Relation between effects of a set of anti-allergic drugs on calcium pathways and membrane structure in Fc∈RI activated signal transduction

The antigen induced stimulation of mast cells by aggregation of Fc∈RI receptors activates a signal transduction cascade leading to release of mediators of inflammation like histamine, arachidonic acid metabolites and cytokines. In this study we investigated a series of structurally related anti-allergic drugs, containing a common lipophilic diphenylmethyl piperazinyl tail and head groups that differ in lipophilicity. Effects of these drugs on various steps of the signal transduction cascade was investigated to gain insight into the mechanism of action of these drugs. It appeared that addition of the drugs to resting cells induced changes in the tyrosine phosphorylation of cellular proteins. The most active anti-allergics in inhibiting exocytosis, AL3264 and oxatomide, also induced the largest changes in phosphorylation. The effects of the drugs on tyrosine phosphorylation after cell activation was complex. Additionally, Ca²⁺ fluxes were investigated. Ca²⁺ efflux from the cells was negligibly influenced by the active drugs. However, the drugs inhibited influx from extracellular Ca²⁺, which was correlated with the effects of the drugs on inhibition of exocytosis and on membrane stabilization induced by the drugs, measured as haemolysis of erythrocytes. It is concluded that inhibition of Ca²⁺ influx is the major mechanism with which these drugs inhibit exocytosis and that for this effect drug-membrane interactions, possibly affecting the function of membrane embedded proteins, are of importance. Possible mechanisms including drug-membrane interactions, phosphorylation and inhibition of Ca²⁺ influx are discussed.     

Effects of protein phosphorylation on the regulation of capacitative calcium influx in xenopus oocytes

The regulation of capacitative Ca²⁺ influx in Xenopus oocytes was investigated using both the two electrode voltage-clamp (where Ca²⁺ is monitored through the Ca²⁺-dependent Cl⁻ current) and patch-clamp techniques. Following stimulation of expressed 5-hydroxytryptamine (5-HT) receptors, capacitative Ca²⁺ influx deactivated in around 15 min. Following injection of [adenosine 5′-O-(3-Thiotriphosphate)] (ATP [γ-S]), an ATP analogue that is readily used by protein kinases, capacitative Ca²⁺ influx activated by 5-HT application either did not deactivate or was prolonged around twofold. However, injection of adenylyl 5′-(β,γ-methylene)-diphosphonate (AMP-PCP), another ATP analogue that is not utilised by kinases, did not affect the time-course of Ca²⁺ influx. When capacitative Ca²⁺ influx was activated by readmission of Ca²⁺ to oocytes incubated in thapsigargin/0 Ca²⁺ solution for several hours, Ca²⁺ influx occurred and a weakly saturating relationship between external Ca²⁺ and Ca²⁺ influx was found. Ca²⁺ influx in thapsigargin-treated cells was unaffected by ATP [γ-S]. ATP [γ-s] and several kinases had no effect on the Ca²⁺-dependent Cl⁻ current when the latter was activated by elevation of Ca²⁺ independent of capacitative Ca²⁺ influx. Protein kinase C slowly and partially inhibited the Cl⁻ current. Outside-out patches taken from thapsigargin-treated cells failed to demonstrated any Ca²⁺ current or Ca²⁺-dependent Cl⁻ current on reapplying high Ca²⁺ to the patch, despite the oocyte showing a large capacitative Ca²⁺ influx. The results suggest that a kinase, activated on receptor stimulation, prolongs the activation time-course of capacitative Ca²⁺ influx. Received: 24 October 1995/Received after revision and accepted: 21 December 1995     

Efficiency of T‐cell costimulation by CD80 and CD86 cross‐linking correlates with calcium entry

Costimulation is a fundamental principle of T‐cell activation. In addition to T‐cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) receptors is required to regulate T‐cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T‐cell proliferation and the ability of CD8⁺ T‐effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single‐cell assay to analyse Ca²⁺ signals following costimulation with bi‐specific antibodies. We found that this stimulation method worked in every human T‐cell that was analysed, making it one of the most efficient T‐cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca²⁺ influx following CD86 costimulation and it was dependent on CD28/CTLA‐4 expression. The enhanced Ca²⁺ influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca²⁺ influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca²⁺ stores but were eliminated by the application of 10 μm 2‐aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)‐dependent Ca²⁺ entry while reducing STIM1‐dependent Ca²⁺ entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca²⁺ entry.     

