Suppressive effects of swainsonine on C6 glioma cell in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, is known for its anti-cancer effects and could prevent metastases. In order to investigate the effects and mechanisms of swainsonine in C6 glioma cells, we carry out correlated experiments in vitro and in vivo. After treatment with swainsonine, the effective dose and IC₅₀ value of swainsonine in the C6 glioma cell were examined using the MTT assay. Cell cycle distribution and apoptotic rates were analyzed using FCM and [Ca²⁺]_i was measured by LSCM. Expressions of p16 and p53 protein were evaluated by immunocytochemical methods. Simultaneously, glioma-bearing rats were administered swainsonine at doses of 2, 4 and 8 mg/kg body wt. The inhibition rate was calculated and pathological sections were observed. The results indicated that the growth of C6 glioma cells is inhibited by swainsonine in vitro, with an IC₅₀ value within 24 h of 0.05 μg/ml. Increases in swainsonine correlate with S phase percentages of 11.3%, 11.6% and 12.4%, respectively. Moreover, the expression of apoptosis inhibiting p53 and p16 protein decreases gradually. Tumor weight in vivo decreased clearly and HE dyeing of tumor tissue showed gray, its texture was soft, with necrosis and hemorrhagic concentrated inward. Swainsonine could inhibit the proliferation of C6 glioma cells in vitro and the growth of C6 glioma in vivo. The mechanisms of swainsonine-induced apoptosis may relate with the expression of apoptosis-related genes and overloading-[Ca²⁺]_i-induced endoplasmic reticulum stress.     

Regulation by clozapine of calcium handling by rat submandibular acinar cells

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.     

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

It is well established that intracellular calcium ([Ca²⁺]_i) controls the inotropic state of the myocardium, and evidence mounts that a “Ca²⁺ clock” controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSC_Ca) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSC_Ca that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca²⁺ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca²⁺ oscillations followed by an overall increase in [Ca²⁺]_i. The latter occurs also in HL-1 cells in Ca²⁺-free solution and after depletion of sarcoplasmic reticulum Ca²⁺ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130–150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca²⁺ oscillations followed by a compensatory increase in [Ca²⁺]_i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.     

The dynamics of mitochondrial Ca fluxes

We have investigated the kinetics of mitochondrial Ca²⁺ influx and efflux and their dependence on cytosolic [Ca²⁺] and [Na⁺] using low-Ca²⁺-affinity aequorin. The rate of Ca²⁺ release from mitochondria increased linearly with mitochondrial [Ca²⁺] ([Ca²⁺]_M). Na⁺-dependent Ca²⁺ release was predominant al low [Ca²⁺]_M but saturated at [Ca²⁺]_M around 400 μM, while Na⁺-independent Ca²⁺ release was very slow at [Ca²⁺]_M below 200 μM, and then increased at higher [Ca²⁺]_M, perhaps through the opening of a new pathway. Half-maximal activation of Na⁺-dependent Ca²⁺ release occurred at 5-10 mM [Na⁺], within the physiological range of cytosolic [Na⁺]. Ca²⁺ entry rates were comparable in size to Ca²⁺ exit rates at cytosolic [Ca²⁺] ([Ca²⁺]_c) below 7 μM, but the rate of uptake was dramatically accelerated at higher [Ca²⁺]_c. As a consequence, the presence of [Na⁺] considerably reduced the rate of [Ca²⁺]_M increase at [Ca²⁺]_c below 7 μM, but its effect was hardly appreciable at 10 μM [Ca²⁺]_c. Exit rates were more dependent on the temperature than uptake rates, thus making the [Ca²⁺]_M transients to be much more prolonged at lower temperature. Our kinetic data suggest that mitochondria have little high affinity Ca²⁺ buffering, and comparison of our results with data on total mitochondrial Ca²⁺ fluxes indicate that the mitochondrial Ca²⁺ bound/Ca²⁺ free ratio is around 10- to 100-fold for most of the observed [Ca²⁺]_M range and suggest that massive phosphate precipitation can only occur when [Ca²⁺]_M reaches the millimolar range.     

Effects of pilocarpine on the secretory acinar cells in human submandibular glands

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.     

