An esterification protocol for cis-parinaric acid-determined lipid peroxidation in immune cells.

Loss of fluorescence from cis-parinaric acid (cPnA) is a sensitive indicator of lipid peroxidation. The purpose of this study was to utilize cPnA to determine, at the level of the intact immune cell, whether enrichment of membranes with polyunsaturated fatty acids (PUFA) increased lipid peroxidation. P388D1 macrophages were labeled by addition of cPnA as an ethanolic solution. Within two minutes of addition, in the absence-of serum, cPnA rapidly intercalated into the plasma membrane. Lipid peroxidation was initiated by addition of Fe(2+)-EDTA resulting in a dose-dependent decrease in fluorescence with increased oxidant concentration. Cells previously enriched with PUFA and labeled by intercalation showed no differences in spontaneous or Fe(2+)-induced lipid peroxidation. In separate experiments, 20 microM cPnA in ethanolic solution was injected into cell culture media containing 0.1% essentially fatty acid free bovine serum albumin (BSA). Cells were resuspended and incubated for 90 min at 37 degrees C. After washing with BSA to remove cPnA which had not incorporated, 0.5% (0.1 microM) of the added cPnA was found esterified within cellular lipids. This level of cPnA provided a 100-fold increase over basal autofluorescence levels. Cells labeled in this manner also lost fluorescence in a dose-dependent manner as levels of oxidant stress increased. Cells enriched with PUFA and labeled by esterification had significantly increased rates and total amounts of lipid peroxidation. Co-incubation with alpha-tocopherol and PUFA resulted in a decrease in lipid peroxidation which was not significantly different from control cells. In conclusion, esterification of cPnA into membrane phospholipids can sensitively detect changes in lipid peroxidation induced by alteration of membrane PUFA and/or vitamin E content.

Potentiation of regulatory volume decrease by P2U purinoceptors in HSG-PA cells.

HSG-PA human salivary gland duct cells exhibit progressively increased regulatory volume decrease (RVD) in response to decreased medium osmolarity. The P2U purinoceptor agonist UTP causes a potentiation of RVD, the extent of which is most pronounced in 220 mosM medium and is least apparent in 180 mosM medium. We examined the underlying mechanisms for this effect. Exposure of HSG-PA cells to UTP promotes Ca2+ mobilization, hyperpolarization, and net K+ efflux, suggesting the participation of Ca(2+)-activated K+ channels in RVD. To delineate the anion counterpart of K+ movement during RVD, cell swelling in the presence of gramicidin, which abolishes the membrane potential, was measured. In response to a sudden dilution in hypotonic media, gramicidin-treated cells swelled immediately, followed by a "secondary swelling" in 180 but not in 220 mosM medium. The results suggest that in 180 mosM cells perform spontaneous RVD mediated by increased anion conductance. In 220 mosM medium in which RVD is minimal, the increase in anion conductance is marginal. In our model of RVD in which cells were challenged by UTP, the ensuing hyperpolarization provides the driving force for net Cl- efflux, which is confirmed by tracer flux studies during purinoceptor-activated RVD. Thus RVD, which has long been regarded as a self-sufficient cellular program, appears to be subject to extracellular control in HSG-PA cells through receptor-mediated processes.

Picomolar platelet-activating factor mobilizes Ca to change platelet shape without activating phospholipase C or protein kinase C; simultaneous fluorometric measurement of intracellular free Ca concentration and aggregation.

The purpose of this study was to investigate signal transduction mechanisms activated by low and high concentrations of platelet-activating factor (PAF) in rabbit platelets and to contrast the responses to those induced by thrombin. We measured changes in intracellular free calcium ([Ca++]i) with fura2, while monitoring light scatter simultaneously as a measure of shape change and aggregation in a dual-excitation dual-emission spectrofluorometer. An abrupt 20% fall in light scatter, coincident with the peak of the [Ca++]i, indicated shape change in Ca-containing or Ca-free medium and was blocked by BAPTA loading and 10 microM cytochalasin B. A secondary decline in light scatter, indicating aggregation, occurred only in Ca-containing medium and only under conditions favoring protein kinase C (PKC) activation. PAF at 10(-12) M did not increase 1,4,5-inositol triphosphate content, which suggested PKC would not be activated. However, PAF at 10(-12) rapidly increased [Ca++]i to 900 nM in 7 sec seemingly by Ca influx through receptor-operated channels inducing shape change. PAF at 10(-9) and 10(-8) M increased [Ca++]i to 2 microM in 12 sec and induced both shape change and aggregation. However, in platelets pretreated with 100 nM staurosporine to inhibit protein kinases, 10(-9) M PAF did not cause aggregation even though [Ca++]i still rose to 2 microM, which indicated that PKC plays a role in aggregation but not in Ca++ mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of extracellular ATP on Fe(2+)-induced cytotoxicity in PC-12 cells.