Increased calcium influx in the presence of ethanol in mouse pancreatic acinar cells

The effects of alcohol on Ca²⁺ signalling remains poorly understood. Here we have investigated the effects of acute ethanol exposure on Ca²⁺ influx in mouse pancreatic acinar cells. Cells were loaded with fura‐2 and the changes in fluorescence were monitored by spectrofluorimetry and imaging analysis. Stimulation of cells with 20 pM cholecystokinin evoked an oscillatory pattern in [Ca²⁺]_c, both in the presence and in the absence of extracellular Ca²⁺. Stimulation of cells with cholecystokinin in the presence of 50 mM ethanol led to a transformation of physiological oscillations into a single transient increase in [Ca²⁺]_c. This effect was observed when Ca²⁺ was present in the extracellular medium, and did not appear in its absence. Addition of 1 mM CaCl₂ to the extracellular medium, following release of Ca²⁺ from intracellular stores by stimulation of cells with 1 nM cholecystokinin or 1 μM thapsigargin in the absence of extracellular Ca²⁺, was followed by an increase in [Ca²⁺]_c. Ca²⁺ influx was increased in the presence of 50 mM ethanol. The anti‐oxidant cinnamtannin B‐1 (10 μM) or inhibition of alcohol dehydrogenase by 4‐MP (1 mM), significantly reduced Ca²⁺ influx evoked by cholecystokinin in the presence of ethanol. In summary, intoxicating concentrations of ethanol may lead to over stimulation of pancreatic acinar cells by cholecystokinin. This might be partially explained by the generation of reactive oxygen species and an increased Ca²⁺ entry in the presence of ethanol. Potentially ethanol might lead to Ca²⁺ overload, which is a common pathological precursor that is implicated in pancreatitis.     

Differential activation of mitogen‐activated protein kinase signalling pathways by isometric contractions in isolated slow‐ and fast‐twitch rat skeletal muscle

Activation of mitogen‐activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre‐type dependent manner. Slow‐twitch (soleus) and fast‐twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non‐stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast‐ and slow‐twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast‐twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow‐twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow‐ and fast‐twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

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.     

Cytoplasmic Ca2+ determines the rate of Ca2+ entry into Mardin-Darby canine kidney-focus (MDCK-F) cells

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca²⁺ oscillations from an inositol 1,4,5-trisphosphate-sensitive cytoplasmic Ca²⁺ store. In this study, Ca²⁺ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca²⁺ video imaging and the Fura-2/Mn²⁺ quenching technique. Oscillations were dependent on extracellular Ca²⁺ concentration and were inhibited by extracellularly applied La³⁺, Co²⁺ and Ni²⁺. Depolarization of the cell membrane with high K⁺ concentrations and the L-type Ca²⁺ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca²⁺ channels. Mn²⁺ quenching experiments disclosed significant Ca²⁺ influx into MDCK-F cells. The rate of this influx was constant between Ca²⁺ spikes, but markedly increased during the spontaneous Ca²⁺ spikes. Similar transient increases in Ca²⁺ entry could be mimicked by agents triggering intracellular Ca²⁺ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca²⁺ permeability permitting Ca²⁺ entry into the cytoplasm. The rate of Ca²⁺ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca²⁺ concentration.     

Calcium signaling in B cells: Regulation of cytosolic Ca increase and its sensor molecules,STIM1 and STIM2

Calcium signals are crucial for diverse cellular functions including adhesion, differentiation, proliferation, effector functions and gene expression. After engagement of the B cell receptor, the intracellular calcium ion (Ca²⁺) concentration is increased promoting the activation of various signaling cascades. While elevated Ca²⁺ in the cytosol initially comes from the endoplasmic reticulum (ER), a continuous influx of extracellular Ca²⁺ is required to maintain the increased level of cytosolic Ca²⁺. Store-operated Ca²⁺ entry manages this process, which is regulated by an ER calcium sensor, stromal interaction molecule (STIM). STIM proteins sense changes in the levels of Ca²⁺ stored within the ER lumen and regulates the Ca²⁺-release activated Ca²⁺ channel in the plasma membrane. This review focuses on the signaling pathways leading to Ca²⁺ influx and the role of Ca²⁺ signals in B cell functions.     

