calcimycin and indo-1

Caffeine is known to stimulate gastric acid secretion, but, the effects of caffeine on gastric mucus secretion have not been clarified. To elucidate the action of caffeine on gastric mucin-producing cells and its underlying mechanism, the effects of caffeine on mucus glycoprotein secretion and agonist-induced [Ca2+]i mobilization were examined in human gastric mucin secreting cells (JR-I cells). The measurement of [Ca2+]i using Indo-1 and the whole cell voltage clamp technique were applied. Mucus glycoprotein secretion was assessed by release of [3H]glucosamine. Caffeine by itself failed to increase [Ca2+]i and affect membrane currents, while it dose-dependently inhibited agonist (acetylcholine (ACh) or histamine)-induced [Ca2+]i rise, resulting in inhibiting activation of Ca2+-dependent K+ current (I(K.Ca)) evoked by agonists. The effect of caffeine was reversible, and the half maximal inhibitory concentration was about 0.5 mM. But, caffeine did not suppress [Ca2+]i rise and activation of I(K.Ca) induced by A23187 or inositol trisphosphate (IP3). Theophylline or 3-isobutyl-1-methyl-xanthine (IBMX) did not mimic the effect of caffeine. Caffeine failed to stimulate mucus secretion, while it significantly decreased ACh-induced mucus secretion. These results indicate that caffeine selectively inhibits agonist-mediated [Ca2+]i rise in human gastric epithelial cells, probably through the blockade of receptor-IP3 signaling pathway, which may affect the mucin secretion.

Topics: Caffeine; Calcimycin; Calcium; Gastric Mucosa; Humans; Indoles; Inositol Phosphates; Mucins; Mucus; Spectrometry, Fluorescence; Tumor Cells, Cultured

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

Topics: Animals; Biological Transport; Calcimycin; Calcium; Cell Compartmentation; Egtazic Acid; Fluorescent Dyes; Humans; Indoles; Intracellular Fluid; Ionophores; KB Cells; Mice; Spectrometry, Fluorescence; Toxoplasma; Vacuoles

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca(++)-ATPase activity and Ca(++)-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle. In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96 +/- 0.1 and 0.99 +/- 0.1 mg/g in WG and RG, respectively. The percent Ca(++)-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca(++)-activated Ca(++)-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P < 0.05) in frozen HOM (5.12 +/- 0.18-3.98 +/- 0.20 mole/g tissue/min in WG and from 5.39 +/- 0.20-4.48 +/- 0.24 mumole/g tissue/min in RG). Ca(++)-uptake was measured at a range of physiological free [Ca++] using the Ca++ fluorescent dye Indo-1. Maximum Ca(++)-uptake rates when corrected for initial [Ca++]f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca++ was reduced (P < 0.05) in quick frozen HOM (1.30 +/- 0.1-0.66 +/- 0.1 mumole/g tissue/min in WG and 1.04 +/- 0.2-0.60 +/- 0.1 mumole/g tissue/min in RG). Linear correlations between Ca(++)-uptake and Ca(++)-ATPase activity measured in the presence of the Ca++ ionophore A23187 were r = +0.25, (P < 0.05) and r = +0.74 (P < 0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca(++)-uptake (r = +0.44, P < 0.05) and between HOM and CM Ca(++)-ATPase activity (r = +0.34, P < 0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca(++)-uptake function and maximal Ca(++)-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage.

Topics: Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Fractionation; Enzyme Activation; Enzyme Stability; Female; Freezing; Indoles; Intracellular Membranes; Microsomes; Mitochondria, Muscle; Muscle, Skeletal; Rats; Rats, Wistar; Sarcoplasmic Reticulum

