Use of La3+ to distinguish activity of the plasmalemmal Ca2+ pump from Na+/Ca2+ exchange in arterial myocytes.

La3+ was tested for its ability to distinguish external Na+ (Nao)-independent Ca2+ efflux via the plasma membrane (PM) Ca2+ pump from Nao-dependent Ca2+ efflux via Na+/Ca2+ exchange. Fura-2 loaded cultured rat aortic myocytes were used with digital imaging to measure the cytosolic free Ca2+ concentration ([Ca2+]cyt) and to monitor La3+ entry. At a La3+ concentration ([La3+]o) of 0.25 mM, but not at lower concentrations, La3+ entered the cells; 0.01 mM verapamil blocked this entry. Transient increases in [Ca2+]cyt were evoked by unloading the sarcoplasmic reticulum with cyclopiazonic acid (CPA)+caffeine (CAF) in Na,Ca-free medium (to inhibit Ca2+ extrusion via Na+/Ca2+ exchange and Ca2+ influx). La3+ (0.03-0.25 mM with verapamil) augmented the Ca2+ transients and slowed Nao-independent [Ca2+]cyt recovery in a dose-dependent manner (IC50 approximately 0.01 mM La3+). This La(3+)-sensitive recovery was apparently mediated by the PM Ca2+ pump. The effects of La3+ were reversible: [Ca2+]cyt returned promptly toward base line when La3+ was washed out in Na,Ca-free medium containing CPA + CAF. Reintroduction of extracellular Na+ during [Ca2+]cyt recovery ([La3+]o = 0.06-0.25 mM) significantly speeded recovery, indicating that the Na+/Ca2+ exchanger was not inhibited by [La3+]o < or = 0.25 mM. The La(3+)-sensitive (Nao-independent) and Nao-dependent [Ca2+]cyt recovery rates were comparable. In Na(+)-loaded cells, < or = 0.25 mM La3+ also did not affect Na+/Ca2+ exchange mediated Ca2+ influx. In medium containing Na+ and Ca2+, 0.125 mM La3+ abolished the serotonin (5-HT) evoked plateau responses that resulted from Ca2+ entry via Ca2+ channels. In Na,Ca-free medium, but not Ca-free medium, however, La3+ converted 5-HT evoked Ca2+ transients into sustained responses. We conclude that low [La3+]o (0.06-0.25 mM) inhibits the PM Ca2+ pump, but spares Na+/Ca2+ exchanger mediated Ca2+ influx and efflux in arterial myocytes.

Blockade of ADP-induced Ca2+-signal and platelet aggregation by lipoxygenase inhibitors.

Stimulation of platelets results in the liberation of arachidonic acid (AA) which is further metabolized via the cyclooxygenase or lipoxygenase (LPG) pathway. We have examined the effect of inhibition of LPG on (i) the ADP-induced increase of cytoplasmic Ca2+ concentration and (ii) platelet aggregation. Lipoxygenase inhibitors, nordigidroguaiaretic acid (NDGA) and BW-755C, both suppressed ADP-induced Ca2+-signals and aggregation in a dose-dependent manner, with an IC50 value of 1 2 microM for NDGA. Qualitatively the same effect was obtained with 4-bromophenylacyl bromide, the inhibitor of phospholipases A2 and C. By contrast, cyclooxygenase inhibitor indomethacin had only a negligible effect on Ca2+-signals and suppressed only the second phase of ADP-induced aggregation. It is concluded that the LPG pathway of AA metabolism in platelets might play a crucial role in ADP-induced Ca2+-signal generation and platelet aggregation.

[Study of molecular mobility in biological membranes by 2-mm band ESR spectroscopy]. / Issledovanie molekuliarnoi podvizhnosti v biologicheskikh membranakh metodom spektroskopii EPR 2-mm diapazona.

Temperature relationship was measured of ESR spectra of spin probes--derivatives of fatty acids whose nitroxyl fragments are incorporated into surface and inner layers of phosphatidylcholine liposomes (T = 180-260 K), as well as of the probe in model ethanol solution (T = 110-220 K). Data on the nature and rotation frequency of probe nitroxyl fragments were obtained from the analysis of the spectra. It was shown that the frequency of the probes movement in the systems under study corresponds to the slow movement region, i.e. it is not above 10(7) s-1.

[Determination of the surface charge of lipoproteins and its changes during lipid peroxidation]. / Opredelenie poverkhnostnogo zariada lipoproteidov i ego izmeneniia pri perekisnom okislenii lipidov.

