Urokinase activates calcium-dependent potassium channels in U937 cells via calcium release from intracellular stores.

The urokinase receptor (uPAR) is highly expressed in the human promyelocytic cell line U937 and contributes to transmembrane signalling. However, the signalling mechanisms are poorly understood. We used the patch-clamp technique to demonstrate that urokinase (uPA) binds to uPAR and thereby stimulates Ca(2+)-activated K+ channels in U937 cells. uPA transiently increased K+ currents within 30 s. The K+ currents were pertussis toxin-sensitive and were also observed in Ca(2+)-free solution. However, when cells were dialysed with EGTA, uPA did not affect K+ currents. The intracellular Ca2+ response to uPA was independent of extracellular Ca2+, was pertussis toxin-sensitive, and was blocked by both thapsigargin and the phospholipase C inhibitor U-73122. The uPA-induced increase in intracellular Ca2+ was independent of uPA proteolytic activity. Furthermore, uPA initiated a rapid formation of inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]. The amino-terminal uPA fragment and uPA inactivated with diisopropyl fluorophosphate or with inhibitory antibody, elicited the same Ca2+ signal. On the other hand, Ca2+ signalling required the intact uPAR because the effects were abrogated by PtdIns-specific phospholipase C, which removes the uPAR from the cell surface. The prevention of glycosyl phosphatidylinositol moiety synthesis and interference with uPAR anchoring to the cell surface using mannosamine also abolished Ca2+ signals. Taken together, our findings indicate that uPA binds to uPAR and stimulates the production of Ins(1,4,5)P3 via a G-protein- and phospholipase C-dependent mechanism. Ins(1,4,5)P3 in turn liberates Ca2+ from intracellular stores, which leads to the stimulation of Ca(2+)-activated K+ channels.     

Clustering of urokinase receptors (uPAR; CD87) induces proinflammatory signaling in human polymorphonuclear neutrophils

Leukocytes use urokinase receptors (uPAR; CD87) in adhesion, migration, and proteolysis of matrix proteins. Typically, uPAR clusters at cell-substratum interfaces, at focal adhesions, and at the leading edges of migrating cells. This study was undertaken to determine whether uPAR clustering mediates activation signaling in human polymorphonuclear neutrophils. Cells were labeled with fluo-3/AM to quantitate intracellular Ca2+ ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by Ab cross-linking. Aggregating uPAR induced a highly reproducible increase in [Ca2+]i (baseline to peak) of 295 +/- 37 nM (p = 0.0002). Acutely treating cells with high m.w. urokinase (HMW-uPA; 4000 IU/ml) produced a response of similar magnitude but far shorter duration. Selectively aggregating uPA-occupied uPAR produced smaller increases in [Ca2+]i, but saturating uPAR with HMW-uPA increased the response to approximate that of uPAR cross-linking. Cross-linking uPAR induced rapid and significant increases in membrane expression of CD11b and increased degranulation (release of beta-glucuronidase and lactoferrin) to a significantly greater degree than cross-linking control Abs. The magnitude of degranulation correlated closely with the difference between baseline and peak [Ca2+]i, but was not dependent on the state of uPA occupancy. By contrast, selectively cross-linking uPA-occupied uPAR was capable of directly inducing superoxide release as well as enhancing FMLP-stimulated superoxide release. These results could not be duplicated by preferentially cross-linking unoccupied uPAR. We conclude that uPAR aggregation initiates activation signaling in polymorphonuclear neutrophils through at least two distinct uPA-dependent and uPA-independent pathways, increasing their proinflammatory potency (degranulation and oxidant release) and altering expression of CD11b/CD18 to favor a firmly adherent phenotype.     

Cytosolic multiple inositol polyphosphate phosphatase in the regulation of cytoplasmic free Ca2+ concentration

