Extracellular Calcium Sources Research Articles

Calcium signaling of primary chondrocytes and ATDC5 chondrogenic cells under osmotic stress and mechanical stimulation

Calcium signaling plays an essential role in chondrocyte mechanotransduction. Guilak and colleagues have revealed the roles of TRPV4 and Piezo channels in chondrocyte calcium signaling and metabolism. This study compared the calcium responses of primary chondrocytes and ATDC5 cells induced by two different stimuli: osmotic stress and intense mechanical stimulus. Roles of three essential calcium signaling pathways, including extracellular calcium source, intracellular ER calcium store and mechanical-sensitive ion channels, were also investigated and compared between cells. Primary chondrocytes showed more vigorous calcium peaks under osmotic stress than under mechanical stimuli, while an opposite trend was found for ATDC5 cells. Extracellular calcium source, intracellular ER store, and PLC/IP3 pathway each played significant roles in the calcium responses of ATDC5 cells under both osmotic and mechanical stimuli. However, high level shear stress can directly cause ER release in primary cells without the presence of extracellular Ca2+ or involvement of PLC-IP3 pathway. TRPV4 channel is essential for the responses of ATDC5 cells, but not for primary chondrocytes. In contrast, inhibition of mechano-sensitive channels had no significant effects on the ATDC5 cells. Therefore, primary chondrocytes and ATDC5 cells rely on distinct calcium sources and ion channels to initiate intracellular calcium signaling. Together, these results contribute to our understanding of stimulation-induced calcium signaling in primary chondrocytes and ATDC5 cells, and the different roles of three essential pathways between the two cell types.

Ultracytochemical visualization of calcium distribution in heart cells and erythrocytes of zebrafish Danio rerio

Detection of patterns of subcellular calcium distribution in the cardiovascular system can contribute to understanding its role in cardiac and blood function. The present study localized calcium in heart atrium, ventricle, and bulbus arteriosus as well as in erythrocytes of zebrafish Danio rerio using an oxalate-pyroantimonate technique combined with transmission electron microscopy. Intracellular calcium stores were detected in caveolae, mitochondria, and the nuclei of several zebrafish cardiac cell types. Melanin pigmentation containing calcium stores was detected in the pericardial cavity. Melanin might be an extracellular source of calcium for heart beating and/or a lubricant to prevent friction during beating process. Calcium deposits were also detected in the plasma membrane, cytoplasm and nucleus of erythrocytes as well as in blood plasma. Possible exchange of calcium between erythrocytes and blood plasma was observed. Interactions of such calcium stores and possible contribution of extracellular calcium stores such as melanin pigmentation to supply calcium for vital functions of heart cells should be addressed in future studies.

The sources of calcium for noradrenaline-induced contraction in the human thoracic internal artery.

The aim of the present study was to examine the contribution of intracellular and extracellular calcium sources in contraction caused by noradrenaline (NA) of the human internal thoracic artery (ITA) in vitro. Distal segments of ITA were obtained from 20 patients (aged 38-73, at the time of routine coronary artery surgical revascularization (CABG)). Contractile responses to 10-6mol/L NA in the physiological salt solution and in Ca2+-free solution without and after incubation with 10-6mol/L thapsigargin (TSG) were recorded under isometric conditions. Responses of ITA rings to 1μM NA without incubation with TSG accounted (% of reaction to 80mM KCl) 224.70±14.06% in PSS solution, 141.30±8.66% in Ca2+-free solution, and 80.03±1.71% after PSS restoration and were statistically significantly different (p<0.0001, one-way ANOVA). Responses of ITA rings to 1μM NA with 1μM TSG accounted (% of reaction to 80mM KCl) 114.50±2.79% in Ca2+-free solution and 36.70±2.38% after PSS restoration. Responses in Ca2+-free solution and after PSS restoration without and with TSG were statistically significantly different (p=0.0257 and p<0.0001, respectively-t test). ITA contraction is caused by calcium derived not only from the SR and the extracellular matrix. The delivery of calcium to the space surrounding tissue does not immediately deliver calcium to the myofilaments.