Surface potential of human plasma lipoproteins was studied by the use of positively charged spin probe. The calculated values of surface potential of high and low density lipoproteins appeared to be -29 +/- 1 and -16 +/- 1 respectively. It was shown that lipid peroxidation process induces an increase of surface potential of both high and low density lipoproteins. Probably, it is connected with the increase of the negative charge density on their surface. This fact can play an important role in pathogenesis of diseases with lipid metabolism and lipid peroxidation level disorders in plasma (atherosclerosis, ischemic heart disease etc.).

[Effect of verapamil on the functional status of thrombocytes in patients with ischemic heart disease]. / Vliianie verapamila na funktsional'noe sostoianie trombotsitov u bol'nykh ishemicheskoi bolezn'iu serdtsa.

A study of 20 anginal patients demonstrated signs of platelet hyperreactivity: increased platelet aggregation, increased mobilization of Ca2+ from platelet membranes, and increased production of malonic dialdehyde. A short-term treatment with isoptin-120 (360 mg daily) produced a considerable drop in those parameters. It is suggested that the drug's basic effect is due to the inhibition of intracellular calcium redistribution in the platelets of coronary patients.

[Effect of tetracaine on thrombocyte aggregation and mobilization of the intracellular calcium]. / Vliianie tetrakaina na agregatsiiu trombotsitov i mobilizatsiiu vnutrikletochnogo kal'tsiia.

Tetracaine (1 mM), a local anesthetics, lowers a degree of aggregation of human thrombocytes which is induced by thrombin (0.15 u/ml) and suppresses its appearance. Aggregation of thrombocytes induced by phorbol ester, TPA (10-8 M), an activator of protein kinase C, is inhibited completely by the mentioned doses of the anesthetics. In the presence of tetracaine the release of intracellular Ca is lower to some extent, but then it surpasses the control level. It is established that under the action of ionophore A23187 tetracaine exerts no effect on mobilization of intracellular Ca2+.

Dual inhibitory effects of dopamine on Na+ homeostasis in rat aorta smooth muscle cells.

Dopamine is an essential catecholamine, which acts not only as a neurotransmitter in sympathetic neurons but also exhibits vasodilating and natriuretic effects in renal tubular cells, blood vessels, etc. This study describes the effect of dopamine on Na+ influx and Na+ efflux and the resulting changes in intracellular Na+ concentration ([Na+]i). [Na+]i was measured in primary cultured vascular smooth muscle cells from rat aorta with digital imaging of cells loaded with the Na+-sensitive fluorescent indicator, SBFI. Na+ influx and Na+ efflux were measured as changes in [Na+]i under the conditions of inhibition of the Na+ flux in the opposite direction. Dopamine inhibited Na+ influx in a dose-dependent manner with a maximal inhibition, approximately 45%, achieved at 10(-4) M. This effect of dopamine, as suggested by several lines of evidence, was mediated by inhibition of Na+/H+ exchange. Besides inhibition of Na+ efflux, dopamine also, with a similar potency, inhibited Na+ efflux. The latter effect was due to inhibition of the Na+ pump-mediated component of Na+ efflux, since it was not observed when Na+ pump was inhibited. Inhibition of the Na+ pump by dopamine was due to the reduction in its maximal flux and not due to the decrease in the Na+ sensitivity of the pump. Similar to dopamine, activation of protein kinase A by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) caused inhibition of both Na+ influx and Na+ pump-mediated Na+ efflux. In contrast, activation of protein kinase C by the phorbol ester, phorbol 12,13-dibutyrate, caused activation of both Na+ influx and Na+ pump-mediated Na+ efflux. H-7, a nonspecific protein kinase inhibitor, abolished the inhibitory effects of either dopamine or 8-BrcAMP on Na+ efflux but did not affect the inhibitory effects of these compounds on Na+ influx. Dopamine either did not change [Na+]i or evoked a slight, 2-3 mM, increase in [Na+]i. Together, these results demonstrate that, in rat aortic smooth muscle cells, 1) dopamine inhibits Na+/H+ exchange-mediated Na+ influx, 2) dopamine inhibits Na+ pump-mediated Na+ efflux, 3) these effects of dopamine are mediated by an increase in cellular cAMP and, at least in the case of inhibition of the Na+ efflux, by the activation of protein kinase A, and 4) dopamine causes either small or no changes in [Na+]i, due to almost equal inhibition of Na+ influx and Na+ efflux.

cAMP evokes a rise in intracellular Na+ mediated by Na+ pump inhibition in rat aortic smooth muscle cells.

