Michael J. Mason

Substance P activates NF-κB independent of elevations in intracellular calcium in murine macrophages and dendritic cells

Professional antigen presenting cells, such as macrophages, can be activated by intracellular calcium-dependent as well as calcium-independent mechanisms, depending upon the stimulus used. In this report, we addressed the mechanism of substance P-induced intracellular signalling in murine macrophages and dendritic cells. While no increases in intracellular calcium concentration were detected in macrophages or dendritic cells using sensitive fluorimetric techniques, substance P did induce rapid enhanced activation of NF-kappaB, a transcriptional activator known to regulate pro-inflammatory cytokines. These data provide an important mechanism by which substance P may augment the production of pro-inflammatory molecules.

Properties of the demarcation membrane system in living rat megakaryocytes.

The demarcation membrane system (DMS) is the precursor of platelet cell membranes yet little is known of its properties in living megakaryocytes. Using confocal microscopy, we now demonstrate that demarcation membranes in freshly isolated rat marrow megakaryocytes are rapidly stained by styryl membrane indicators such as di-8-ANEPPS and FM 2-10, confirming that they are invaginations of the plasma membrane and readily accessible from the extracellular space. Two-photon excitation of an extracellular indicator displayed the extensive nature of the channels formed by the DMS throughout the extranuclear volume. Under whole-cell patch clamp, the DMS is electrophysiologically contiguous with the peripheral plasma membrane such that a single capacitative component can account for the biophysical properties of all surface-connected membranes in the majority of recordings. Megakaryocyte capacitances were in the range of 64-694 pF, equivalent to 500-5500 platelets (mean value 1850). Based upon calculations for a spherical geometry, the DMS results in a 4- to 14-fold (average 8.1-fold) increase in specific membrane capacitance expressed per unit spherical surface area. This indicates a level of plasma membrane invagination comparable with mammalian skeletal muscle. Whole-cell capacitance measurements and confocal imaging of membrane-impermeant fluorescent indicators therefore represent novel approaches to monitor the DMS during megakaryocytopoiesis and thrombopoiesis.

Depolarization‐evoked Ca2+ release in a non‐excitable cell, the rat megakaryocyte

1 The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole‐cell patch clamp and fura‐2 fluorescence recordings. 2 Depolarization from −75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 ± 95 nM) during activation of metabotropic purinoceptors by 1 μM ADP. 3 The depolarization‐evoked Ca2+ increase was present in Ca2+‐free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+‐Ca2+ exchange activity. 4 Intracellular dialysis with heparin blocked the depolarization‐evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5 Under current clamp, ADP caused the membrane potential to fluctuate between −43 ± 1 and −76 ± 1 mV. Under voltage clamp, depolarization from −75 to −45 mV evoked a transient [Ca2+]i increase (398 ± 91 nM) during exposure to ADP. 6 We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non‐excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase.

Voltage‐dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors

Using simultaneous whole‐cell patch‐clamp and fluorescence measurements of [Ca2+]i in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5‐trisphosphate (IP3) receptors in Ca2+ release induced by membrane depolarization during agonist stimulation. Voltage‐dependent Ca2+ release was observed during application of the IP3‐generating agonists U46619 (a thromboxane A2 analogue) and ADP. Furthermore, voltage‐dependent Ca2+ release was observed in the absence of exogenous agonist following sensitization of IP3 receptors with thimerosal. Depolarization‐induced Ca2+ release was not detected during depletion of intracellular Ca2+ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca2+ release mechanism. Block of IP3 receptors by carbacyclin‐stimulated elevations in cAMP, uncaging of cAMP or exposure to a high concentration of caffeine reversibly abolished Ca2+ increases stimulated by both ADP and depolarization. The cAMP‐dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity leading to modulation of protein kinase A activity does not underlie the control of Ca2+ release by voltage. These results are consistent with the requirement for functional IP3 receptors for voltage control of Ca2+ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a voltage sensor downstream of surface membrane receptors in the depolarization‐evoked Ca2+ response.