α9α10 Acetylcholine receptors: structure and functions

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits assemble as pentameric complexes with a central ligand-gated cation-selective pore. However, these subunits do not assemble with other nAChR subunits. The general topology of each subunit is similar to that of other Cys-loop ligand-gated ion channels. Although the sequence and genomic organization of the α9 and α10 subunits determined their classification as nicotinic subunits, α9α10 nAChRs exhibit peculiar pharmacological properties that are not common to other nicotinic or muscarinic receptors. α9α10 nAChRs are expressed in diverse tissues, including auditory sensory hair cells, afferent neurons in the vestibular sensory organs, neurons of the dorsal root ganglion, skin, and bronchial epithelia. Furthermore, it has become apparent that nAChRs containing α9 and α10 subunits play essential roles in various physiological processes. For example, α9 is required for the normal development of the efferent synapses in the inner ear, and after the onset of hearing α9α10 nAChRs serve as the unique source of extracellular calcium for Ca 2+ -activated potassium channels in mature auditory hair cells. The development of drugs that selectively target α9/α10 receptors promises to be of therapeutic benefit since these receptors have distinct physiologic functions. They hold promise as pharmacological targets to treat complex auditory and vestibular pathologies, neuropathic pain, and pathologies of the skin and bronchia.

Store-Operated Calcium Entry through Orai Is Required for Transcriptional Maturation of the Flight Circuit in Drosophila.

The specificity of an animal's response to an environmental stimulus is determined in part by the release of neurotransmitters, which are sensed by responding neurons through cognate receptors on their surface. One way by which neurons respond is through release of calcium from intracellular stores followed by store refilling from extracellular calcium sources. This mechanism is called store-operated calcium entry (SOCE). The function of SOCE in neurons has been debated. Here we describe a new function for SOCE in the regulation of neurotransmitter levels in Drosophila flight neurons. This cell-signaling mechanism is required to maintain optimal levels of a key enzyme for dopamine synthesis and may serve as a mechanism for restoring dopamine levels in relevant pathological conditions.

Calcium response in osteocytic networks under steady and oscillatory fluid flow

The fluid flow in the lacunar–canalicular system of bone is an essential mechanical stimulation on the osteocyte networks. Due to the complexity of human physical activities, the fluid shear stress on osteocyte bodies and processes consists of both steady and oscillatory components. In this study, we investigated and compared the intracellular calcium ([Ca2+]i) responses of osteocytic networks under steady and oscillatory fluid flows. An in vitro osteocytic network was built with MLO-Y4 osteocyte-like cells using micro-patterning techniques to simulate the in vivo orderly organization of osteocyte networks. Sinusoidal oscillating fluid flow or unidirectional steady flow was applied on the cell surface with 2Pa peak shear stress. It was found that the osteocytic networks were significantly more responsive to steady flow than to oscillatory flow. The osteocytes can release more calcium peaks with higher magnitudes at a faster speed under steady flow stimulation. The [Ca2+]i signaling transients under the steady and oscillatory flows have significantly different spatiotemporal characters, but a similar responsive percentage of cells. Further signaling pathway studies using inhibitors showed that endoplasmic reticulum (ER) calcium store, extracellular calcium source, ATP, PGE2 and NO related pathways play similar roles in the [Ca2+]i signaling of osteocytes under either steady or oscillating flow. The spatiotemporal characteristics of [Ca2+]i transients under oscillating fluid flow are affected more profoundly by pharmacological treatments than under the steady flow. Our findings support the hypothesis that the [Ca2+]i responses of osteocytic networks are significantly dependent on the profiles of fluid flow.

