Oxidant-induced pHi/Ca2+ changes in rat aortic smooth muscle cells. The role of atrial natriuretic peptide.

The aim of this study was to investigate the effects of oxidative stress on PLD activity, [Ca2+]i and pHi levels and the possible relationship among them. Moreover, since atrial natriuretic peptide (ANP) protects against oxidant-induced injury, we investigated the potential protective role of the hormone in rat aortic smooth muscle (RASM) cells exposed to oxidative stress. Water-soluble 2,2'-Azobis (2-amidinopropane) dihydrochloride (AAPH) was used as free radical generating system, since it generates peroxyl radicals with defined reaction and the half time of peroxyl radicals is longer than other ROS. A significant increase of PLD activity was related to a significant decrease in pHi, while [Ca2+]i levels showed an increase followed by a decrease after cell exposure to AAPH. [Ca2+]i changes and pHi fall induced by AAPH were prevented by cadmium which inhibits a plasma membrane Ca2+ ATPase coupled to Ca2+/H+ exchanger, that operates the efflux of Ca2+ coupled to H+ influx. The involvement of PLD in pHi and [Ca2+]i changes was confirmed by calphostin-c treatment, a potent inhibitor of PLD, which abolished all AAPH-induced effects. Pretreatment of RASM cells with pharmacological concentrations of ANP attenuated the AAPH effects on PLD activity as well as [Ca2+]i and pHi changes, while no effects were observed with physiological ANP concentrations, suggesting a possible role of the hormone as defensive effector against early events of the oxidative stress.

Involvement of phospholipids in the mechanism of insulin action in HEPG2 cells.

The mechanism of action by which insulin increases phosphatidic acid (PA) and diacylglycerol (DAG) levels was investigated in cultured hepatoma cells (HEPG2). Insulin stimulated phosphatidylcholine (PC) and phosphatidyl-inositol (PI) degradation through the activation of specific phospholipases C (PLC). The DAG increase appears to be biphasic. The early DAG production seems to be due to PI breakdown, probably through phosphatidyl-inositol-3-kinase (PI3K) involvement, whereas the delayed DAG increase is derived directly from the PC-PLC activity. The absence of phospholipase D (PLD) involvement was confirmed by the lack of PC-derived phosphatidylethanol production. Experiments performed in the presence of R59022, an inhibitor of DAG-kinase, indicated that PA release is the result of the DAG-kinase activity on the DAG produced in the early phase of insulin action.

Differential sensitivity of human monocytes and macrophages to ANP: a role of intracellular pH on reactive oxygen species production through the phospholipase involvement.

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present work was to study the effect of ANP on the intracellular pH (pHi) of human monocytes and macrophages and to investigate whether pHi changes could play a role on phospholipase activities and reactive oxygen species (ROS) production. Human macrophages isolated by peripheral blood mononuclear cells and THP-1 monocytes, which were shown to express all three natriuretic peptide receptors (NPR-A, NPR-B, and NPR-C), were treated with physiological concentrations of ANP. A significant decrease of pHi was observed in ANP-treated macrophages with respect to untreated cells; this effect was paralleled by enhanced phospholipase activity and ROS production. Moreover, all assessed ANP effects seem to be mediated by the NPR-C. In contrast, no significant effect on pHi was observed in THP-1 monocytes treated with ANP. Treatment of macrophages or THP-1 monocytes with 5-(N-ethyl-N-isopropyl)amiloride, a specific Na(+)/H(+) antiport inhibitor, decreases pHi in macrophages and monocytes. Our results indicate that only macrophages respond to ANP in terms of pHi and ROS production, through diacylglycerol and phosphatidic acid involvement, pointing to ANP as a new modulator of ROS production in macrophages.

Atrial natriuretic factor inhibits mitogen-induced growth in aortic smooth muscle cells.