Multiple inositol polyphosphate phosphatase (MIPP) is an enzyme that, in vitro, has the interesting property of degrading higher inositol polyphosphates to the Ca2+ second messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), independently of inositol lipid breakdown. We hypothesized that a truncated cytosolic form of the largely endoplasmic reticulum-confined MIPP (cyt-MIPP) could represent an important new tool in the investigation of Ins(1,4,5)P3-dependent intracellular Ca2+ homeostasis. To optimize our ability to judge the impact of cyt-MIPP on intracellular Ca2+ concentration ([Ca2+]i) we chose a poorly responsive beta-cell line (HIT M2.2.2) with an abnormally low [Ca2+]i. Our results show for the first time in an intact mammalian cell that cyt-MIPP expression leads to a significant enhancement of Ins(1,4,5)P3 concentration. This is achieved without a significant interference from other cyt-MIPP-derived inositol phosphates. Furthermore, the low basal [Ca2+]i of these cells was raised to normal levels (35 to 115 nm) when they expressed cyt-MIPP. Noteworthy is that the normal feeble glucose-induced Ca2+ response of HIT M2.2.2 cells was enhanced dramatically by mechanisms related to this increase in basal [Ca2+]i. These data support the use of cyt-MIPP as an important tool in investigating Ins(1,4,5)P3-dependent Ca2+ homeostasis and suggest a close link between Ins(1,4,5)P3 concentration and basal [Ca2+]i, the latter being an important modulator of Ca2+ signaling in the pancreatic beta-cell.     

Ca2+ Mobilized by Caffeine from the Inositol 1,4,5-Trisphosphate-Insensitive Pool of Ca2+ in Somatic Regions of Sympathetic Neurons Does Not Evoke [3H]Norepinephrine Release

The effects of electrical stimulation, muscarinic and serotonergic agonists, and caffeine on [3H]inositol 1,4,5-trisphosphate ([3H]Ins(1,4,5)P3) content, intracellular free Ca2+ concentration ([Ca2+]i), and release of [3H]norepinephrine ([3H]NE) were studied in cultured sympathetic neurons. Neuronal cell body [Ca2+]i was unaffected by muscarinic or serotonergic receptor stimulation, which significantly increased [3H]Ins(1,4,5)P3 content. Stimulation at 2 Hz and caffeine had no effect on [3H]Ins(1,4,5)P3, but caused greater than two-fold increase in [Ca2+]i. Only 2-Hz stimulation released [3H]NE. Caffeine had no effect on the release. When [Ca2+]i was measured in growth cones, only electrical stimulation produced an increase in [Ca2+]i. The other agents had no effect on Ca2+ at the terminal regions of the neurons. We conclude that Ins(1,4,5)P3-insensitive, but caffeine-sensitive Ca2+ stores in sympathetic neurons are located only in the cell body and are not coupled to [3H]NE release.     

Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry

The relationships between receptor-mediated endocytosis and the generation of intracellular signals were analyzed in angiotensin II (AII)-stimulated adrenal glomerulosa cells. In cells equilibrated with 125I-AII analogs at 4 degrees C, specifically bound agonist but not antagonist AII derivatives were rapidly internalized at 37 degrees C. AII-induced internalization was not influenced by the presence or absence of extracellular Ca2+ but was inhibited by treatment with phenylarsine oxide (PAO) or by arresting coated pit formation with hypotonic shock and potassium depletion. Inhibition of internalization by PAO was prevented by the bifunctional sulfhydryl reagent dithiothreitol but only partially reversed by mercaptoethanol, and readdition of K+ restored internalization in K(+)-depleted cells. Treatment with PAO did not impair the initial AII-induced elevations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and cytoplasmic calcium [( Ca2+]i) but reduced the sustained phase of the Ins(1,4,5)P3 response by 85% and abolished the second phase of the cytoplasmic Ca2+ response; these responses were restored by concomitant treatment with dithiothreitol. Inhibition of AII-receptor internalization by K+ depletion also caused selective loss of the sustained phase of the AII-induced Ca2+ response. Thus, blockade of AII-receptor internalization has similar effects as extracellular Ca2+ deficiency, which abolishes the sustained but not the early AII-induced increases in Ins(1,4,5)P3 production and [Ca2+]i. The close correlations between AII-induced internalization and the generation of Ins(1,4,5)P3 and [Ca2+]i responses suggest that endocytosis of the agonist-receptor complex is necessary to maintain the production of these intracellular signals. It is also possible that receptor-operated vesicular uptake of extracellular Ca2+ makes a significant contribution to the sustained [Ca2+]i responses of certain agonist-stimulated target cells.     

Roles for Ca2+ stores release and two Ca2+ influx pathways in the Fc epsilon R1-activated Ca2+ responses of RBL-2H3 mast cells.