Osteocytic network is more responsive in calcium signaling than osteoblastic network under fluid flow

Osteocytes, regarded as the mechanical sensor in bone, respond to mechanical stimulation by activating biochemical pathways and mediating the cellular activities of other bone cells. Little is known about how osteocytic networks respond to physiological mechanical stimuli. In this study, we compared the mechanical sensitivity of osteocytic and osteoblastic networks under physiological-related fluid shear stress (0.5 to 4 Pa). The intracellular calcium ([Ca(2+)](i)) responses in micropatterned in vitro osteoblastic or osteocytic networks were recorded and analyzed. Osteocytes in the network showed highly repetitive spikelike [Ca(2+)](i) peaks under fluid flow stimulation, which are dramatically different from those in the osteoblastic network. The number of responsive osteocytes in the network remained at a constant high percentage (>95%) regardless of the magnitude of shear stress, whereas the number of responsive osteoblasts in the network significantly depends on the strength of fluid flow. All spatiotemporal parameters of calcium signaling demonstrated that osteocytic networks are more sensitive and dynamic than osteoblastic networks, especially under low-level mechanical stimulations. Furthermore, pathway studies were performed to identify the molecular mechanisms responsible for the differences in [Ca(2+)](i) signaling between osteoblastic and osteocytic networks. The results suggested that the T-type voltage-gated calcium channels (VGCC) expressed on osteocytes may play an essential role in the unique kinetics of [Ca(2+)](i) signaling in osteocytic networks, whereas the L-type VGCC is critical for both types of cells to release multiple [Ca(2+)](i) peaks. The extracellular calcium source and intracellular calcium store in ER-, ATP-, PGE₂-, NO-, and caffeine-related pathways are found to play similar roles in the [Ca(2+)](i) signaling for both osteoblasts and osteocytes. The findings in this study proved that osteocytic networks possess unique characteristics in sensing and processing mechanical signals.

An ATP-dependent mechanism mediates intercellular calcium signaling in bone cell network under single cell nanoindentation

To investigate the roles of intercellular gap junctions and extracellular ATP diffusion in bone cell calcium signaling propagation in bone tissue, in vitro bone cell networks were constructed by using microcontact printing and self-assembled monolayer technologies. In the network, neighboring cells were interconnected through functional gap junctions. A single cell at the center of the network was mechanically stimulated by using an AFM nanoindenter. Intracellular calcium ([Ca 2+] i) responses of the bone cell network were recorded and analyzed. In the untreated groups, calcium propagation from the stimulated cell to neighboring cells was observed in 40% of the tests. No significant difference was observed in this percentage when the intercellular gap junctions were blocked. This number, however, decreased to 10% in the extracellular ATP-pathway-blocked group. When both the gap junction and ATP pathways were blocked, intercellular calcium waves were abolished. When the intracellular calcium store in ER was depleted, the indented cell can generate calcium transients, but no [Ca 2+] i signal can be propagated to the neighboring cells. No [Ca 2+] i response was detected in the cell network when the extracellular calcium source was removed. These findings identified the biochemical pathways involved in the calcium signaling propagation in bone cell networks.

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca 2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca 2+] i) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382–6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca 2+] i. We observed small, brief, Ca 2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd 3+) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca 2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells.