Atrial natriuretic factor (ANF) is a polypeptide able to affect cardiovascular homeostasis exhibiting diuretic, natriuretic, and vasorelaxant activities. ANF shows antimitogenic effects in different cell types acting through R(2) receptor. Excessive proliferation of smooth muscle cells is a common phenomenon in diseases such as atherosclerosis, but the role of growth factors in the mechanism which modulate this process has yet to be clarified. The potential antimitogenic role of ANF on the cell growth induced by growth factors appears very intriguing. Aim of the present study was to investigate the possible involvement of ANF on rat aortic smooth muscle (RASM) cells proliferation induced by known mitogens and the mechanism involved. Our data show that ANF, at physiological concentration range, inhibits RASM cell proliferation induced by known mitogens such as PDGF and insulin, and the effect seems to be elicited through the modulation of phosphatidic acid (PA) production and MAP kinases involvement.

PLA(2) stimulation of Na(+)/H(+) antiport and proliferation in rat aortic smooth muscle cells.

The proliferative properties and the ability to stimulate the Na(+)/H(+) antiport activity of a secretory phospholipase A(2) were studied in rat aortic smooth muscle cells in culture. The requirement of the enzymatic activity of phospholipase A(2) to elicit mitogenesis was assessed by the use of ammodytin L, a Ser(49) phospholipase A(2) from the venom of Vipera ammodytes, devoid of hydrolytic activity. We propose that the proliferative effect is mediated by the same transduction pathway for both proteins. In particular, 1) both secretory phospholipase A(2) and ammodytin L stimulated thymidine incorporation in a dose-dependent manner; 2) both proteins affected the cell cycle, as assessed by cell growth and fluorescence-activated cell sorting experiments; 3) both phospholipase A(2) and ammodytin L increased intracellular pH, a permissive factor for cell proliferation, through activation of the Na(+)/H(+) antiport; 4) ammodytin L was able to displace the (125)I-labeled phospholipase A(2) from specific binding sites in a concentration range consistent with that capable of eliciting a cellular response; and 5) the inhibition by heparin was similar for both proteins, taking into account the ratio of heparin to protein. In conclusion, the enzymatic activity of phospholipase A(2) is not required for the stimulation of mitogenesis. The inhibitory effect of heparin combined with its therapeutic potential could help to clarify the role of phospholipase A(2) in the pathogenesis of several preinflammatory situations.

Short-term effects of thyroid hormones on the Na/H antiport in L-6 myoblasts: high molecular specificity for 3,3',5-triiodo-L-thyronine.

The thyroid hormones L-T3 and L-T4 were shown to activate the Na/H antiport in L-6 cells from rat skeletal muscle by a rapid, nongenomic mechanism. Under pH equilibrium conditions, a significant rise in the intracellular pH, measured by the fluorescent pH indicator 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein was observed after the addition of physiological concentrations (10(-10) M) of either L-T3 or L-T4, but with different time courses. L-T3 at all concentrations increased the pH after a delay of 2 min, whereas L-T4 showed a concentration-dependent lag time, going from 11 min at 10(-11) M down to 5 min for a hormone concentration of 10(-6) M. The effect of L-T4 was blocked in the presence of the 5'-deiodinase inhibitor 6-n-propyl-2-thiouracil, suggesting that the difference in lag time between L-T3 and L-T4 was due to the 5'-deiodination process that transforms L-T4 into the bioactive L-T3. In short term studies (<5 min), a high molecular specificity for L-T3 was found, as L-T4, rT3, the D-isomer of T3, and the deaminated analogues were ineffective at physiological concentrations. In analogy with the results found at equilibrium, intracellular pH recovery from an acid load and set-point were increased after 2 min for L-T3 (10(-9) M) and after 10 min for L-T4 (10(-9) M). The effect of the hormones on the intracellular pH was completely blocked by the specific antiport inhibitor 5-(ethyl-N-isopropyl)amiloride. These findings suggest that thyroid hormones may play an active role in the recovery from muscular acidosis through direct stimulation of the Na/H antiport.

Dual modulation of Na/H antiport by atrial natriuretic factor in rat aortic smooth muscle cells.