The effect of adenosine 3',5'-cyclic monophosphate (cAMP) on intracellular Na+ concentration ([Na+]i) was studied in primary cultured vascular smooth muscle cells from rat aorta. [Na+]i was measured using digital imaging of cells loaded with the Na(+-)sensitive fluorescent dye sodium-bonding benzofuran isophthalate. The cAMP level was raised by 1) the membrane-permeable cAMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate, 2) the combination of the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 3) the beta-adrenoceptor agonist isoproterenol. All three methods caused a dose-dependent continuous rise in [Na+]i during 40-60 min of observations. A rise in [Na+]i may be caused by stimulation of the Na+ influx and/or inhibition of Na+ efflux; therefore, the involvement of both mechanisms was studied. Elevation of the cAMP level had no effect on Na+ influx, measured as the rate of rise of [Na+]i when Na+ efflux was inhibited with 1 mM ouabain. In contrast, elevation of the cAMP level attenuated Na+ efflux, measured as the rate of decline of [Na+]i in Na(+)-loaded cells exposed to Na(+)-free medium. cAMP-induced inhibition of Na+ efflux was not observed when the Na+ pump was inhibited; therefore, cAMP inhibits the Na+ pump-mediated component of Na+ efflux. Agents that raise the cAMP level also inhibited, in a dose-dependent fashion, ouabain-sensitive 86Rb uptake in rat aortic rings. The latter observation confirms that the cAMP-induced inhibition of the Na+ pump occurs both in cultured cells and in the native tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular free Na+ in resting and activated A7r5 vascular smooth muscle cells.

Regulation of intracellular Na+ ([Na+]i) in cultured vascular smooth muscle cells (A7r5 line) was studied with Na(+)-sensitive fluorescent dye sodium-binding benzofuran isophthalate. Digital imaging microscopy was used to study single-cell fluorescence. Na+ was distributed uniformly in cytoplasm and nucleus; mean Na+ concentration in resting cells was 4.4 +/- 0.3 mM in cytoplasmic areas ([Na+]cyt) and 4.5 +/- 0.4 mM in nuclear areas ([Na+]n). Na+ pump inhibition and cell activation evoked uniform changes in [Na+]cyt and [Na+]n. Inhibition of Na+ pump with 1 mM ouabain or K(+)-free medium caused a rise in [Na+]cyt; in the latter case, [Na+]cyt fell rapidly when external K+ was later restored. Exposure to Ca(2+)-free medium also caused [Na+]cyt to rise; this effect was augmented by Na+ pump inhibition and was reversed by 10(-5) M verapamil or nitrendipine or by restoration of external Ca2+. The implication is that this Na+ entry in absence of external Ca2+ is mediated by Ca2+ channels. Activation by 10(-9) M arginine vasopressin (AVP) and 10(-6) M serotonin (5-HT) caused [Na+]cyt to increase, but response to 5-HT was small (0.6 mM on average) and transient, whereas response to AVP was larger (2.4 mM on average) and was maintained as long as AVP was present (to 20 min). AVP and, to a much smaller extent, 5-HT stimulated Na+ influx; this could be detected when Na+ pump was inhibited by ouabain. Both AVP and 5-HT activated the Na+ pump, as detected by ouabain-sensitive decrease in [Na+]cyt when Na+ influx was inhibited. Agonist-evoked increases in [Na+]cyt were dependent on a rise in cytosolic Ca2+ concentration ([Ca2+]cyt); these [Na+]cyt responses were abolished by prolonged exposure to Ca(2+)-free media, when cytoplasmic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or when Ca2+ mobilization was blocked with thapsigargin. Raising [Ca2+]cyt with 40 mM K+ or with thapsigargin did not increases in [Na+]cyt. We conclude that 1) AVP- and 5-HT-evoked increases in [Na+]cyt are agonist specific and depend on the balance between stimulated Na+ influx and efflux; 2) AVP and 5-HT activate the Na+ pump; this is, at least in part, independent of agonist-induced rise in [Na+]cyt; and 3) a rise in [Ca2+]cyt is necessary but not sufficient to trigger agonist-evoked rise in [Na+]i.

Functionally and spatially distinct Ca2+ stores are revealed in cultured vascular smooth muscle cells.