Extracellular ATP is known to cause a variety of changes, including the alteration of ion fluxes, cell growth, and other physiological activities. Recently, it has been suggested that ATP acts as an excitatory synaptic transmitter, which may produce a Ca2+ influx via the activation of a P2y purinoceptor. Rat pheochromocytoma (PC-12) cells are known to resemble rat sensory neurons and to possess a P2y purinoceptor. In this study, we demonstrated that extracellular ATP dose-dependently increased PC-12 cell death in the presence of ferrous ions. Voltage-sensitive calcium channel blockers and calpain and xanthine oxidase inhibitors were found to be effective at protecting PC-12 cells from Fe2+/ATP-induced lipid peroxidation and cell death. These results suggest that xanthine oxidase activation induced by calpains and subsequent free radical formation may be responsible for Fe2+/ATP-induced neuronal cell death.

Quick and accurate method to convert BCECF fluorescence to pHi: calibration in three different types of cell preparations.

A rapid, easy, and accurate method for converting the fluorescence of BCECF to pH, as an alternative to the nigericin method, is described. The ratio of the fluorescence intensities for BCECF can be converted to pH between 4 and 9 by a formula similar to the one used to calculate [Ca2+]i from the fluorescence of fura2. The formula is inverted because H+ binding to BCECF causes a decrease in fluorescence, whereas Ca2+ binding to fura2 causes an increase in fluorescence. The ratio of the fluorescence intensities is a sigmoidal function of the [H+] between pH 4 and 9 with an essentially linear mid region from pH 6 to 8. This calibration procedure in cells is similar to the popular method for fura2 where ionomycin, Ca2+, and an alkaline EGTA solution are added in succession to change the intracellular pCa from 4 to 9. For BCECF in cells, a protonophore, FCCP or CCCP, is added and the cells are titrated with acid to an intracellular pH of 4 and then back to pH 9 with base by observing the gradual change in fluorescence as it asymptotically reaches its limiting minimum and maximum values. This method does not require changing the medium to one with high KCl to depolarize the membrane potential nor does the proton concentration need to be equilibrated across the plasma membrane. The technique can be used to calibrate BCECF in sheets of cells, as well as suspensions of cells over a wide range of pH sensitivities.

Direct relationship between intracellular calcium mobilization and phospholipase D activation in prostaglandin E-stimulated human erythroleukemia cells.

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.

Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts.

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady-state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.

Regulation of the neurotensin receptor and intracellular calcium mobilization in HT29 cells.

Regulation of the neurotensin receptor-inositol phosphate-intracellular Ca2+ ((Ca2+]i) pathway was studied in HT29 cells. Preincubation with neurotensin or phorbol 12-myristate, 13-acetate decreased the number of cell surface neurotensin receptors and neurotensin-induced increases of inositol trisphosphates and [Ca2+]i. The phorbol 12-myristate, 13-acetate-(43 +/- 1% at 1 microM) but not the neurotensin (65 +/- 9% at 10 nM)-induced decrease in receptors was blocked by staurosporine. The decrease in cell surface receptors was accompanied by a 55 +/- 7% shift of specifically bound 125I-neurotensin from the plasma membrane fraction to the light vesicle fraction of sucrose density gradients if a 37 degrees C incubation step was included. The time course for desensitization of [Ca2+]i mobilization was more rapid (maximal at approximately 1 min) than for loss of receptors (maximal at 45 min). After a 5-min exposure to neurotensin, the cell surface receptor number rapidly returned to control levels in the absence of agonist, but [Ca2+]i sensitivity to neurotensin recovered only partially. Incubation with carbachol, ATP or phorbol 12-myristate, 13-acetate desensitized the subsequent [Ca2+]i response to neurotensin. These results demonstrate a polyphosphoinositide-[Ca2+]i pathway in HT29 cells stimulable by neurotensin and other agents. The results from pre-incubation studies indicate that the neurotensin receptor-signaling pathway is homologously and heterologously regulated. Finally, differences in time courses for loss and recovery of cell surface receptors and desensitization of the [Ca2+]i response, as well as the lack of effect on 125I-neurotensin binding of other agonists that desensitize the neurotensin [Ca2+]i response, suggest that receptor internalization alone does not account for desensitization of the system.

Measurement of cytoplasmic free Ca2+ concentration in cultured muscle cells by aequorin and quin 2.