Cross-linking the high affinity IgE receptor, Fc epsilon R1, with multivalent antigen induces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent release of intracellular Ca2+ stores, Ca2+ influx, and secretion of inflammatory mediators from RBL-2H3 mast cells. Here, fluorescence ratio imaging microscopy was used to characterize the antigen-induced Ca2+ responses of single fura-2-loaded RBL-2H3 cells in the presence and absence of extracellular Ca2+ (Ca2+o). As antigen concentration increases toward the optimum for secretion, more cells show a Ca2+ spike or an abrupt increase in [Ca2+]i and the lag time to onset of the response decreases both in the presence and the absence of Ca2+o. When Ca2+o is absent, fewer cells respond to low antigen and the lag times to response are longer than those measured in the presence of Ca2+o, indicating that Ca2+o contributes to Ca2+ stores release. Ins(1,4,5)P3 production is not impaired by the removal of Ca2+o, suggesting that extracellular Ca2+ influences Ca2+ stores release via an effect on the Ins(1,4,5)P3 receptor. Stimulation with low concentrations of antigen can lead, only in the presence of Ca2+o, to a small, gradual increase in [Ca2+]i before the abrupt spike response that indicates store release. We propose that this small, initial [Ca2+]i increase is due to receptor-activated Ca2+ influx that precedes and may facilitate Ca2+ stores release. A mechanism for capacitative Ca2+ entry also exists in RBL-2H3 cells. Our data suggest that a previously undescribed response to Fc epsilon R1 cross-linking, inhibition of Ca2+ stores refilling, may be involved in activating capacitative Ca2+ entry in antigen-stimulated RBL-2H3 cells, thus providing the elevated [Ca2+]i required for optimal secretion. The existence of both capacitative entry and Ca2+ influx that can precede Ca2+ release from intracellular stores suggests that at least two mechanisms of stimulated Ca2+ influx are present in RBL-2H3 cells.     

Ethanol-induced increases in [Ca2+]i and inositol (1,4,5) triphosphate in rat hepatocytes

Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.     

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.     

Neuropeptide Y-induced intracellular Ca2+ increases in vascular smooth muscle cells

The effect of neuropeptide Y (NPY) on cytosolic free Ca2+ concentration ([Ca2+]i) was studied in cultured smooth muscle cells from porcine aorta (PASMC) and compared with the effect of bradykinin (BK) and angiotensin II (ATII) on [Ca2+]i. All peptides induced dose-dependent and transient rises in [Ca2+]i which were not blocked by extracellular EGTA, but the NPY response was different from the others' as follows. First, the [Ca2+]i rise induced by NPY was not as rapid as that induced by BK or ATII. Second, pertussis toxin abolished the [Ca2+]i rise induced by NPY, but not by BK or ATII. Third, following initial treatment with BK, PASMC were able to respond to NPY, but not to ATII. Finally, BK and ATII, but not NPY, significantly increased inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation. Although NPY attenuated forskolin-induced accumulation of cyclic AMP, forskolin- and 3-isobutyl-1-methyl-xanthine-induced alterations in intracellular cyclic AMP did not affect the NPY-induced [Ca2+]i rise. These results suggest that NPY increases [Ca2+]i by a pertussis toxin-sensitive GTP binding protein-involved mechanism which is not mediated by the intracellular messengers such as Ins(1,4,5)P3 and cyclic AMP.     

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.     

Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate

The ability of epidermal growth factor (EGF) and angiotensin II to stimulate production of inositol trisphosphate and mobilize intracellular Ca2+ in hepatocytes was compared using quin2 fluorescence to monitor changes in Ca2+ levels and high performance liquid chromatography to resolve the inositol trisphosphate (InsP3) isomers. Both EGF and angiotensin II stimulated an increase in free intracellular Ca2+ concentration ([Ca2+]i) as well as a rapid increase in the production of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Concentrations of angiotensin II which gave a rise in [Ca2+]i equivalent to that seen with maximal doses of EGF produced an equivalent increase in Ins(1,4,5)P3 formation. Both EGF and angiotensin II stimulated the formation of the Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate isomers. The formation of the Ins(1,3,4)P3 isomer lagged behind production of Ins(1,4,5)P3 but eventually reached higher levels in the cell. The initial rise in [Ca2+]i and InsP3 levels stimulated by EGF and angiotensin II was not affected by reducing the external Ca2+ concentration below 30 nM with an excess of [ethylenebis(oxyethylenenitrilo)] tetraacetic acid. Treatment of hepatocytes for 30-180 s with 1 micrograms/ml phorbol 12-myristate 13-acetate prior to the addition of EGF blocked the EGF-stimulated production of Ins(1,4,5)P3 and the increase in [Ca2+]i. Phorbol 12-myristate 13-acetate attenuated the production of Ins(1,4,5)P3 generated by angiotensin II over the concentration range of 10(-10) to 10(-8) M; however, the Ca2+ signal was only inhibited at the 10(-10) M dose of angiotensin II. Treatment of rats with pertussis toxin for 72 h prior to isolating hepatocytes blocked the ability of EGF to increase Ins(1,4,5)P3 and Ins(1,3,4)P3 but did not inhibit the ability of any concentration of angiotensin II to stimulate formation of InsP3 or inositol tetrakisphosphate. The observation that pertussis toxin selectively abolishes EGF-stimulated inositol lipid breakdown suggests that EGF and angiotensin II use different mechanisms to activate phospholipase C in hepatocytes.     

Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: which route?

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca2+]i. The rise in [Ca2+]i is biphasic with the peak phase being due to release from an intracellular Ins(1,4,5)P3-sensitive site and the plateau phase being due to Ca2+ entry across the plasma membrane. Ca2+ entry does not appear to involve voltage sensitive Ca2+ channels, a pertussis toxin sensitive G-protein-operated Ca2+ channel or Ins(1,4,5)P3/Ins(1,3,4,5)P4-operated Ca2+ channel. We suggest that carbachol-stimulated Ca2+ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca2+ channels and through capacitive refilling.     

Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry.

Changes in intracellular Ca2+ concentration ([Ca2+]i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40-48 h. In these cells, the carbachol-induced sustained increase in [Ca2+]i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca2+ and a corresponding further enhancement of Ca2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P3 or changes in the generation of Ins(1,3,4,5)P4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P3, or a desensitization of the Ins(1,4,5)P3 receptor/Ca(2+)-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca2+ entry specifically on the status of the Ca(2+)-uptake component of the agonist-sensitive store, rather than the Ca(2+)-release component.     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

Receptor-activated calcium entry in exocrine cells does not occur via agonist-sensitive intracellular pools.

Currently, most models describing receptor-activated Ca2+ entry in exocrine cells invoke a pathway for the entry of extracellular Ca2+ directly linking the agonist-sensitive intracellular Ca2+ pools with the plasma membrane. In the avian nasal gland, a model exocrine ion-secreting tissue, we have found that Ca2+ entry during refilling of the intracellular pools following termination of receptor activation (by atropine) occurs via the cytoplasm and not directly into the empty pools. Under appropriate conditions this can be demonstrated as a transient increase in [Ca2+]i (intracellular Ca2+ concn.) seen on restoration of normal extracellular Ca2+ concentrations after atropine to stimulated cells whose intracellular stores have been prevented from refilling by incubation in a low-extracellular-Ca2+ medium. The magnitude of these [Ca2+]i transients decays with time, but with a time course markedly slower than for the corresponding decrease in intracellular Ins(1,4,5)P3. Further experiments have revealed that Ca2+ entry into the cytoplasm during the initial stimulation phase is also direct and not via the intracellular pools. Thus the initial rates of increase in [Ca2+]i during stimulation are always faster in conditions where both Ca2+ entry and Ca2+ release occur (i.e. they are additive). These differences could not be explained by any effects of extracellular Ca2+ on the initial increases in intracellular Ins(1,4,5)P3 after addition of carbachol. These data are therefore inconsistent with the current models in which the rate of Ca2+ entry through the agonist-sensitive pools cannot exceed the rate of Ca2+ release. It appears therefore that Ca2+ entry and Ca2+ release must occur via separate pathways operating in parallel, and not in series as previously predicted.     

Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.     

Role of Thapsigargin-Sensitive Intracellular Ca2+ Pools in Secretion Induced by Muscarinic Agonists in Porcine Adrenal Chromaffin Cells