Adenosine triphosphate (ATP) is a potent agonist of surfactant secretion from type II pneumocytes. The extracellular ATP signal is transduced by both P1- and P2-purinergic pathways, which respectively initiate cyclic adenosine monophosphate formation and phosphatidyl inositol hydrolysis to inositol phosphates (Ins P). We investigated the role of inositol triphosphate (Ins P3) isomer formation in this signal transduction pathway. Primary cultures of rat type II pneumocytes were labeled with 30 microCi [3H]myoinositol/5 x 10(6) cells for 48 h. After preincubation with 10 mM LiCl for 20 min, the cells were stimulated with ATP (10(-4) M) and then were rapidly frozen with liquid N2. The Ins P3 isomers were analyzed by high performance liquid chromatography. A 4-fold increase in Ins 1,4,5 P3 occurred within 2 s after stimulation with ATP, decreased to half maximum by 60 s, and returned close to baseline values by 2 min. In contrast, Ins 1,3,4 P3 did not increase until 15 s, peaked by 60 s with a 4-fold increase, and returned to baseline values by 2 min. Intracellular calcium [( Ca2+]i), measured as Indo-1 fluorescence, also increased 3-fold within 2 s of exposure to ATP (10(-4)M) and fell to a plateau level 25% above baseline values by 10 s. We conclude that Ins 1,4,5 P3 formation and [Ca2+]i release both occur rapidly after exposure of type II pneumocytes to extracellular ATP. We speculate that these early events in type II pneumocyte signal transduction play a role in the initiation of stimulation of surfactant secretion by extracellular ATP.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cells, Cultured; Chlorides; Chromatography, High Pressure Liquid; Egtazic Acid; Fluorescent Dyes; Indoles; Inositol; Inositol 1,4,5-Trisphosphate; Kinetics; Lithium; Lithium Chloride; Lung; Male; Rats; Signal Transduction

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.

Topics: Animals; Binding Sites; Calcimycin; Calcium; Calibration; Cells, Cultured; Chick Embryo; Digitonin; Fluorescent Dyes; Fura-2; Indoles; Mice; Myocardium; Protein Binding; Spectrometry, Fluorescence

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha-thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.

Topics: Adenosine Diphosphate; Blood Platelets; Calcimycin; Calcium; Cytosol; Egtazic Acid; Flow Cytometry; Humans; In Vitro Techniques; Indoles; Platelet Activation; Temperature; Thrombin

Membrane assembly of the C5b-9 proteins on gel-filtered human platelets has been shown to initiate the nonlytic release of alpha-granule contents and expression of membrane prothrombinase sites, suggesting cellular activation by these ostensibly cytolytic plasma proteins (Wiedmer, T., Esmon, C. T., and Sims, P. J. (1986) J. Biol. Chem. 261, 14587-14592). We now examine the mechanism of the C5b-9-induced release reaction. The release of alpha-granule contents upon C5b-9 assembly is accompanied by expression of alpha-granule membrane glycoprotein 140 on the platelet surface, confirming that the complement-mediated release reaction occurs by secretory fusion of the alpha-granule with the plasma membrane. C5b-9 binding initiates the phosphorylation of both 40- and 20-kDa platelet proteins, indicative of activation of protein kinase C and myosin light chain kinase, respectively. Activation of cellular protein kinases under these conditions was not accompanied by the formation of inositol phosphates and was found to strictly depend upon extracellular Ca2+, suggesting that the platelet's secretory response to the C5b-9 proteins is triggered directly by the influx of Ca2+ across the plasma membrane. measurement of intracellular Ca2+ confirmed that elevation of this ion in the cytosol was strictly dependent upon increased plasma membrane permeability due to C5b-9 assembly and was not accompanied by mobilization of this ion from internal storage pools. The C5b-9-mediated secretory response was blocked by sphingosine, a potent inhibitor of protein kinase C, but was unaffected by the cyclooxygenase inhibitor indomethacin, suggesting that feedback (receptor-linked) by thromboxane is not required for platelet activation after C5b-9 insertion.

Topics: Adult; Blood Platelets; Blood Proteins; Calcimycin; Calcium; Complement Membrane Attack Complex; Complement System Proteins; Cytoplasmic Granules; Enzyme Activation; Fluorescent Dyes; Humans; Indoles; Inositol Phosphates; Kinetics; Phosphorylation; Protein Kinases; Spectrometry, Fluorescence; Thrombin

The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.

Topics: Animals; Calcimycin; Calcium; Cell Movement; Cells, Cultured; Chick Embryo; Fluorescent Dyes; Indoles; Myocardial Contraction; Myocardium; Spectrometry, Fluorescence