Phosphoproteomics analyses show subnetwork systems in T‐cell receptor signaling

A key issue in the study of signal transduction is how multiple signaling pathways are systematically integrated into the cell. We have now performed multiple phosphoproteomics analyses focused on the dynamics of the T‐cell receptor (TCR) signaling network and its subsystem mediated by the Ca²⁺ signaling pathway. Integration of these phosphoproteomics data sets and extraction of components of the TCR signaling network dependent on Ca²⁺ signaling showed unexpected phosphorylation kinetics for candidate substrates of the Ca²⁺‐dependent phosphatase calcineurin (CN) during TCR stimulation. Detailed characterization of the TCR‐induced phosphorylation of a novel CN substrate, Itpkb, showed that phosphorylation of this protein is regulated by both CN and the mitogen‐activated protein kinase Erk in a competitive manner. Phosphorylation of additional CN substrates was also found to be regulated by Erk and CN in a similar manner. The combination of multiple phosphoproteomics approaches thus showed two major subsystems mediated by Erk and CN in the TCR signaling network, with these subsystems regulating the phosphorylation of a group of proteins in a competitive manner.     

Function follows form: how fibronectin matrix architecture controls cell behaviour

Introduction Fibronectin (FN) matrix assembly is a tightly regulated stepwise process that is initiated by interactions between FN and cell‐surface integrin receptors. Assembly is affected by extracellular factors including availability of FN and the presence of other matrix molecules. In turn, FN matrix activates intracellular signalling pathways via integrin receptors, and these signals regulate cell adhesion, migration and survival. Two kinases immediately downstream of integrins are focal adhesion kinase (FAK) and pp60‐Src. Our results show that activation of these kinases regulates accumulation of FN matrix fibrils and that this process is affected by tenascin‐C, an ECM protein that modulates cell interactions with FN. Methods FN assembly was monitored by indirect immunofluorescence and by immunoblotting of deoxycholate (DOC)‐insoluble matrix material with anti‐FN antibodies. Wild‐type mouse fibroblasts and cells lacking FAKs or Src family kinases (SYF cells) were used. Kinase and phosphatase inhibitors were used to regulate enzyme activities. Results Mouse fibroblasts lacking FAK assemble significantly reduced amounts of FN fibrils and DOC‐insoluble matrix. FAK is phosphorylated by Src family kinases and fibroblasts lacking Src family kinases (SYF cells) or cells treated with PP1, an inhibitor of SYF kinases, also lack FN matrix. The effects of Src activity are most dramatic during the early stages of de novo assembly by fibroblasts implicating this kinase in the initiation process. While FN stimulates FAK, tenascin‐C, an ECM protein that is expressed at sites of cell movement, has the opposite effect on FAK activity. In fibroblasts on a 3D FN matrix, FAK is constitutively phosphorylated. In contrast, FAK is only transiently activated in the presence of tenascin‐C. Concomitant with the absence of FAK phosphorylation is an inability to assemble FN matrix fibrils. Conclusion Our results show that FAKs and Src kinases, which lie downstream of integrins, are essential for efficient initiation of FN matrix assembly. The effects of tenascin‐C on FN matrix and FAK phosphorylation suggest that this protein limits the extent of matrix deposition by regulating FAK activity. Thus, extracellular and intracellular events co‐operate to control FN matrix assembly.     

The Role of the Protein Tyrosine Phosphatase CD45 in Regulation of B Lymphocyte Activation

The transmembrane protein tyrosine phosphatase CD45 is expressed throughout B cell development and differentiation, with the exception of terminally differentiated plasma cells on which its expression is down regulated. Numerous studies using CD45-deficient B cell lines and CD45-deficient mice have clearly demonstrated that CD45 plays an important role in modulating the signal that is transduced via the B cell antigen receptor by regulating the phosphorylation state of Src family kinases. Spatial and temporal controls enable CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining Src family kinases in a partially active state, such that the B cell is able to effectively respond to an antigenic challenge. Moreover, CD45 is required for optimal activation of Ca²⁺-dependent and MAP kinase-dependent signal transduction pathways in the B cell. The net result is that CD45 affects the B cell response by controlling the relative threshold of sensitivity to a given antigenic stimulus. Thus, CD45 expression and function is required for normal B cell development, tolerance induction, and responsiveness to antigen.     