The aim of the present work was to study the effect of the atrial natriuretic factor (ANF) on the Na/H antiport in rat aorta smooth muscle cells, evaluated as intracellular pH (pHi) recovery after an acid load with ammonium chloride. The Na/H antiport was studied using a fluorescent probe, sensitive to pHi, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Our data indicate that ANF modulates the activity of the Na/H antiport in both a dose- and time-dependent manner. Hormone concentrations of 10(-10) M activate the antiport, increasing both the rate of recovery and the set point by approximately 0.2 pH units. This effect is mediated by diacylglycerol as a result of phospholipid hydrolysis by a phospholipase C, even if an involvement of adenosine 3',5'-cyclic monophosphate (cAMP) cannot be ruled out. ANF (10(-7) M) inhibits the antiport, decreasing both the rate of recovery and the set point by approximately 0.3 pH units, because of guanosine 3',5'-cyclic monophosphate production. Both inhibition and stimulation of pHi by ANF were more pronounced when the hormone was given before the acid load, perhaps because of the longer time exposure. We present new hypotheses on the mechanism of action of this paracrine/autocrine factor.

Insulin stimulation of Na/H antiport in L-6 cells: a different mechanism in myoblasts and myotubes.

Insulin modulation of the Na/H antiport of L-6 cells, from rat skeletal muscle was studied in both myoblasts and myotubes using the fluorescent, pH sensitive, intracellular probe 2',7' bis (carboxyethyl)-5(6)-carboxyfluorescein. Insulin stimulated the Na/H antiport activity in L-6 cells, showing a bell-shaped dose response typical of other insulin responses: a maximum at 10 nM (delta pH of 0.132 +/- 0.007 and 0.160 +/- 0.040 over basal value, for myoblasts and myotubes, respectively; means +/- SD, n = 6-8) and smaller effects at higher and lower concentrations. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated the antiport in myoblasts but not in myotubes. Surprisingly the rapid increase in intracellular pH was not observed when insulin and PMA were added simultaneously to myoblasts; apparently these two activators mutually excluded each other. Downregulation of protein kinase C, obtained by preincubation of cells with PMA for 20 hr, totally abolished both hormone and PMA effects in myoblasts, whereas in myotubes insulin stimulation was not affected. Inhibitors of tyrosine kinase activity, such as erbstatin analog and genistein abolished insulin effect on the Na/H antiport, both in myoblasts and in myotubes. Different sensitivity to pertussis toxin in the two cell types suggests that the differentiation process leads to a change in the signal pathways involved in the physiological response to insulin.

Phosphatidylinositol- and phosphatidylcholine-dependent phospholipases C are involved in the mechanism of action of atrial natriuretic factor in cultured rat aortic smooth muscle cells.

We have investigated the involvement of specific phospholipase systems and their possible mutual relationship with the mechanism by which atrial natriuretic factor (ANF) increases phosphatidate (PA) and diacylglycerol (DAG) in rat aortic smooth muscle cells (RASMC), one of the major targets of this hormone. Our results indicate that ANF initially stimulates a phosphatidylinositol-dependent phospholipase C (PI-PLC) with a significant increase of DAG, enriched in arachidonate, and inositol trisphosphate (IP3) and then a phosphatidylcholine-dependent phospholipase C (PC-PLC) with formation of DAG, enriched in myristate, and phosphocholine (Pcho). Moreover, ANF stimulates PA formation at an intermediate stage between early and late DAG formation. The transphosphatidylation reaction, as well as its labeling ratio, demonstrate that phosphatidylcholine-dependent phospholipase D (PC-PLD) is not involved. Our experiments with R59022, a DAG kinase (DAGK) inhibitor, indicate that such an increase may be due to the phosphorylation of DAG derived from phosphatidylinositol (PI) hydrolysis. Our results show that phorbol 12-myristate 13 acetate (PMA) plays a significant role in late DAG formation and that Pcho is released concomitantly, suggesting there is a relationship between the two phospholipase Cs (PLCs) that occurs through a protein kinase C (PKC) translocation from cytosol to the plasma membrane. These findings are confirmed by the use of PKC inhibitors calphostin, H7, and staurosporine. The involvement of membrane phospholipid hydrolysis and the ensuing production of second messengers might explain the vasorelaxant effect of ANF.

Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture.