Effects of chemical ischemia in cerebral cortex slices: Focus on nitric oxide

Superfused rat cerebral cortex slices were submitted to a continuous electrical (5 Hz) stimulation and treated with sodium azide (1–10 mM) in the presence of 2 mM 2-deoxyglucose (“chemical ischemia”). Presynaptic cholinergic activity, evaluated as acetylcholine release, was inhibited depending on the sodium azide concentrations and on the length of application (5–30 min). Following a 5-min treatment with 10 mM sodium azide, acetylcholine release was reduced to 45 ± 2.3%; simultaneously, there was a 15- and 10-fold increase in glutamate and nitric oxide effluxes, respectively. After restoring normal superfusion conditions, acetylcholine release recovered to 70 ± 3.1% of the controls; the N-methyl- d-aspartate receptor antagonist MK-801 (10 μM) as well as the nitric oxide scavengers, haemoglobin (20 μM) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (150 μM), improved the recovery in presynaptic activity, showing that both glutamate and nitric oxide play detrimental roles in chemical ischemia. On the other hand, the post-ischemic recovery was worsened by the guanylylcyclase inhibitor 1H-[l,2,4]oxadiazolo[4,3,- a]quinoxalin-1-one (10 μM), suggesting that the activation of such a pathway plays a neuroprotective role and that the nitric oxide-induced harmful effects depend on different mechanisms. Chemical ischemia-evoked nitric oxide efflux partly derived from its calcium-dependent endogenous synthesis, since both the intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (1 mM), and the nitric oxide synthase inhibitor, N ω-nitro- l-arginine methyl ester (100 μM), substantially prevented sodium azide effects. Nitric oxide efflux was only weakly reduced by MK-801 and was not modified by either the L-type calcium channel blocker, nifedipine (10 μM) or the N-type calcium channel blocker ω-conotoxin (0.5 μM), thus suggesting a prevailing intracellular calcium-dependence of nitric oxide production, although a partial extracellular calcium source cannot be ruled out. These findings show that sodium azide plus 2-deoxyglucose treatment is a useful protocol to induce brain ischemia in vitro and underline the involvement of nitric oxide in the complex events following the ischemic insult.

TRPV1 Antagonists Elevate Cell Surface Populations of Receptor Protein and Exacerbate TRPV1-Mediated Toxicities in Human Lung Epithelial Cells

TRPV1 mediates cell death and pro-inflammatory cytokine production in lung epithelial cells exposed to prototypical receptor agonists. This study shows that NHBE, BEAS-2B and TRPV1 over-expressing BEAS-2B cells pre-treated with various TRPV1 antagonists become sensitized to the prototypical TRPV1 agonist, nonivamide, via a mechanism that involves translocation of existing receptor from the endoplasmic reticulum to the plasma membrane. As such, typical cellular responses to agonist treatment, as measured by calcium flux, inflammatory cytokine gene induction, and cytotoxicity were exacerbated. These data were in contrast to the results obtained when TRPV1 antagonists were co-administered with nonivamide; conditions which inhibited TRPV1-mediated effects. The antagonists LJO-328, SC0030, and capsazepine increased the cytotoxicity of nonivamide by approximately 20-fold and agonist-induced calcium flux by approximately 6-fold. Inflammatory-cytokine gene induction by nonivamide was also increased significantly by pre-treatment with the antagonists. The enhanced responses were inhibited by the co-administration of antagonists with nonivamide, confirming that increases in sensitivity were attributable to increased TRPV1-associated activity. Sensitization was attenuated by brefeldin A (a golgi transport inhibitor), but not cycloheximide (a protein synthesis inhibitor), or actinomycin D (a transcription inhibitor). Sensitized cells exhibited increased calcium flux from extracellular calcium sources, while unsensitized cells exhibited calcium flux originating primarily from intracellular stores. These results demonstrate the presence of a novel mechanism for regulating the sub-cellular distribution of TRPV1 and subsequent control of cellular sensitivity to TRPV1 agonists.

Localization of functional calcitonin gene-related peptide binding sites in a subpopulation of cultured dorsal root ganglion neurons

In this study we investigated whether cultured dorsal root ganglion (DRG) neurons from the adult rat express binding sites for calcitonin gene-related peptide (CGRP). These were identified on fixed cells by using CGRP labeled at the N-terminal site with 1.4-nm gold particles. After 1 day in culture, about 20% of small to medium-sized DRG neurons showed CGRP-gold binding. Binding of CGRP-gold was dose-dependently reduced by coadministration of CGRP. The calcium imaging technique in living cells revealed that the bath administration of CGRP evoked an increase of the intracellular calcium in up to 30% of the DRG neurons tested. Both depletion of intracellular calcium stores by thapsigargin or using a calcium-free medium blocked the CGRP-mediated increase of cytosolic calcium in most neurons. Thus intracellular and extracellular sources of calcium are relevant for the CGRP response. Using the whole-cell patch-clamp technique, about 30% of the neurons were found to exhibit an inward current and a depolarization upon administration of CGRP close to the neurons. Immunocytochemical double-labeling techniques showed that most of the CGRP-gold binding sites were expressed in unmyelinated (neurofilament 200-negative) DRG neurons. Most of the neurons with CGRP-gold binding sites also expressed the tyrosine kinase A receptor, and all of them showed CGRP-like immunoreactivity. This study shows, therefore, that a subpopulation of unmyelinated, peptidergic primary afferent neurons express CGRP binding sites that can be activated by CGRP in an excitatory direction. The binding sites may serve as autoreceptors because all of these neurons also synthesize CGRP. The activation of CGRP binding sites may sensitize primary afferent neurons and influence the release of mediators.