Ca2+ has been proposed to regulate expression of the gene for the Ca2+ pump of the sarcoplasmic reticulum in developing chicken myoblasts (A. N. Martonosi, L. Dux, R. L. Terjung, and D. Roufa. Ann. NY Acad. Sci. 402: 485-514, 1982. In the present study, intracellular free Ca2+ ([Ca2+]i) was measured with the photoprotein, aequorin, incorporated by a reversible hyperpermeabilization technique, and with the fluorescent probe, quin 2. Because aequorin reacts irreversibly with Ca2+ and is inactivated by heat, at 37 degrees C the active aequorin content declined markedly. The resting glow (1/10(6) of the maximum light) disappeared after a few hours, whereas the light from the total active aequorin remaining was still detectable when cells were lysed 3 days later. A new approach, which compared the rate of disappearance of aequorin in treated and control cells, was developed on the basis that aequorin would be inactivated more quickly in cells with higher [Ca2+]i. A23187, ionomycin, and trifluoperazine, all of which accelerated gene expression (A. N. Martonosi et al.), increased the rate of decay of active aequorin and therefore increased [Ca2+]i. "Ca shock", and ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, which also increased the gene expression (A. N. Martonosi et al.), caused small increases of [Ca2+]i not detectable by aequorin but detectable by quin 2. X537A and ouabain had no effect on the gene expression (A. N. Martonosi et al.) and did not raise [Ca2+]i. The results support the proposal that Ca2+ may regulate expression of this gene.

Voltage dependence of membrane charge movement and calcium release in frog skeletal muscle fibres.

Voltage dependent membrane charge movement (gating current) and the release of Ca2+ from intracellular stores have been measured simultaneously in intact frog skeletal muscle fibres. Charge movement was measured using the three microelectrode voltage clamp technique. Ca2+ release was measured using the metallochromic indicator dye arsenazo III. Fibres were bathed in 2.3 X hypertonic solutions to prevent contraction. Rb+, tetraethylammonium and tetrodotoxin (TTX) were used to eliminate voltage-dependent ionic currents. The maximum rate of Ca2+ release from the sarcoplasmic reticulum in response to voltage-clamp step depolarizations to 0 mV was calculated using the dye-related parameters of model 2 of Baylor et al. (1983) and a method described in the Appendix for calculating a scaling factor (1 + p) that accounts for the additional Ca2+ buffering power of the indicator dye. The estimates of the maximum rate of Ca2+ release at 5-6 degrees C ranged from 3 to 19 microM ms-1 in the 17 fibres examined. The mean value was 8.9 +/- 1.1 microM ms-1 (S.E.M.) The maximum rate of Ca2+ release was linearly related to the magnitude of the nonlinear membrane change moved during suprathreshold depolarizing steps. The voltage dependence of charge movement and the maximum rate of Ca2+ releases were nearly identical at 6 degrees C. The voltage-dependence of the delay between the test step and the onset of Ca2+ release could be adequately described by an equation having the same functional form as the voltage dependence of nonlinear charge movement. The relationship between the test pulse voltage and the delay was shifted to more negative voltages and to shorter delays as the temperature was raised from 6 degrees C to 15 degrees C. The inactivation of Ca2+ release was found to occur at more negative holding voltages and to be more steeply voltage dependent than the immobilization of nonlinear membrane charge movement. The above data are discussed using the 'hypothetical coupler' model of excitation-contraction coupling (Miledi et al., 1983b) applied to the specific case in which each mobile charge group controls the gating of one Ca2+ release site in the sarcoplasmic reticulum.

Actions of epimers of 12-(OH)-reduced saxitoxin and of 11-(OSO3)-saxitoxin on squid axon.

The actions of the 12 alpha-saxitoxinol, 12 beta-saxitoxinol and a C-12 ethylene thioketal derivative of saxitoxin, as well as those of 11 alpha-(OSO3)-saxitoxin, 11 beta-(OSO3)-saxitoxin and 11 alpha-(OH)-saxitoxin, have been examined on the isolated squid giant axon. Each of these analogues acted similarly to saxitoxin in blocking specifically the sodium channel. The relative potencies are: STX (1); 11 beta-(OSO3)-STX (gonyautoxin III) (0.42); 11 alpha-(OSO3)-STX (gonyautoxin II) (0.20); 11 alpha-(OH)-STX (0.10); 12 alpha-saxitoxinol (0.0021); 12 beta-saxitoxinol (0.0005). Thus, the presence of a bulky and negatively charged sulphate group on C-11 does not materially affect the biological activity of STX. Hydrogen bonding at the C-12 position is probably an important means of binding of STX to the membrane receptor site. The difference between the epimers of saxitoxinol suggests that the H in one of them may be geometrically better aligned than that in the other, with the hydrogen acceptor group in the receptor.

The active guanidinium group of saxitoxin and neosaxitoxin identified by the effects of pH on their activities on squid axon.

