Cytoplasmic Ca2+ concentrations in intact Merkel cells of an isolated, functioning rat sinus hair preparation

An isolated, functioning sinus hair preparation was developed to investigate cytoplasmic Ca²⁺ concentrations in intact Merkel cells using microfluorimetric techniques. Intracellular Ca²⁺ levels were monitored by means of photon counters in small groups of Merkel cells loaded with the calcium fluorescent indicators fura-2 or fluo-3. Mechanical stimulation of Merkel cells with fine glass rods resulted in small transient increases in intracellular Ca²⁺ levels (by about 20%) in the group of Merkel cells around the stimulating probe. A rise in Ca²⁺ is presumed to be essential for the postulated synaptic transmission to the afferent nerve terminal. Depolarization with a high concentration of potassium chloride (100 mM) caused increases in intracellular Ca²⁺ concentrations in Merkel cells (by about 70%) only in the presence of extracellular Ca²⁺, indicating an influx of Ca²⁺ through voltage-gated channels. The Ca²⁺ response was abolished neither by (+)-BayK8644 nor -conotoxin, suggesting that the Ca²⁺ channels are different from the classical L- or N-type channels. Extracellular application of ATP (10 M to 5 mM) caused dose-dependent increases in intracellular Ca²⁺ levels in Merkel cells of up to sevenfold from the basal level of about 100 nM. Similar responses to ATP were also measured during superfusion with Ca²⁺-free medium, suggesting intracellular stores as the main Ca²⁺ source. Pre-incubation of Merkel cells with the purinoceptor antagonist suramin (100 M) for 30 min reduced the Ca²⁺ responses to ATP by about 50% compared with control conditions. In conclusion, the results have demonstrated that a rise in intracellular Ca²⁺ in Merkel cells can be evoked by mechanical stimulation, membrane depolarization and chemical stimulation by ATP. These observations strongly suggest a possible contribution of Ca²⁺ to the normal responsiveness of Merkel cell mechanoreceptors, in turn supporting the hypothesis that Merkel cells are involved in the mechano-electric transduction process in sinus hair type I mechanoreceptors.     

Activation of different pathways for calcium elevation by bradykinin and ATP in rat pheochromocytoma (PC 12) cells

We have studied the pathways by which extra-cellular bradykinin and adenosine 5-triphosphate (ATP) elicit changes in intracellular free calcium ([Ca²⁺]_i) in nerve-growth-factor(NGF)- treated rat pheochromocytoma (PC 12) cells. Both substances caused a significant rise in [Ca²⁺]_i as assessed by fura-2 based micro-fluorimetry. The bradykinin-induced response consisted of an initial Ca²⁺ mobilization from an internal pool followed by a sustained increase in [Ca²⁺]_i, which was due to activation of a small inward current. The initial response always started at a localized site opposite to the cell nucleus. The inward current was partially carried by Ca²⁺ and began with a time lag of about 4 s after the start of the initial transient signal. Stepwise hyperpolarization of the plasma membrane, after activation of the inward current by bradykinin, caused a simultaneous increase in current amplitude and in [Ca²⁺]_i, due to an increase in the driving force for Ca²⁺ influx. With ATP as an agonist the onset of inward current coincided with an increase in [Ca²⁺]_i. Inward current and [Ca²⁺]_i were enhanced during hyperpolarizing steps indicating a substantial Ca²⁺ influx through ATP-activated channels. No release of Ca²⁺ from internal stores, but a large Na⁺ inward current, was observed in Ca²⁺-free external solution after addition of ATP. While the bradykinin-induced responses were much more pronounced in cell bodies than in growth cones, the ATP effects were somewhat variable in cell bodies and more homogeneous in growth cones.     

Activation of Ca2+-dependent K+ and Cl− currents by UTP and ATP in CFPAC-1 cells

Activation of Cl^– and K⁺ conductances by nucleotide receptor-operated mobilization of intracellular Ca²⁺ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5-triphosphate (ATP) and uridine 5-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltageclamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl^– ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1±0.3 min. Its amplitude was about 2.7 nA at –100 mV with 100 mol/l of either ATP or UTP. The second current was carried by K⁺ ions and was blocked by Cs⁺. This current peaked more slowly and had a mean duration of 4.6±0.7 min. Its amplitude was 0.9 nA and 0.5 nA at –20 mV with 100 umol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) that was similar in the presence or absence of extracellular Ca²⁺. The ED₅₀ for UTP was 24 umol/l and that for ATP was 94 mol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ store by thapsigargin prevented both the nucleotide-induced [Ca²⁺]_i increase and the activation of membrane currents. Addition of 2 mmol/l Ca²⁺ to thapsigargin-treated cells produced a sustained increase of Cl^– and K⁺ currents, which was reversed by Ca²⁺ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca²⁺]_i that stimulates Ca²⁺-dependent Cl^– and K⁺ currents. This phenomenon is probably mediated by inositol 1,4,5-trisphosphate-dependent Ca²⁺ stores.     