Protein Phosphatase Inhibitors Exert Specific and Nonspecific Effects on Calcium Influx in Thapsigargin-Treated human Neutrophils

C2-ceramide but not inhibitors of phosphatase types 1 and 2A (okadaic acid, calyculin A, tautomycin) blocked store-regulated Ca²⁺ entry induced in human neutrophils by thapsigargin. This contrasts with previous results showing that both types of compounds inhibit Ca²⁺ influx in fmet-leu-phe-treated cells. In present studies, phosphatase inhibitors increased the rate of secondary Ca²⁺ influx in a temperature-dependent manner. Their mechanism of action appeared to be independent of phosphatase inhibition since the inactive congeners, norokadaone and tetraacetyl okadaic acid, also potentiated Ca²⁺ influx at similar concentrations. When Ca²⁺ stores were predischarged by thapsigargin, okadaic acid but not norokadaone acted synergistically with fMLP to inhibit subsequent Ca²⁺ entry. Results suggest that blockade of Ca²⁺ influx in neutrophils is mediated by a phosphorylation reaction that is prolonged by phosphatase inhibitors. The requisite phosphorylation occurs in fMLP-activated cells but may be absent in cells incubated with thapsigargin.     

Calcium entry is regulated by Zn in relation to extracellular ionic environment in human airway epithelial cells

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). This elevation of [Ca²⁺]_i and activation of Ca²⁺-dependent Cl⁻ channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca²⁺]_i in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na⁺ and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca²⁺ signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca²⁺ entry was either inhibited or stimulated by Zn²⁺ depending on the extracellular Na⁺ concentration. In Na⁺-free environment, Zn²⁺ and other divalent cations elicited a biphasic Ca²⁺ signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca²⁺ entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.     

Role for protein kinase in Ca2+-dependent feedback modulation of divalent cation influx in internal-Ca2+-store-depleted rat parotid gland cells

 Divalent cation (Ca²⁺ and Mn²⁺) influx, stimulated by internal Ca²⁺ store depletion, into rat parotid acinar cells is inhibited by conditions which increase protein phosphorylation [T. Sakai and I.S. Ambudkar (1996) Am J Physiol 271:C284–C294]. The present study examines the involvement of this protein phosphorylation and Ca²⁺ in the store-dependent inactivation of divalent cation entry. Internal Ca²⁺ store depletion, achieved by incubation (30 min) of cells in nominally Ca²⁺-free medium containing either carbachol or thapsigargin, stimulated Ca²⁺, and Mn²⁺, influx into cells. In either case, inclusion of 1.5 mM Ca²⁺ for the last 5 min of incubation resulted in a decrease in Ca²⁺ (33–41%) and Mn²⁺ (50%) influx, which could not be accounted for by internal Ca²⁺ store refill. The inhibition was prevented when internal-store-depleted cells were treated (prior to incubation with Ca²⁺) with either staurosporine or K-252a, but not with H-7 or KN-93. Refilling of internal Ca²⁺ store(s) in carbachol-treated cells (incubation with Ca²⁺+atropine) induced complete inhibition of divalent cation influx, which was not prevented by treatment with protein kinase inhibitors. These data suggest the staurosporine-sensitive (and K-252a-sensitive) protein phosphorylation is not involved in Ca²⁺-store-refilling-dependent inactivation of Ca²⁺ influx but mediates a Ca²⁺-dependent feedback modulation of divalent cation influx in rat parotid gland acinar cells. Received: 24 July 1996 / Received after revision: 26 September 1996 / Accepted: 2 October 1996     

A tyrosine kinase regulates propofol‐induced modulation of the β‐subunit of the GABAA receptor and release of intracellular calcium in cortical rat neurones

Propofol, an intravenous anaesthetic, has been shown to interact with the β‐subunit of the γ‐amino butyric acid_A (GABA_A) receptor and also to cause changes in [Ca²⁺]_i. The GABA_A receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre‐incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca²⁺]_i were investigated. Propofol (3 μg mL^?1, 16.8 μM) increased intracellular calcium levels by 122 ± 34% (mean ± SEM) when applied to neurones in calcium free medium. This rise in [Ca²⁺]_i was lowered by 68% when the cells were pre‐incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 ± 10%) in tyrosine phosphorylation, with maximum at 120 s, of the β‐subunit of the GABA_A‐receptor. This tyrosine phosphorylation was decreased after pre‐treatment with herbimycin A (44 ± 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the β‐subunit of the GABA_A‐receptor.     