A key role for dense granule secretion in potentiation of the Ca2+ signal arising from store-operated calcium entry in human platelets

Recent work has demonstrated a role for Na(+)/Ca(2+) exchange in potentiation of the Ca(2+) entry elicited through the human platelet store-operated channel by controlling a Mn(2+)-impermeable Ca(2+) entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na(+)/Ca(2+) exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn(2+)-impermeable Ca(2+) entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca(2+) signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca(2+) signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion.

A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes

1 The effect of membrane potential (Vm) on ADP‐evoked [Ca2+]i oscillations was investigated in rat megakaryocytes, a non‐excitable cell type recently shown to exhibit depolarisation‐evoked Ca2+ release from intracellular stores during metabotropic purinoceptor stimulation. 2 Hyperpolarising voltage steps caused a transient fall in [Ca2+]i and either abolished Ca2+ oscillations or reduced the oscillation amplitude. These effects were observed in both the presence and absence of extracellular Ca2+ and also in Na+‐free saline solutions, suggesting that hyperpolarisation leads to a reduction in the level of ADP‐dependent Ca2+ release without a requirement for altered transmembrane Ca2+ fluxes. 3 In the presence of Ca2+ oscillations, depolarising voltage steps transiently enhanced the amplitude of Ca2+ oscillations. Following run‐down of Ca2+ oscillations, depolarisation briefly restimulated oscillations. 4 Simultaneous [Ca2+]i and current‐clamp recordings showed that Ca2+ and Vm oscillate in synchrony, with an average fluctuation of approximately 30–40 mV, due to activation and inactivation of Ca2+‐dependent K+ channels. Application of a physiological oscillating Vm waveform to non‐oscillating cells under voltage clamp stimulated [Ca2+]i oscillations. 5 Analysis of the relationship between [Ca2+]i and Vm showed a threshold for activation of hyperpolarisation at about 250–300 nM. The implications of this threshold in the interaction between Vm and Ca2+ release during oscillations are discussed. 6 We conclude that the ability of voltage to control release of endosomal Ca2+ in ADP‐stimulated megakaryocytes is bipolar in nature. Our data suggest that Vm changes are active components of the feedback/feedforward mechanisms contributing to the generation of Ca2+ oscillations.

Differential kinetics for induction of interleukin-6 mRNA expression in murine peritoneal macrophages: evidence for calcium-dependent and independent-signalling pathways

It is presently unclear what role elevations in intracellular calcium concentration ([Ca2+]i) play in the control of monokine secretion, or whether such alterations underlie the ability of physiologic stimuli to induce production of these important signalling molecules. To address these issues, we have performed experiments in murine peritoneal macrophages to determine whether lipopolysaccharide (LPS) or interferon gamma (IFN‐γ) initiate production of the proinflammatory monokine interleukin 6 (IL‐6) concomitant with elevations in [Ca2+]i and with kinetics similar to that seen with known Ca2+ mobilizing agents. Alterations in [Ca2+]i after treatment with LPS, IFN‐γ, platelet activating factor (PAF), or thapsigargin were measured by fluorimetric methods. These effects were compared with the ability of each to induce IL‐6 mRNA expression as measured by semiquantitative reverse‐transcribed polymerase chain reactions. We report that neither LPS nor IFN‐γ elicited detectable elevations in [Ca2+]i but that both up‐regulated expression of IL‐6 mRNA expression within 60 min. In contrast, experiments using either thapsigargin or PAF showed rapid and dramatic elevations in [Ca2+]i with marked increases in IL‐6 mRNA expression, as quickly as 15 min after initial exposure. Elevations in mRNA encoding IL‐6 by thapsigargin and PAF were found to occur in a dose‐dependent manner, mirroring their ability to elicit elevations in [Ca2+]i. These data demonstrate that LPS and IFN‐γ induce IL‐6 message expression by means of Ca2+‐independent signalling pathways. Furthermore, Ca2+‐mobilizing agents that evoke monokine message expression do so far more rapidly than do LPS or IFN‐γ. Taken in concert, these data are consistent with the hypothesis that multiple signalling pathways exist by which production of proinflammatory monokines are initiated. J. Cell. Physiol. 177:232–240, 1998.