Outside-in Signaling Pathway Linked to CD146 Engagement in Human Endothelial Cells

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Wound Healing in Jellyfish Striated Muscle Involves Rapid Switching between Two Modes of Cell Motility and a Change in the Source of Regulatory Calcium

Small wounds (1.2 mm in diameter) made in the sheet of myoepithelial cells forming the “swimming” muscle of the jellyfish, Polyorchis penicillatus, were closed within 10 h by epithelial cells migrating centripetally to the wound center. Some 24 to 48 h later these cells redifferentiated into fully contractile muscle cells. Labeling with bromodeoxyuridine failed to reveal any cell proliferation during this process. Phenotype switching (within 1 h) from contractile muscle cells to migratory cells did not require synthesis of new protein as shown by treatment with 40 μM cycloheximide. Excitation–contraction coupling in undamaged muscle depended on entry of Ca2+ through voltage-gated ion channels, as shown by a block of contractility by 40 μM nitrendipine and also on calcium released from intracellular stores since caffeine (10 mM) caused a 25% reduction in contractile force. In contrast, migratory cells did not require a source of extracellular calcium since migration was unimpeded by low (1 μM) free Ca2+ or nitrendipine. Instead, modulatory calcium was derived from intracellular stores since caffeine (10 mM) and thapsigargin (10 μM) slowed migration. This lack of dependence on calcium influx in migratory cells was further confirmed by a dramatic down-regulation in voltage-gated inward current as shown by whole-cell patch recordings.

Coordinating Calcium Release

Several mechanisms exist for releasing intracellular stores of calcium: activation of inositol trisphosphate (IP3) receptors, activation of ryanodine receptors, and stimulation of Ca2+ release by nicotinic acid adenine dinucleotide phosphate (NAADP+). Santella et al. studied calcium fluxes in starfish oocytes in response to injected caged NAADP+. Photolysis of the caged NAADP+ led to an increase in intracellular calcium [Ca2+]i that was partially blocked by the addition of both heparin (to block Ca2+ release from IP3 receptors) and 8-NH2-cyclic ADPribose (to block Ca2+ release from ryanodine receptors). The authors suggest that NAADP+ initiates an increase in [Ca2+]i that triggers further release from IP3 and ryanodine receptor-operated stores. Additionally, NAADP+ stimulated an increase in [Ca2+]i from intracellular stores in immature oocytes, but the NAADP+-stimulated increase in [Ca2+]i in mature oocytes was derived from extracellular sources of calcium, suggesting that the NAADP+-sensitive compartment fused with the plasma membrane as a consequence of oocyte maturation.Santella, L., Kyozuka, K., Genazzani, A.A., De Riso, L., and Carafoli, E. (2000) Nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release. J. Biol. Chem. 275: 8301-8306.[Abstract][Full Text]