The CD8β polypeptide is required for the recognition of an altered peptide ligand as an agonist

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca²⁺ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 α and β chains. With a video imaging system, we showed that the intracellular Ca²⁺ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca²⁺ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8β in the CD8 co-receptor dimers expressed at the T cell surface.     

Role of protein kinase C in the regulation of inositol phosphate production and Ca2+ mobilization evoked by ATP and acetylcholine in rat lacrimal acini

Stimulation of rat lacrimal acinar cells with ATP and acetylcholine (ACh) induced a rapid accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] and its degradation products, resulting in an initial release of Ca²⁺ from intracellular stores. However, after pretreating the acini with U73122 no increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) or Ins(1,4,5)P ₃ production was observed. A short pre-treatment with the phorbol ester 4--phorbol-12--myristate-13--acetate (PMA) significantly attenuated the ATP- and ACh-induced increase in [Ca²⁺]_i and over- all inositol phosphate production. In contrast, staurosporine enhanced Ins(1,4,5)P ₃ and inositol 1,3,4-trisphosphate [Ins(1,3,4)P ₃] production and [Ca²⁺]_i above control values in ATP- and ACh-stimulated cells. Stimulation of phospholipase C by iono-mycin-evoked changes in [Ca²⁺]_i were unaltered by pretreatment with staurosporine and PMA. The data show that a change in protein kinase C activity during cell stimulation affects the inositol phosphate metabolism and thereby the cellular Ca²⁺ signalling processes in lacrimal acinar cells.     

Functional expression of P2Y purinoceptor in Xenopus oocyte injected with brain mRNA

Xenopus oocytes injected with embryonic guinea-pig brain mRNA expressed functional P_2Y purinoceptors. Extracellular ATP stimulated in a dose-dependent manner a delayed Ca²⁺-dependent Cl^– current component. Analysis of the interactions of ATP with compounds that affect Ca²⁺ fluxes through the plasma membrane or Ca²⁺ release from internal stores indicates that ATP raises [Ca²⁺]_i by a mechanism that involves activation of voltage-dependent Ca²⁺ channels, which leads to influx of extracellular Ca²⁺ into the cells, as well as release of Ca²⁺ from intracellular stores. Since this phenomenon was not found in control oocytes, it is suggested that brain mRNA encoded for a newly synthesized Ca²⁺-release process stimulated by purinoceptor activation. This mechanism could be largely involved in the short-term regulation of intracellular Ca²⁺ level involved in ATP neuromodulation functions.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

Differential STIM1 expression in T and B cell subsets suggests a role in determining antigen receptor signal amplitude

Ca²⁺ acts ubiquitously as a second messenger in transmembrane signal transduction. In lymphocytes, calcium mobilization is triggered by antigen and chemokine receptors, among others, and controls cell functions ranging from proliferation to migration. The primary mechanism of extracellular Ca²⁺ entry in lymphocytes is the CRAC influx. STIM1 is a crucial component of the CRAC influx mechanism in lymphocytes, acting as a sensor of low Ca²⁺ concentration in the ER and an activator of the Ca²⁺ selective channel ORAI1 in the plasma membrane. While STIM1 function has been studied extensively, little is known regarding whether it is differentially expressed and thereby affects the magnitude of calcium mobilization responses. We report here that STIM1 expression differs in murine T and B lymphocytes, and in respective subsets. For example, mature T cells express ∼4 times more STIM1 than mature B cells. Furthermore, we show that through the physiologic range of expression, STIM1 levels determine the magnitude of Ca²⁺ influx responses that follow BCR-induced intracellular store depletion. Considered in view of previous reports that differences in amplitude of lymphocyte Ca²⁺ mobilization determine alternate biological responses, these findings suggest that differential STIM1 expression may be important determinant of biological responses.     

Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization

Ligation of very late antigen (VLA)-4 (α₄β₁ integrin) with a cross-linked anti-α₄ subunit monoclonal antibody (mAb) triggered a biphasic Ca²⁺ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca²⁺ response. Tumor necrosis factor-α-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca²⁺ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca²⁺ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca²⁺ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (～ 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca²⁺ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca²⁺ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca²⁺.     

Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells

ATP and adenosine(5)tetraphospho(5)adenosine (Ap₄A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca²⁺]_i rises (EC₅₀=3.8 M) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca²⁺ concentration to around 100 nM suppressed the [Ca²⁺]_i transient evoked by ATP but not the [Ca²⁺]_i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC₅₀= 12.5 M) and UTP, but did not respond to either adenosine 5-[-thio]diphosphate (ADP[S]), a P_2Y receptor agonist, adenosine 5-[,-methylene]triphosphate (pp[CH₂]pA), a P_2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca²⁺]_i responses when stimulated with ATP (EC₅₀=0.86 M), UTP (EC₅₀=1.6 M) and 2MeSATP (EC₅₀= 0.38 M). 2MeSATP behaved as a partial agonist. Ap₄A and ADP[S] also raised the [Ca²⁺]_i in endothelial cells, whereas AMP and pp[CH₂]pA were ineffective. Lowering extracellular free Ca²⁺ to around 100 nM did not affect the peak ATP-evoked [Ca²⁺]_i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca²⁺-releasing P_2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P_2X, non-P_2Y subtype exclusively coupled to Ca²⁺ influx.     

Endothelial cell intracellular Ca concentration is increased upon breast tumor cell contact and mediates tumor cell transendothelial migration

Tumor cell extravasation is a determinant step in the process of hematogenous metastasis. The signal transduction pathways involved in the interactions between tumor cells and the vascular endothelium during transendothelial migration are still undefined. In the present study, we have investigated the influence of human breast adenocarcinoma cells (MCF7) on human umbilical vein endothelial cell (HUVEC) intracellular Ca²⁺ concentration ([Ca²⁺]i). We show that the contact between MCF7 cells and a confluent HUVEC monolayer induces an immediate and transient increase in HUVEC [Ca²⁺]i. This [Ca²⁺]i rise could not be elicited by tumor cell-conditioned medium, isolated tumor cell membranes, inert beads or normal breast epithelial cells, demonstrating the involvement of specific recognition mechanisms between MCF7 cells and HUVEC. Depletion of HUVEC intracellular Ca²⁺ stores by the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin as well as the selective depletion of inositol 1,4,5-tri phosphate (IP3)-sensitive Ca²⁺ stores by prior activation of HUVEC using histamine resulted in a complete inhibition of tumor cell-induced [Ca²⁺]_i elevation. Similar results were obtained when HUVEC monolayers were treated with the tyrosine kinase inhibitor herbimycin A, suggesting a role for tyrosine kinase-associated cell surface receptors in tumor cell-endothelial cell interactions. The depletion of HUVEC intracellular Ca²⁺ stores by thapsigargin was also shown to delay MCF7-induced endothelial cell disjunction, to prevent their spreading on the subendothelial extracellular matrix and transendothelial migration in vitro. These results suggest that transient changes in endothelial [Ca²⁺]_i may govern multiple steps of tumor cell extravasation. © Rapid Science 1998     

Involvement of CD44 variant isoforms in hyaluronate adhesion by human activated T cells

The standard, 85–95-kDa form of the hyaluronic acid (HA) receptor CD44 and a number of CD44 mRNA splice variants play important roles in immune responses and tumor metastasis. Variants carrying exon 6 (v6), or 9 (v9) products are transiently expressed on activated human T cells. Here, modulation experiments with specific monoclonal antibodies (mAb) indicate that v6 and v9 are expressed independently on distinct sets of CD44 molecules, and that their combined expression is necessary for HA adhesion. Moreover, the finding that mAb-mediated cross-linking of v6 and v9 promoted cytosolic free Ca²⁺ mobilization and co-stimulated CD3-triggered T cell proliferation indicates that v6 and v9 possess signaling and effector function activation ability. Finally, HA-mediated signaling appears to be required for variant-dependent adhesion to HA. The observation that soluble HA promoted cytosolic free Ca²⁺ mobilization indicates that HA-induced Ca²⁺ mobilization can occur during T cell-HA interaction. Since Ca²⁺ mobilization was inhibited by pretreatment of cells with an anti-CD44 mAb directed against the HA-binding domain of CD44, CD44 receptors appear to be involved in HA-mediated signal transduction. The requirement of cytosolic free Ca²⁺ for adhesion is shown by the fact that ionomycin (a Ca²⁺ ionophore) stimulated, and EGTA (a Ca²⁺ chelator), inhibited HA adhesion. In addition, cytoskeletal functional activation is required for cell adhesion to HA, since drugs that block actin polymerization, such as cytochalasin B, or actomyosin contraction, such as the calmodulin antagonist W-7, inhibited cell adhesion to HA. As this adhesion is also ADP ribosylation-sensitive, it may involve a GTP-dependent function of CD44v, i.e. ankyrin binding. Our data indicate that there is a functional hierarchy among the CD44 molecules expressed on human peripheral blood T cells and that the splice variants, as compared to the standard form, exhibit a greater HA binding ability which involves CD44-mediated signaling and effector function activation.     