Sarcoplasmic reticulum Ca2+ in vascular smooth muscle may be separated into at least two types of Ca2+ stores, one releasable by inositol 1,4,5-trisphosphate and the other releasable by caffeine and ryanodine. We employed digital imaging with fura-2 to study the effects of thapsigargin (which blocks Ca2+ sequestration in the inositol trisphosphate-sensitive store) and caffeine on the cytosolic free Ca2+ concentration ([Ca2+]cyt) in cultured arterial myocytes. These agents increased [Ca2+]cyt by depleting different Ca2+ stores in the absence of extracellular Ca2+. Moreover, Ca2+ could be transferred between the two stores, as prior application of caffeine, which alone evoked little or no rise in [Ca2+]cyt, significantly augmented the response to thapsigargin. Conversely, a substantial caffeine-induced rise in [Ca2+]cyt was observed only after the ability of the thapsigargin-sensitive Ca2+ store to sequester Ca2+ was inhibited. This suggests that the caffeine-sensitive store may have a thapsigargin-insensitive Ca(2+)-sequestration mechanism. To complement these fura-2 experiments, chlortetracycline was used to visualize the Ca2+ stores directly. The latter studies revealed spatial differences in the location of the thapsigargin-sensitive and caffeine-sensitive Ca2+ stores (measured as thapsigargin-sensitive and caffeine-sensitive chlortetracycline fluorescence). Thus, these two types of stores appear to be both functionally and spatially distinct.

Increased intracellular Na+ augments mobilization of Ca2+ from SR in vascular smooth muscle cells.

The effect of a rise in intracellular Na+ concentration ([Na+]cyt) on the amount of Ca2+ in intracellular stores was studied in vascular smooth muscle cells from the A7r5 line. The relative amount of stored Ca2+ was estimated in fura 2-loaded cells by the rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) evoked by Ca2+ release from the sarcoplasmic reticulum (SR). To improve the detection of released Ca2+, extrusion of Ca2+ from the cytosol was minimized by using nominally Na+/Ca(2+)-free medium containing 0.5 mM La3+ [for vasoconstrictor experiments, the medium contained 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and no La3+]. Ca2+ release was triggered by thapsigargin (TG), an SR Ca(2+)-ATPase inhibitor, and by the vasoconstrictors arginine vasopressin (AVP) and serotonin (5-HT). Incubation with 1-3 mM ouabain for 20 min, which raises [Na+]cyt from 4.4 to 9.0 mM, increased "resting" [Ca2+]cyt only slightly (from 87 to 122 nM). However, ouabain greatly augmented the release of Ca2+ evoked by TG [from 639 nM (control) to 1,021 nM], by AVP (from 993 to 1,597 nM), and by 5-HT (from 559 to 1,486 nM). Ouabain-induced augmentation of TG-evoked Ca2+ release was not affected by 10 microM verapamil; this implies that the effect of ouabain was not due to Ca2+ entry through voltage-gated Ca2+ channels. The response to TG was not augmented when ouabain was applied for 20 min in Na(+)-free medium (Na+ replaced by equimolar N-methyl-D-glucamine) to prevent [Na+]cyt from rising.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of adenylate cyclase system activation on Na+/H+-exchange in human platelets]. / Vliianie aktivatsii adenilattsiklaznoi sistemy na Na+/H+-obmen v trombotsitakh cheloveka.

In experiments with human platelets it has been shown, that stimulation of adenylate cyclase by carbacycline (CC)--a stable analog of prostacyclin, does not affect the initial pHi decrease caused by thrombin and PAF, but it abolishes the second phase of pHi changes, a pHi increase resulted from Na+/H+ exchange activation. CC also abolishes pHi increase induced by ionophore A23187 and the activator of protein kinase C, phorbol ester (TPA). The results obtained suggest that cAMP exerts inhibitory action on the agonist induced activation of Na+/H+ exchange but does not affect its pHi-sensitivity in the resting cell.

[Effects of phospholipase A2 and alpha-tocopherol on the activity of adenylate cyclase of rat brain synaptosomes]. / Vliianie fosfolipazy A2 i alfa-tokoferola na aktivnost' adenilattsiklazy sinaptosom mozga krysy.

The action of phospholipase A2 and alpha-tocopherol on adenylate cyclase system functioning and on the lipid bilayer microviscosity of the rat brain synaptosome membranes was investigated. It was shown that the exposure of the synaptosomes to phospholipase A2 increases the adenylate cyclase activity stimulated by guanylyl imidotriphosphate (GITP), decreases the adenylate cyclase activity stimulated both by isoproterenol and by isoproterenol with GITP. The preincubation of synaptosomes in medium containing alpha-tocopherol does not change the character of the phospholipase action on the adenylate cyclase activity stimulated by isoproterenol but normalizes the adenylate cyclase activity stimulated both by GITP and by GITP with isoproterenol. In the last case the normalizing action of alpha-tocopherol is not caused by alteration of the microviscosity of the lipid bilayer. It appears to be due to the modification of the lipid-protein interactions of annular lipids with activated complex of catalytic subunit and guanyl nucleotide-binding protein.