Coordinating Calcium Release

Several mechanisms exist for releasing intracellular stores of calcium: activation of inositol trisphosphate (IP 3 ) receptors, activation of ryanodine receptors, and stimulation of Ca 2+ release by nicotinic acid adenine dinucleotide phosphate (NAADP + ). Santella et al. studied calcium fluxes in starfish oocytes in response to injected caged NAADP + . Photolysis of the caged NAADP + led to an increase in intracellular calcium [Ca 2+ ] i that was partially blocked by the addition of both heparin (to block Ca 2+ release from IP 3 receptors) and 8-NH 2 -cyclic ADPribose (to block Ca 2+ release from ryanodine receptors). The authors suggest that NAADP + initiates an increase in [Ca 2+ ] i that triggers further release from IP 3 and ryanodine receptor-operated stores. Additionally, NAADP + stimulated an increase in [Ca 2+ ] i from intracellular stores in immature oocytes, but the NAADP + -stimulated increase in [Ca 2+ ] i in mature oocytes was derived from extracellular sources of calcium, suggesting that the NAADP + -sensitive compartment fused with the plasma membrane as a consequence of oocyte maturation. Santella, L., Kyozuka, K., Genazzani, A.A., De Riso, L., and Carafoli, E. (2000) Nicotinic acid adenine dinucleotide phosphate-induced Ca 2+ release. J. Biol. Chem. 275 : 8301-8306. [Abstract] [Full Text]

Effects of kinins upon cytosolic calcium concentrations in mouse mesangial cells

Bradykinin (BK) induces increases in cytosolic calcium concentration [Ca++]i in several cell lines. Because the role of BK in the renal system, particularly in mesangial cell (MC), is not clear, we investigated the effects of kinins on [Ca++]i in mouse-immortalized MC. [Ca++]i was evaluated by spectrofluorometry and expressed as a ratio between the obtained and basal [Ca++]i. BK (0.1 μM) induced a non-sustained increase in [Ca++]i (4.70±0.27; N=28). A similar effect was observed with the B2 receptor agonist, Tyr8-BK (0.1 μM, 3.34±0.48; N=7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 μM, N=11) and des-Arg9-BK (1 μM, N=8), exhibited only discrete responses (1.45±0.08 and 1.12±0.04, respectively). Cross-desensitization was seen between BK and Tyr8-BK, but not between BK and des-Arg10-Kal. The BK response was decreased (5.09±0.30, N=6 to 1.57±0.12, N=7, P<0.001) by the B2 receptor antagonist HOE 140 (0.1 μM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 μM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 μg/ml, 24 h) alone or in association with tumor necrosis factor-α (TNF-α) (10 ng/ml, N=6) did not enhance B1 agonist responses. BK was inhibited by repeated cell washouts in zero Ca++ solution (2.04±0.19, N=6 P<0.001), and the residual response was almost abolished by thapsigargin (Thaps) a sarcoplasmic reticulum (SR) calcium-ATPase inhibitor (1 μM) (1.18±0.08, N=5 P<0.001). Additionally, BK was not inhibited by verapamil (50 μM), nifedipine (30 μM), Ni++ (300 μM) or La+++ (10 μM). In conclusion, BK induces [Ca++]i in mouse MC mainly by B2 receptor activation. B1 receptors have a minor role in this phenomenon even in the presence of known B1 receptor synthesis inducers. Finally, BK mobilizes extracellular calcium sources and, to a lesser extent, intracellular Thaps-sensitive calcium stores. The ion channels involved in calcium influx remain to be detected.

Particulate Matter Initiates Inflammatory Cytokine Release by Activation of Capsaicin and Acid Receptors in a Human Bronchial Epithelial Cell Line

