mitochondrial Ca2+ store site of various types of cells. To study the mechanisms of the Ca2+ release from the store site, the effect of InsP3 on the passive Ca2+ release and influx, and the active Ca2+ uptake in the presence of oxalate, was examined using saponin-treated guinea pig peritoneal macrophages. InsP3 stimulated the passive Ca2+ release and influx. Although InsP3 slightly inhibited the active Ca2+ uptake in the presence of oxalate, it seems unlikely that the Ca2+ release by this agent is caused by the inhibition of the Ca2+ uptake, because the addition of apyrase or hexokinase (which removes ATP within 30 s, so that no more Ca2+ can be accumulated) or vanadate (which inhibits the Ca2+ uptake) resulted in very slow release of Ca2+. These results suggest that the Ca2+ permeability of the Ca2+ store membrane is increased by InsP3. InsP3 did not cause an increase in the Ca2+ permeability of phospholipid vesicles (liposomes), indicating that this agent may bring about Ca2+ release by a specific effect on the physiologically relevant Ca2+ channels or carriers in the non-mitochondrial Ca2+ store site. The passive Ca2+ release by InsP3 was enhanced by ATP and an unhydrolyzable ATP analogue, 5'-adenylyimidodiphosphate, but not by ADP or AMP. The passive Ca2+ release by InsP3 was observed even at 0 degree C.     

Receptor-operated calcium influx in rat hepatocytes. Identification and characterization using manganese

Agonist-stimulated divalent cation entry was studied in fura-2-loaded hepatocytes. In the presence of extracellular Mn2+, the Ca2(+)-mobilizing hormone vasopressin produced a severalfold stimulation of the basal rate of fura-2 fluorescence quenching as a result of Mn2+ influx; this effect was blocked by the presence of Ni2+ in the incubation medium. Half-maximum and maximum stimulation of Mn2+ influx was observed with 0.1 and 0.8 nM vasopressin, respectively. Agonist-stimulated Mn2+ influx was also seen with angiotensin II, ATP, phenylephrine, and the combination of AlCl3 and NaF. The stimulation of Mn2+ influx did not occur immediately after addition of Ca2(+)-mobilizing agents, but was characterized by a latency period of 20-30 s. In contrast to vasopressin, glucagon did not stimulate Mn2+ influx into hepatocytes, but produced both a 3-fold enhancement of the rate of vasopressin-stimulated Mn2+ entry and the abolishment of the latency period. The effects of glucagon were mimicked by forskolin and dibutyryl cAMP. Pretreatment of hepatocytes with pertussis toxin or depolarization of the cells altered neither the basal rate of Mn2+ entry nor the ability of vasopressin to stimulate this rate. Emptying of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by treatment with 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) did not enhance Mn2+ entry into hepatocytes; however, exposure of the cells to tBuBHQ for 2 min markedly enhanced the ability of vasopressin, alone or in combination with glucagon, to increase the rate of Mn2+ influx. Furthermore, pretreatment with tBuBHQ for 2 min abolished the latency of vasopressin-stimulated Mn2+ influx. It is concluded that Ca2(+)-mobilizing hormones stimulate Ca2+ influx in hepatocytes, possibly through receptor-operated Ca2+ channels. The stimulation of divalent cation entry is transduced by a G protein, and the rate of influx appears to be controlled both by the intracellular level of cAMP and the empty state of an intracellular Ca2+ pool that may be inositol 1,4,5-trisphosphate-insensitive.     

Intracellular alkalinization induces Ca2+ influx via non-voltage-operated Ca2+ channels in rat aortic smooth muscle cells

In smooth muscle, the cytosolic Ca2+ concentration ([Ca2+](i)) is the primary determinant of contraction, and the intracellular pH (pH(i)) modulates contractility. Using fura-2 and 2',7'-biscarboxyethyl-5(6) carboxyfluorescein (BCECF) fluorometry and rat aortic smooth muscle cells in primary culture, we investigated the effect of the increase in pH(i) on [Ca2+](i). The application of the NH(4)Cl induced concentration-dependent increases in both pH(i) and [Ca2+](i). The extent of [Ca2+](i) elevation induced by 20mM NH(4)Cl was approximately 50% of that obtained with 100mM K(+)-depolarization. The NH(4)Cl-induced elevation of [Ca2+](i) was completely abolished by the removal of extracellular Ca2+ or the addition of extracellular Ni2+. The 100mM K(+)-induced [Ca2+](i) elevation was markedly inhibited by a voltage-operated Ca2+ channel blocker, diltiazem, and partly inhibited by a non-voltage-operated Ca2+ channel blocker, SKF96365. On the other hand, the NH(4)Cl-induced [Ca2+](i) elevation was resistant to diltiazem, but was markedly inhibited by SKF96365. It is thus concluded that intracellular alkalinization activates the Ca2+ influx via non-voltage-operated Ca2+ channels and thereby increases [Ca2+](i) in the vascular smooth muscle cells. The alkalinization-induced Ca2+ influx may therefore contribute to the enhancement of contraction.     