Monitoring the intracellular store Ca2+ concentration in agonist-stimulated, intact human platelets by using Fluo-5N.

Summary.  Background: Most Ca2+ signaling research in platelets has relied solely on monitoring the cytosolic Ca2+ concentration ([Ca2+]cyt). Changes in [Ca2+]cyt constitute the net effect of Ca2+ fluxes into the cytosol across the plasma membrane (PM) and from intracellular stores, and Ca2+ sequestration into the stores and Ca2+ removal across the PM. This makes interpretation of the effects of pharmacologic or genetic interventions on Ca2+ signaling difficult and subject to error. Objectives:  To validate the use of the low‐affinity Ca2+ indicator Fluo‐5N to monitor the concentration of Ca2+ in the intracellular stores ([Ca2+]st) of human platelets as a first step in developing assays for a systems‐level analysis of platelet Ca2+ signaling. Methods:  Fluo‐5N‐loaded and Fura‐2‐loaded human platelets were used to observe the effects of agonist stimulation and other manipulations on [Ca2+]cyt and [Ca2+]st. Results:  Fluo‐5N fluorescence changed appropriately in response to compounds that induce passive depletion of intracellular Ca2+ stores and to physiologic agonists. Ca2+ reuptake inhibitors and blockers of Ca2+ release channels had the expected effects on Fura‐2 and Fluo‐5N fluorescence. Agonist‐evoked Ca2+ release was reversed by Ca2+ addition to the medium, and required intact Ca2+ reuptake mechanisms. Store refilling was observed in the presence of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitors and ionomycin, suggesting the presence of a non‐SERCA Ca2+ reuptake mechanism. Evidence for a role for Ca2+‐induced Ca2+ release in agonist‐evoked responses was obtained. Conclusions:  Our data provide a validation of the use of Fluo‐5N as a method for monitoring changes in [Ca2+]st in human platelets.

Depolarisation-evoked Ca2+ waves in the non-excitable rat megakaryocyte.

1 A combination of patch clamp, confocal microscopy and immunohistochemistry was used to examine the spatial properties of Ca2+ signalling in the rat megakaryocyte, a non‐excitable cell type in which membrane potential can markedly modulate agonist‐evoked Ca2+ release. 2 Intracellular calcium ion concentration ([Ca2+]i) increases, stimulated by both ADP and depolarisation, frequently originated from a peripheral locus and spread as a wave throughout the cell. Spatially restricted [Ca2+]i increases, consistent with elementary Ca2+ release events, were occasionally observed prior to ADP‐evoked waves. 3 ADP‐ and depolarisation‐evoked Ca2+ waves travelled approximately twice as fast around the periphery of the cell compared to across its radius, leading to a curvilinear wavefront. There was no sigificant difference between wave velocities generated by the two stimuli. 4 Immunohistochemical staining of type III IP3 receptors, the endoplasmic reticulum‐specific protein GRP78/BiP and calreticulin indicated a major peripheral location of the cellular Ca2+ stores which probably accounts for the accelerated wave velocity at the cell periphery. 5 These data demonstrate that [Ca2+]i increases, stimulated by depolarisation or the agonist ADP, have indistinguishable spatial properties, providing evidence that similar underlying mechanisms are responsible for their generation.

Phorbol ester-evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors, not a novel non-capacitative Ca2+entry

Summary.  Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca2+entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca2+entry in human platelets for Na+/Ca2+ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca2+entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca2+signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca2+or Na+ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na+, significantly reduced PMA‐evoked Ca2+entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca2+entry. Ro‐0437626 or chelation of extracellular Ca2+ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P2X1 receptor activation but is accelerated by P2X1‐mediated Ca2+entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca2+entry results from an NCX3‐dependent dense granule secretion and subsequent P2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.