[Changes in the surface potential of plasma lipoproteins in ischemic heart disease. Studied with spin probes]. / Izmenenie potentsiala poverkhnosti lipoproteidov plazmy pri ishemicheskoi bolezni serdtsa. Issledovaniia s pomoshch'iu spinovykh zondov.

Changes in lipoprotein surface potentials were studied by a positively charged analog as a spin probe. Low density lipoproteins (LDL) and high density lipoproteins (subfractions HDL2 and HDL3) of patients with coronary heart disease (CHD) were studied. CHD patients have revealed a significant decrease (by 14.4 +/- 0.3 mV) in LDL and an increase (by 6.3 +/- 2.0 mV) in HDL3 negative surface potential, as compared to the control. The increase in HDL2 surface potential in CHD patients was insignificant (1.9 +/- +/- 2.5 mV). The possible role of LDL and HDL3 surface potential changes in the mechanism of interaction of these types of lipoproteins with vascular wall and blood cellular membranes and in pathogenesis of CHD and atherosclerosis is discussed.

[Changes in the surface charge of plasma lipoproteins during their auto-oxidation]. / Izmenenie poverkhnostnogo zariada lipoproteidov plazmy pri ikh autookislenii.

Low density lipoproteins (LDL) and high density lipoproteins (HDL) surface potential (charge) changes were studied upon autooxidation, using positively charged spin probes. Lipid peroxidation product accumulation in LDL and HDL suspensions was found to be accompanied by a significant reduction in their surface area associated with a decreased negative surface charge, and probably, deposition of lipid peroxidation polar products and/or surface charge redistribution as a result of lipoprotein autooxidative modification.

Alterations of surface charges of plasma lipoproteins in ischemic heart disease.

For a charged and an uncharged long chain spin probe the partition between the aqueous phase and the lipoproteins LDL, HDL2 and HDL3 was measured by use of ESR spectroscopy. The partition coefficients were compared for lipoproteins from normal donors and lipoproteins from patients with ischemic heart disease. The partition coefficients of the uncharged spin probe are not different. However, the charged spin probe has a significantly different partition for LDL and HDL3. This difference results from changes in the surface charge. Patients with ischemic heart disease have LDL which is more electropositively charged and HDL3 is more electronegatively charged compared to the corresponding lipoproteins of normal subjects. The surface charge of HDL2 is not changed. The results are discussed in the light of current concepts of the pathogenesis of atherosclerosis.

[Relation between the changes in Na+/H+-exchange and cytoplasmic Ca levels in platelet activation]. / Vzaimosviaz' mezhdu izmeneniiami Na+/H+-obmena i kontsentratsiei tsitoplazmaticheskogo Ca pri aktivatsii trombotsitov.

In experiments on human and rat platelets the changes in cytoplasmic pH (pHi) and Ca2+ concentration (Ca2+) have been studied by the use of fluorescent probes BCECF and quin-2, respectively. Inhibition of Na+/H+ exchange resulted in removal of external Na+ (equimolar substitution by cholin) induced a considerable reduction of Ca2+-signal caused by 10 mMPAF, and a slight decrease in Ca2+-signal elicited by 0.1 mu/ml thrombin. In the control Na+ and Ca2+ containing medium both PAF and thrombin induced first a decrease then an increase of pHi above its original level. The latter phase being much more pronounced in the case of thrombin action. Removal of Ca2+ from the external solution suppressed pHi increase and correspondingly it enhanced initial decrease. Addition of Ni2+ also suppressed stimulus-induced pHi increase. A treatment of platelets by Ca-ionophore A23187 caused a rise of pHi without its initial decrease; in medium without Ca2+ the changes of pHi were inhibited. The results obtained suggest that in platelets there exist a mutual interdependence between Ca2+ influx and change in pHi: Ca2+ influx enhanced the activation of Na+/H+ exchange by agonist; in turn Na+/H+ exchange activation enhances the stimulus-induced Ca2+ influx.

[Na+/H+ metabolic activity in the thrombocytes of spontaneously hypertensive rats]. / Aktivnost' Na+/H+-obmena v trombotsitakh spontanno gipertenzivnykh krys.

The sodium-proton exchange was determined in platelets of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The platelets were suspended in sodium propionate; the cytoplasmic acidification activated the exchanger and intracellular pH (the increasing) and volume of the platelets (the swelling) were registered. The activity of Na+/H+ exchange was inhibited by isopropyl amiloride. The platelets' volume and the exchange rate constant of SHR were increased on 30-40% as compared with those of WKY.