The relative potencies of saxitoxin at pH 7.25 and 8.25 have been determined on the squid giant axon under voltage-clamp conditions or by Vmax of the propagated action potential. Of the two guanidinium groups in saxitoxin, the 7, 8, 9 group has been identified as the biologically active group. The evidence lies in the demonstration of a quantitative agreement between the relative abundance of the protonated, positively charged form of that group at pH's 7.25 and 8.25 (ratio 1.80) with the relative potencies (ratio 1.79) of the toxin. The 1, 2, 3 group is excluded by the lack of agreement between the relative abundance of the protonated form (ratio 1.00) and the relative potencies at these pH's. The 1, 2, 3 group is further excluded by the observation that neosaxitoxin is equally potent at pH 6.50 and 7.25, in spite of a difference of 6-fold in the abundance of a deprotonated hydroxyl group on N-1 which should have influenced the potency.

Alteration of smooth muscle contractility after muscarinic agonist-induced K+ loss.

1. After stimulation of the longitudinal smooth muscle of the guinea-pig ileum by an optimal dose (2 x 10(-7) M) of a muscarinic agent, cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), the muscles failed to regain their normal spontaneous activity for 20 to 30 min. During the recovery period, subsequent contractions induced by either CD or 60 mM KCl were altered, particularly when only short times (15 min or less) were allowed between exposures. 2. Altered responses to CD had depressed phasic but increased tonic tensions and were characteristic of responses induced by lower doses of CD. The altered responsiveness probably represented an early phase of muscle 'densensitization'. 3. In contrast to muscarinic stimulation, the smooth muscles gave identical responses after repeated stimulation by 60 mM KCl, even when only 2 min were allowed between exposures. 4. Whereas K+ levels increased in muscles exposed to 60 mM KCl, they decreased during contractions to CD. The K+ levels remained low until the muscles recovered their normal responsiveness. 5. Increasing the extracellular K+ concentration (5 to 13 mM) hastened the recovery of the muscle responsiveness after CD, whereas lowering external K+ concentration to 1.35 mM or the addition of ouabain (5 x 10(-7) M) delayed the recovery. The results suggested that the Na+, K+-pump is rate-limiting in the recovery of the normal ionic balance of the muscles after stimulation by muscarinic agonists.

Identification of a vasoactive substance (vasopressin) in a brain extract containing an unknown inhibitor of Na,K-ATPase.

An endogenous inhibitor of the Na,K pump, postulated to be involved in the etiology of some hypertensive states, has been reported in extracts of mammalian brain. This encouraged us to test its effects on arterial muscles. An acid-acetone extract of guinea pig brain inhibited Na,K-ATPase derived from canine kidney and evoked responses in arterial strips similar to those produced by ouabain. Unlike ouabain, however, it did not prevent muscles in K-free solutions from relaxing when K was re-added. Bioassays on strips of arteries, uterus and portal vein indicated that the extract did not contain sufficient concentrations of norepinephrine, dopamine, serotonin, prostaglandins, angiotensin II, oxytocin or the Na,K-ATPase inhibitor to account for the observed vascular effects. This could not be said of vasopressin. Furthermore, vasopressin and the vasoactive component of the extract were equally sensitive to several peptidases, and conditions which cleave disulfide bridges. A radioimmunoassay verified that the extract contained sufficient vasopressin to cause contractions. Vasopressin did not inhibit the kidney Na,K-ATPase activity. Finally, the Na,K-ATPase inhibitor, but not the vasoactive substance, was present in extracts of vasopressin-deficient Brattleboro rat brains. Therefore, the Na,K-ATPase inhibitor and the vasoactive substance in these extracts were distinctly different.

Electron probe analysis of cultured vascular smooth muscle.

Mitochondrial and cytoplasmic composition were determined with electron probe analysis in freeze-dried guinea pig aortic smooth muscle cells cultured on stainless steel grids. The mitochondrial calcium content in normal cells was low: not significantly different from that detected in the cytoplasm. Mitochondrial calcium granules were found in less than 3% of the cells, and in these the cytoplasmic K/Na ratio was reduced, indicating that they were damaged. There were no major differences between the cytoplasmic concentrations of K, Cl, Ca, Mg, and S of cultured cells and those previously found in adult vascular smooth muscle (Somlyo et al '79). There was no evidence of nuclear Na or Ca sequestration in cultured cells, and the transmitochondrial Na, Cl, and K gradients were small. Attempts to selectively remove adhering, extracellular ions with a 2-second wash with isotonic ammonium acetate were unsuccessful because they were accompanied by loss of cell K.

Lysosomal composition in cultured vascular smooth muscle cells: electron probe analysis.

Spherical electron-dense organelles in the perinuclear region of cultured guinea pig aortic smooth muscle cells were identified as lysosomes by their ability to accumulate acridine orange and by cytochemical demonstration of their acid phosphatase content. The number and size of lysosomes increased in subcultured cells. The elemental composition of the lysosomes was quantitated by electron probe analysis of whole freeze-dried cells and of cryosections. In lysosomes at this stage in their development, the sulfur concentration was higher than that in the cytoplasm and the K/Na concentration ratio was similar to that in the cytoplasm.