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive Ca2+ pools in secretion, induced by muscarinic agonists in porcine adrenal chromaffin cells, was studied. Activation of muscarinic receptors, as in other species, was found to increase inositol phosphate production including that of Ins(1,4,5)P3. Treatment of cells with thapsigargin, which is known to deplete Ins(1,4,5)P3-sensitive Ca2+ pools, eliminated the initial transient component of increases in the cytosolic free Ca2+ concentration ([Ca2+]in) induced by the muscarinic agonist, methacholine, in both the presence and the absence of extracellular Ca2+. Thapsigargin treatment also decreased methacholine-induced secretion by about 30% in the presence of extracellular Ca2+ and essentially eliminated secretion that occurred independently of extracellular Ca2+ (which was about 30% of the secretory response that occurred in the presence of extracellular Ca2+). Thapsigargin itself had no effect on inositol phosphate production. These results indicate that about 30% of muscarinic agonist-induced secretion is mediated by the release of Ca2+ from Ins(1,4,5)P3- and thapsigargin-sensitive intracellular Ca2+ pools. These results also suggest that Ca2+ influx activated by muscarinic agonists is not due to depletion of intracellular Ca2+ pools, as prior depletion of these pools had no effect on the portion of the methacholine-induced secretory response and [Ca2+]in signal that was dependent on extracellular Ca2+.     

Relationship between agonist- and thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. Thapsigargin-induced Ca2+ mobilization and entry

The relationships between agonist-sensitive calcium pools and those discharged by the Ca(2+)-ATPase inhibitor thapsigargin were studied in intact bovine adrenal glomerulosa cells and a subcellular adrenocortical membrane fraction. In Fura-2-loaded glomerulosa cells, angiotensin II (AII) stimulated a rapid increase in cytoplasmic Ca2+ concentration ([Ca2+]i) followed by a smaller plateau phase that was dependent on extra-cellular Ca2+. In such cells thapsigargin caused a sustained and dose-dependent increase in [Ca2+]i which was diminished in Ca(2+)-deficient medium. The contribution of an influx component to the thapsigargin-induced [Ca2+]i response was demonstrated by measurement of 45Ca influx rate in glomerulosa cells. Thapsigargin-induced Ca2+ entry was significantly less than that evoked by AII, and its kinetics were similar to those of the concomitant increase in [Ca2+]i. The rate of emptying of the agonist-responsive Ca2+ pool after thapsigargin treatment, as indicated by the progressive decrease in the size of the AII-induced Ca2+ transient, showed a rapid initial (t1/2 = 1.7 min) component that accounted for about 80% of the response and a slowly decreasing phase with t1/2 = 112 min. The latter thapsigargin-resistant component was abolished by the removal of extracellular Ca2+. Pretreatment with AII dose-dependently attenuated but did not abolish the subsequent Ca2+ response to thapsigargin and also increased the rate of the Ca2+ rise induced by thapsigargin. In bovine adrenocortical microsomes, thapsigargin inhibited the ATP-dependent filling of Ca2+ pools and caused a dose-dependent rise in extravesicular Ca2+ levels when added to previously loaded microsomes. The thapsigargin-releasable Ca2+ pool in adrenal microsomes was larger than the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ pool but only slightly greater than the GTP-releasable pool. Ins(1,4,5)P3-induced Ca2+ release was reduced markedly when ATP-dependent Ca2+ loading of the microsomes was prevented by prior addition of thapsigargin. However, the subsequent Ca2+ response to Ins(1,4,5)P3 was consistently better preserved after the addition of thapsigargin to microsomes preloaded with Ca2+. This difference suggests that although Ca2+ uptake by the Ins(1,4,5)P3-responsive pool is also sensitive to thapsigargin, once filled, this pool shows a slower passive leakage than other thapsigargin-sensitive pools. These findings indicate that thapsigargin increases [Ca2+]i by inhibiting Ca2+ uptake into multiple intracellular Ca2+ pools and by also promoting entry of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation between plasma membrane potential and second messenger generation in the promyelocytic cell line HL-60.

The effects of plasma membrane depolarization on cytosolic free calcium ([Ca2+]i) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) generation were investigated in the human promyelocytic cell line HL-60 differentiated with either dimethyl sulfoxide or retinoic acid into neutrophil-like cells. Increases in [Ca2+]i and accumulation of Ins(1,4,5)P3 were triggered by two chemoattractants fMet-Leu-Phe and leukotriene B4. Plasma membrane potential was depolarized by isoosmotic substitution of NaCl with KCl, by the pore-forming ionophore gramicidin D, or by long term treatment with ouabain. Both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane were reduced by prior depolarization of plasma membrane potential regardless of the procedure employed to collapse it. Agonist-induced generation of Ins(1,4,5)P3 was also reduced in parallel in pre-depolarized HL-60 cells. The present findings provide further evidence suggesting that plasma membrane potential can be an important modulator of agonist-activated second messenger generation in myelocytic cells.