Combination use of immune complexes and a Ca2+ channel blocker azelnidipine enhances interleukin‐12 p40 secretion without T helper type 17 cytokine secretion in human monocyte‐derived dendritic cells

Immune complexes (ICs) improve the capacity of priming specific CD8⁺ cytotoxic T cell responses of dendritic cells (DCs). ICs induce phosphorylation of mitogen‐activated protein kinases (MAPK) and calcium influx, although the precise regulating mechanism still remains unclear. In the present study, we investigated the effect of a Ca2⁺ channel blocker on the phosphorylation of p38 MAPK and extracellular signal‐regulated kinase (ERK) in immature monocyte‐derived DCs stimulated with lipopolysaccharide (LPS) or LPS‐ICs, and the production of interleukin (IL)‐12 family members (p40, p70, IL‐23), T helper type 17 (Th17) cytokines (IL‐6 and IL‐23), tumour necrosis factor (TNF)‐α and IL‐10 were also investigated. In comparison with LPS stimulation, LPS‐ICs stimulation enhanced p38 MAPK phosphorylation significantly, which was associated with an increase in IL‐12 p40 monomer/homodimer secretion. LPS‐ICs also enhanced TNF‐α and IL‐6 secretion, but suppressed IL‐23 secretion. The use of azelnidipine (Aze), a long‐acting L‐type Ca2⁺ channel blocker with a high lipid solubility, suppressed p38 MAPK phosphorylation stimulated with LPS or LPS‐ICs, but surprisingly enhanced IL‐12 p40 monomer/homodimer secretion stimulated with LPS‐ICs. This IL‐12 p40 secretion‐enhancing effect was not accompanied by IL‐10 or IL‐23 production, but was associated with ERK phosphorylation. The use of Aze did not affect IL‐12 p70 production. These results suggest that the use of Aze enhances ICs‐mediated IL‐12 p40 secretion without additional IL‐23 secretion. Therefore, the use of Aze and ICs could be a new therapeutic approach to immunomolecular therapy, as it does not cause Th17 differentiation which induces autoimmunity or reduces anti‐tumour immunity.     

A myosin phosphatase modulates contractility in skinned smooth muscle

The influence of a purified holoenzyme form of polycation-modulable (PCM-) myosin phosphatase on Ca²⁺-dependent actin-myosin interactions was studied in detergent-skinned smooth muscle fibers from chicken gizzard. The concentration of Ca²⁺ required for half maximal isometric contraction (A_0.5; 0.26 M) of fibers incubated in the absence of phosphatase was increased 2-fold when PCM-phosphatase (13 U/ml) was included in the medium. Removal of the phosphatase restored A_0.5 to control level showing that the enzyme-mediated decrease in Ca²⁺-sensitivity was reversible. Two-dimensional electrophoresis of fiber homogenates revealed that PCM-phosphatase decreased Ca²⁺-sensitivity for phosphorylation of the regulatory myosin light chains in parallel fashion. Ca²⁺-dependent increases in isometric force were directly correlated to increases in the extent of light chain phosphorylation up to about 0.35 mol PO₄/mol light chain; further increases in phosphorylation were not associated with further increases in force. Addition of PCM-phosphatase to fibers which had been contracted with a suboptimal concentration of Ca²⁺ (0.35 M) resulted in rapid relaxation. Unloaded shortening velocity, reflecting cross-bridge cycling rate, was reduced by 92% in the presence of PCM-phosphatase and light chain phosphorylation was decreased by 50%. These data show that both tension and unloaded shortening velocity may be related to Ca²⁺-dependent phosphorylation of the light chains. The results indicate that the level of phosphorylation attained in the fiber preparations studied probably reflects the ratio of myosin kinase to phosphatase activities. Since protein phosphatases are regulated enzymes the results also suggest that modulation of phosphatase activity may participate in control of smooth muscle contractility.     

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.