Capacitative cation entry in human myometrial cells and augmentation by hTrpC3 overexpression

Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca2+ influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca2+ entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca2+ stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca2+ entry, which was inhibited both by SKF 96365 and gadolinium (Gd3+). Whereas unstimulated cells did not exhibit Sr2+ entry, oxytocin and thapsigargin enhanced Sr2+ entry that was also inhibited by SKF 96365 and Gd3+. In contrast, Ba2+, a poor substrate for Ca2+ pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd3+. The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca2+ that were dependent on extracellular Ca2+. These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca2+ entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca2+ entry can potentially contribute to Ca2+ dynamics controlling uterine smooth muscle contractile activity.     

Soluble E-selectin acts in synergy with platelet-activating factor to activate neutrophil beta 2-integrins. Role of tyrosine kinases and Ca2+ mobilization

Selectins play a critical role in neutrophil recruitment to sites of inflammation, in tethering and rolling of neutrophils on vascular endothelium, as well as triggering beta(2)-integrin-mediated adhesion. We have previously demonstrated potential pro-inflammatory effects of soluble E-selectin upon neutrophil effector functions, using a soluble recombinant molecule (E-zz), which increased beta(2)-integrin-mediated adhesion, decreased beta(2)-integrin-dependent migration, and triggered reactive oxygen species generation and release. In this study, we have examined the intracellular signals following neutrophil activation by soluble E-selectin. We show that exposure of neutrophils to E-selectin and platelet-activating factor (PAF) in combination induced a synergistic effect upon beta(2)-integrin-mediated adhesion. Although soluble E-selectin did not induce Ca(2+) mobilization in neutrophils by itself, elevation of intracellular Ca(2+) was specifically prolonged in response to PAF but not leukotriene B(4) or N-formyl-Met-Leu-Phe. The prolonged Ca(2+) mobilization observed in the presence of E-selectin was dependent on Ca(2+) influx from intracellular stores rather than influx of extracellular Ca(2+) through SKF 96365-sensitive channels. The specific alteration of Ca(2+) mobilization reported here appears not to have a role in the synergistic effects of E-selectin and PAF upon neutrophil O(2) release but may be involved in augmentation of beta(2)-integrin-mediated adhesion.     

Characterisation of Distinct Inositol 1,4,5-Trisphosphate-Sensitive and Caffeine-Sensitive Calcium Stores in Digitonin-Permeabilised Adrenal Chromaffin Cells

The effect of inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3] and caffeine on Ca2+ release from digitonin-permeabilised bovine adrenal chromaffin cells was examined by using the Ca2+ indicator fura-2 to monitor [Ca2+]. Permeabilised cells accumulated Ca2+ in the presence of ATP and addition of either Ins(1,4,5)P3 or caffeine released 17% or 40-50%, respectively, of the accumulated Ca2+, indicated by sustained rises in [Ca2+] in the cell suspension. Prior addition of Ins(1,4,5)P3 had no effect on the magnitude of the response to a subsequent addition of caffeine. The response to Ins(1,4,5)P3 was prevented by prior addition of caffeine or CaCl2, indicating that the Ins(1,4,5)P3 response was blocked by elevated [Ca2+]. The responses were essentially identical in the presence of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, indicating that the Ca2+ release was not from mitochondria or secretory granules and that a proton gradient was not required for Ca2+ accumulation into the Ins(1,4,5)P3- or caffeine-sensitive stores. Ca2+ release from the caffeine-sensitive store was selectively blocked by ryanodine. The Ins(1,4,5)P3-sensitive store was emptied by thapsigargin, which had no effect on caffeine responses. These data suggest that permeabilised chromaffin cells possess two distinct nonoverlapping Ca2+ stores sensitive to either Ins(1,4,5)P3 or caffeine and support previous conclusions that these stores possess different Ca2(+)-ATPases.