Bcl-2 acts subsequent to and independent of Ca fluxes to inhibit apoptosis in thapsigargin- and glucocorticoidmtreated mouse lymphoma cells

The mechanism by which Bcl-2 inhibits apoptosis is unknown. One proposal is that Bcl-2 regulates intracellular Ca²⁺ fluxes thought to mediate apoptosis. In the present study, we investigated Bcl-2's mechanism of action by determining the effect of Bcl-2 on intracellular Ca²⁺ fluxes in the WEH17.2 mouse lymphoma cell line, which does not express Bcl-2, and its stable transfectant, which expresses a high level of Bcl-2. Treatment with the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin produced marked alterations in intracellular Ca²⁺ homeostasis in both WEH17.2 and W.Hb12 cells, including elevation of free cytosolic Ca²⁺, endoplasmic reticulum Ca²⁺ pool depletion, capacitative entry of extracellular Ca²⁺, and increased loading of Ca²⁺ into mitochondria. Similar changes in intracellular Ca²⁺ occurred spontaneously in both cell lines following exponential growth. In both situations, W.Hb12 cells maintained optimal viability despite marked alterations in intracellular Ca ²⁺' whereas WEH17.2 cells underwent apoptosis. Treatment with the glucocorticoid hormone, dexamethasone, induced apoptosis in WEH17.2 cells, but not in W.HB12 cells, even though dexamethasone treatment did not alter intracellular Ca²⁺ homeostasis in either cell line. These findings indicate that Bcl-2 acts downstream from intracellular Ca ²⁺ fluxes in a pathway where Ca²⁺-dependent and Ca²⁺-independent death signals converge.     

Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells

The formation of macrophage foam cells, which is the key event in atherosclerosis, occurs by the uptake of oxidized low-density lipoprotein (Ox-LDL) via the scavenger receptor (CD36) pathway. Ca(2+) plays an important role in atherosclerosis. However, in the spatiotemporal view, the correlation between kinetic changes of intracellular-free calcium ([Ca(2+)](i)) and the cellular dysfunctions in the formation of macrophage foam cells has not yet been studied in detail. By the use of confocal laser scanning microscope and flow cytometer, we have detected Ca(2+) dynamics, the assembly of F-actin, and the expression of CD36 under the exposure of U937-derived macrophages to Ox-LDL. The uptake of Ox-LDL significantly increased [Ca(2+)](i) in U937-derived macrophages in both acute and chronic treatments (P<0.01). In particular, the increases of the induced [Ca(2+)](i) were different in the presence or absence of extracellular Ca(2+) under acute exposure. A time-dependent rise in F-actin assembly and CD36 expression at 12 and 24h was induced, respectively, by Ox-LDL. The spatiotemporal increases of [Ca(2+)](i) induced by Ox-LDL probably have the key effect on the early phrase in the formation of macrophage foam cells.     

Release of the Ca(2+) oscillation-inducing sperm factor during mouse fertilization

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.     

Mechanisms of signal transduction in photo-stimulated secretion in Phaeocystis globosa

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.     

Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.     

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5′-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5′-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca²⁺]_i) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca²⁺]_i transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca²⁺]_i transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca²⁺]_i response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca²⁺]_i transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-015-9468-1) contains supplementary material, which is available to authorized users.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Pioglitazone acutely influences glucose-sensitive insulin secretion in normal and diabetic human islets

We have studied acute effects of the PPARgamma agonist pioglitazone in vitro on human islets from both non-diabetic and type 2 diabetic subjects. In 5 mM glucose, pioglitazone caused a transient increase in insulin secretion in non-diabetic, but not diabetic, islets. Continuous presence of the drug suppressed insulin release in both non-diabetic and diabetic islets. In islets from non-diabetic subjects, both high glucose and tolbutamide-stimulated insulin secretion was inhibited by pioglitazone. When islets were continuously perifused with 5 mM glucose, short-term pretreatment with pioglitazone caused approximately 2-fold increase in insulin secretion after drug withdrawal. Pioglitazone pretreatment of diabetic islets restored their glucose sensitivity. Examination of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in non-diabetic islets revealed slight Ca(2+) transient by pioglitazone at 3 mM glucose with no significant changes at high glucose. Our data suggest that short-term pretreatment with pioglitazone primes both healthy and diabetic human islets for enhanced glucose-sensitive insulin secretion.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

A locally-induced increase in intracellular Ca propagates cell-to-cell in the presence of plasma membrane Ca ATPase inhibitors in non-excitable cells

Intercellular Ca²⁺ waves are commonly observed in many cell types. In non-excitable cells, intercellular Ca²⁺ waves are mediated by gap junctional diffusion of a Ca²⁺ mobilizing messenger such as IP₃. Since Ca²⁺ is heavily buffered in the cytosolic environment, it has been hypothesized that the contribution of the diffusion of Ca²⁺ to intercellular Ca²⁺ waves is limited. Here, we report that in the presence of plasma membrane Ca²⁺ ATPase inhibitors, locally-released Ca²⁺ from the flash-photolysis of caged-Ca²⁺ appeared to induce further Ca²⁺ release and were propagated from one cell to another, indicating that Ca²⁺ was self-amplified to mediate intercellular Ca²⁺ waves. Our findings support the notion that non-excitable cells can establish a highly excitable medium to communicate local responses with distant cells.