Fluoride interactions with stimulus-secretion coupling of normal and pathological parathyroid cells

Effects of the GTP binding protein (G-protein) activator NaF on parathyroid hormone (PTH) release, cytoplasmic Ca²⁺ concentration ([Ca²⁺]₁) and cAMP content of bovine as well as normal and pathological human parathyroid cells were studied using precautions to avoid CaF₂ precipitation. In 0.5 mm external Ca²⁺, NaF inhibited PTH release and lowered the cAMP content by 50–70% of the effects attained with 3.0 mm Ca²⁺. The NaF-induced increase of [Ca²⁺]₁ was considerably smaller than that obtained with rise of external Ca²⁺. It seems likely that NaF activates the inhibitory G_i-protein involved in the regulation of cAMP generation. However, it is unclear whether the sluggish rise of [Ca²⁺]₁ induced by NaF is due to a direct effect of a G-protein on Ca²⁺ entry, or somehow related to the G-protein mediated formation of inositol 1,4,5-trisphosphate, which is part of the signal transduction pathway normally initiated by Ca²⁺ binding to its receptor on the parathyroid cell surface. Inhibition of PTH release by NaF probably results from the combined effects on [Ca²⁺]₁ and cAMP content. In hyperparathyroidism (HPT) the actions of NaF were not markedly affected despite severe impairments of Ca²⁺-inhibited PTH release and Ca²⁺ triggered increase of [Ca²⁺]₁. Consistent with observations of down regulation of the parathyroid Ca²⁺ receptor in HPT, the present results indicate that the disease perturbs signal transduction at a level proximal to the site of action for NaF.     

Initiation sites for Ca2+ signals in endothelial cells

Intracellular Ca²⁺ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca²⁺ oscillations and propagating Ca²⁺ waves originating at discrete initiation sites. We studied the spatial organization of the Ca²⁺ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca²⁺ concentration and rate of wave propagation than the cell body. Ca²⁺ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca²⁺ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca²⁺ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca²⁺ release in the individual store units to produce a Ca²⁺ wave.     

Induction of Ca2+ oscillations by selective,U73122-mediated,depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca²⁺ sequestering and releasing properties of internal Ca²⁺ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca²⁺ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P ₃]-sensitive, but not the Ins(1,4,5)P ₃-insensitive, Ca²⁺ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca²⁺ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av), which was largely independent of external Ca²⁺. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonistevoked [Ca²⁺]_i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca²⁺-ATPase activity resulted in a [Ca²⁺]_i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P ₃- casu quo agonist-sensitive internal Ca²⁺ store, whereas thapsigargin affects both the Ins(1,4,5)P ₃-sensitive and -insensitive Ca²⁺ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca²⁺]_i. The U73122-evoked oscillations were abolished in the absence of external Ca²⁺. The ability of U73122 to generate external Ca²⁺-dependent Ca²⁺ oscillations suggests that depletion of the agonistsensitive store leads to an increase in Ca²⁺ permeability of the plasma membrane and that the Ins(1,4,5)P ₃-insensitive Ca²⁺ pool is necessary for the Ca²⁺ oscillations.     

Desensitization of the bradykinin-induced rise in intracellular free calcium in cultured endothelial cells

We studied the cellular mechanism involved in the desensitization of cultured endothelial cells to bradykinin. Bradykinin (10 nmol/l) evoked a rise in the intracellular free calcium concentration ([Ca _i ²⁺ ]), measured with the fluorescent probe indo-1, from 137±30 (±SEM) to 623±101 nmol/l. Cells were desensitized to bradykinin by repetitive stimulation with the peptide over 10 min, after which they no longer responded to bradykinin. However, purinergic stimulation with ATP (10 mol/l) elicited the same increase in [Ca _i ²⁺ ] in endothelial cells desensitized to bradykinin as in cells never exposed to bradykinin. The initial peak of [Ca _i ²⁺ ] after stimulation with bradykinin or ATP was not affected by removal of extracellular calcium ions, indicating mobilization of Ca²⁺ from intracellular stores. Since GTP-binding proteins (G-proteins) are probably involved in the receptor-mediated stimulation of endothelial cells, we also tested the effects of sodium fluoride (NaF), a reported direct stimulator of G-proteins, on endothelial [Ca _i ²⁺ ]. NaF (5 mmol/l) increased [Ca _i ²⁺ ] to 412±88 nmol/l in control cells and was equally effective in cells desensitized to bradykinin. We conclude that the homologous desensitization to bradykinin does not occur at the level of intracellular signal transduction but at the level of membrane receptors.     