Recent experiments have shown that human bronchial epithelial cells (i.e., BEAS-2B) release pro-inflammatory cytokines (i.e., IL-6 and TNFα) in a receptor-mediated fashion in response to the neuropeptides, substance P (SP), calcitonin gene-related protein (CGRP), and the prototype botanical irritant capsaicin. In the present experiments, we examined the relevance of these receptors to particulate matter (PM)-associated cellular inflammation. BEAS-2B cells, exposed to residual oil fly ash particles (ROFA), responded with an immediate (<30 s) increase in intracellular calcium levels ([Ca2+]i), increases of key inflammatory cytokine transcripts (i.e., IL-6, IL-8, TNFα) within 2 h exposure, and subsequent release of IL-6 and IL-8 cytokine protein after 4 h exposure. Pretreatment of BEAS-2B cells with pharmacological antagonists selective for the SP or CGRP receptors reduced the ROFA-stimulated IL-6 cytokine production by ∼25 and 50%, respectively. However, pretreatment of these cells with capsazepine (CPZ), an antagonist for capsaicin (i.e., vanilloid) receptors, inhibited the immediate increases in [Ca2+]i, diminished transcript (i.e., IL-6, IL-8, TNFα) levels and reduced IL-6 cytokine release to control levels. BEAS-2B cells exposed to ROFA in calcium-free media failed to demonstrate increases of [Ca2+]iand showed reduced levels of cytokine transcript (i.e., IL-6, IL-8, TNFα) and IL-6 release, suggesting that ROFA-stimulated cytokine formation was partially dependent on extracellular calcium sources. A final set of experiments compared the inflammatory properties of the soluble and acidic insoluble components of ROFA. BEAS-2B cells, exposed to ROFA or ROFA that had been filtered through a 0.2-μm pore filter, produced equivocal IL-6. BEAS-2B cells exposed to pH 5.0 media for 15 min released moderate amounts of IL-6, 4 h later. This cytokine release could be blocked by amiloride, a pH receptor antagonist, but not by CPZ. BEAS-2B cells, pretreated with amiloride before ROFA exposure, showed a partial (∼25%) reduction of IL-6. Together, these data indicate that the acidic, soluble components of ROFA initiate cytokine release in BEAS-2B cells through activation of both capsaicin- and pH-sensitive irritant receptors.

Influence of nimodipine on vasoconstrictor responses in the hindquarters vascular bed of the cat.

The beneficial effects of the calcium-entry blocking agent, nimodipine, on the cerebral circulation have been extensively studied but less is known about its peripheral vascular effects. In the present study, the effects of nimodipine on vasoconstrictor responses were investigated in the hindquarters vascular bed of the cat under constant-flow conditions. Nimodipine decreased hindquarters vascular resistance and inhibited vasoconstrictor responses to BAY K8644 (methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine-5-carboxylate) and noradrenaline, to the alpha1-adrenoceptor agonists phenylephrine and methoxamine, and the alpha2-adrenoceptor agonists BHT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazolo[5,4-d]azepine dihydrochloride) and UK 14304 (5-bromo-6-(2-imidazoline-2-yl-amino)quinoxaline). In addition to inhibiting alpha-adrenoceptor-mediated responses, nimodipine decreased responses to the vasoactive peptides angiotensin II and endothelin-1. Both the vasodilatory actions and inhibitory effects of nimodipine on vasoconstrictor responses were dose-dependent when the calcium antagonist was infused at rates of 0.1 and 1 microg min(-1). The results of the present study suggest that vasoconstrictor responses to alpha-adrenoceptor agonists and to the vasoactive peptides are dependent, in part, on an extracellular source of calcium. It is concluded that nimodipine and related dihydropyridine calcium-entry blocking agents may be effective in the treatment of peripheral vascular disorders in which adrenergic tone is increased or plasma levels of angiotensin II or endothelin-1 are elevated.

HLA Class II Molecule Signal Transduction Leads to either Apoptosis or Activation via Two Different Pathways

The class II human leukocyte antigens (HLA class II) are constitutively expressed on antigen presenting cells (APC) and are essential for peptide presentation to helper T lymphocytes. Signal transduction by HLA class II molecules on B lymphocytes has been described and has been shown in many cases to induce cellular proliferation. However, since signalling via HLA class II can also lead to apoptosis, it has not been clear how the outcome of the signals is determined. We have distinguished two separate HLA class II-initiated pathways leading to either proliferation or apoptosis of primary human B lymphocytes. Proliferation requires new gene transcription and activation of src family tyrosine kinases. In contrast, apoptosis is significantly increased in the absence of transcription/translation. It is dependent on serine/threonine phosphatases and cytoskeletal mobility. An extracellular source of calcium was essential for apoptosis, suggesting the need for a sustained high level of intracellular calcium. Activation of iso-enzymes of the protein kinase C family was needed for both pathways. We therefore conclude that HLA class II molecules can initiate two distinct signalling pathways leading to either proliferation or apoptosis of APC.