Phospholipase Cγ1 is required for pre‐TCR signal transduction and pre‐T cell development

Pre‐T cell receptor (TCR) signaling is required for pre‐T cell survival, proliferation, and differentiation from the CD4 and CD8 double negative (DN) to the double positive (DP) stage. However, the pre‐TCR signal transduction pathway is not fully understood and the signaling molecules involved have not been completely identified. Phospholipase Cγ (PLCγ) 1 is an important signaling molecule that generates two second messengers, diacylglycerol and inositol 1,4,5‐trisphosphate, that are important to mediate PKC activation and intracellular Ca²⁺ flux in many signaling pathways. Previously, we have shown that PLCγ1 is important for TCR‐mediated signaling, development and T‐cell activation, but the role of PLCγ1 in pre‐TCR signal transduction and pre‐T cell development is not known. In this study, we demonstrated that PLCγ1 expression level in pre‐T cells was comparable to that in mature T cells. Deletion of PLCγ1 prior to the pre‐TCR signaling stage partially blocked the DN3 to DN4 transition and reduced thymic cellularity. We also demonstrated that deletion of PLCγ1 impaired pre‐T cell proliferation without affecting cell survival. Further study showed that deficiency of PLCγ1 impaired pre‐TCR mediated Ca²⁺ flux and Erk activation. Thus our studies demonstrate that PLCγ1 is important for pre‐TCR mediated signal transduction and pre‐T cell development.     

Rapid refilling of Ca2+ stores in macrophages stimulated by ATP involves the sequential activation of phospholipase D and protein kinase C

Ca²⁺ movements between intracellular stores, the cytoplasm and external solution were analysed in murine peritoneal macrophages stimulated by various agonists. The Ca²⁺ content of intracellular stores was estimated from the amplitude of Ca²⁺-transients elicited by ionomycin applied in Ca²⁺-free solution. Both uridine 5-triphosphate (UTP) and platelet-activating factor (PAF) triggered the release of Ca²⁺ followed by a sustained influx, during which intracellular stores remained totally empty. In contrast, in the continuous presence of adenosine 5-triphosphate (ATP), Ca²⁺ was initially released and then rapidly sequestered again by the stores. ATP-induced store refilling was not related to cell depolarization or to an increase in the intracellular Na⁺ concentration (two specific consequences of ATP stimulation which are not induced by PAF and UTP). Store refilling was not caused by a signal that ATP would fail to induce (e.g. as a result of receptor desensitization), but was positively controlled by ATP, even in the simultaneous presence of a concentration of PAF which, on its own, would have caused a persistent store depletion. The hypothesis that the signal delivered by ATP involves the sequential activation of phospholipase D and protein kinase C is consistent with the present pharmacological evidence. However, although we found conditions in which Ca²⁺ stores did not refill in the presence of ATP, this maintained store depletion was not accompanied by a sustained Ca²⁺ response similar to that elicited by PAF or UTP, suggesting that store depletion is a condition which is necessary, but not sufficient, for inducing Ca²⁺ influx.     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

Effect of Melafen on Structure and Function of Liposome and Ehrlich Ascitic Carcinoma Cell Membranes

The effects of melafen (plant growth stimulant) on membrane structure and functions of animal cells were studied. The process of signal transduction from cell surface to intracellular structures and conformation changes in membranes in the presence of this substance were studied by light scatter and differential scanning microcalorimetry. Melafen in a wide range of concentrations (10⁻¹³-10⁻³ M) inhibited Ca²⁺ signal system involved in the function of Ca²⁺-dependent K⁺ and Cl⁻ channels in Ehrlich ascitic carcinoma cells. Being a hydrophilic substance, melafen had little effect on the lipid phase of artificial membranes, but impaired the function state of transformed cell. The importance of studies of transformed cells causes no doubt because of increasing incidence of diseases associated with uncontrolled cell division.