Ammodytin L, purified from the venom of Vipera ammodytes, triggers a rapid and dramatic lytic process in myotubes in vitro, as well as in differentiated muscle cells in vivo, through a mechanism that is not well understood. Despite its great sequence similarity to phospholipase A2, it is devoid of any enzyme activity. Data on artificial membranes demonstrating a direct interaction between this toxin and the hydrophobic core of the lipid bilayer suggest that the toxin also acts on the lipid microenvironment in cell membranes. Recent experiments on living cells do not confirm this hypothesis, and a more intricate mechanism is proposed. In vitro, ammodytin L has necrotic effects only in well-differentiated myogenic cells, whereas other cell types such as platelets, red blood cells and lymphocytes show neither morphological nor functional alterations. In this work we demonstrate that rat 208F fibroblasts in culture after ammodytin L challenge increase [3H]thymidine incorporation, indicating that this toxin has a myogenic effect. Moreover, ammodytin L increases intracellular Ca2+ by acting on intracellular stores probably by activating a phosphatidylinositol-specific phospholipase C. Preincubation of the cells with ammodytin L did not prevent the massive Ca2+ release evoked by bradykinin, a phenomenon observed when fibroblasts were incubated with both thapsigargin and ionomycin. Heparin, an agent that inhibits the necrotic effect of the myotoxin in myotubes, also reduces the effect of ammodytin L on DNA synthesis. Heparin inhibits only the late sustained increase in intracellular Ca2+ induced by the toxin.

Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

Effect of ammodytin L from Vipera ammodytes on L-6 cells from rat skeletal muscle.

Ammodytin L (AMDL) is a myotoxic phospholipase-like protein from the venom of Vipera ammodytes with a serine in position 49 instead of an aspartate, therefore this toxin is devoid of phospholipase activity, and the membrane-damaging effect does not involve any step of phospholipase activity. The aim of the present study was to analyze the effect of AMDL on L-6 cells from rat skeletal muscle to investigate its mechanism of action and the role of calcium ions in its muscle-damaging activity. Our data indicate that the effect of ammodytin L is strongly dependent on the degree of cell differentiation. Low doses of myotoxin gave rise to a marked release of creatine kinase in myotubes differentiated from L-6 myoblasts and the presence of calcium ions plays a role in the cytotoxic effect. The presence of EGTA in the incubation buffer reduced by 50% the release of creatine kinase. No membrane damage was observed in myoblasts, but there was a significant increase of intracellular calcium concentration measured with Fura-2. A non-specific membrane effect of AMDL was ruled out using platelets as reference cells: no platelet aggregation pattern and no increase in intracellular calcium were observed.

Selective activation by atrial natriuretic factor of phosphatidylcholine-specific phospholipase activities in purified heart muscle plasma membranes.

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10(-11) M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10(-10) M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10(-10) M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTP-gamma-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.

Modulation of the Na-H antiport by insulin: interplay between protein kinase C, tyrosine kinase, and protein phosphatases.

The insulin modulation of Na-H antiport in rat hepatocytes was studied using the fluorescent, pH-sensitive intracellular probe, 2',7' bis (carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Our data show that insulin stimulates the Na-H antiport. The dose-response of insulin effect shows a behavior typical of other insulin responses: a maximum in the physiological range (1 nM) and smaller effects at higher and lower hormone concentrations. The time-course of activation is very fast at high hormone concentrations and slow, but reaching a higher value, for the physiological concentrations (0.26 +/- 0.05 and 0.18 +/- 0.022 pH units for 1 nM and 1 microM insulin respectively). The use of phorbol, 12-myristate, 13-acetate (PMA), a potent activator of protein kinase C and its inhibitor staurosporine, and the inhibitor of tyrosine kinase erbstatin analog, suggests that both protein kinase C and tyrosine kinase could be involved in the mechanism leading to Na-H antiport activation by insulin. We suggest that the activation of the antiport involves the two pathways depending on the hormone concentration. In particular, protein kinase C would mediate the effects of high hormone concentrations, acting as a growth factor, since staurosporine fully inhibited insulin 1 microM, but only partially 1 nM effects, and tyrosine kinase would mediate the effect of insulin 1 nM and only partially 1 microM. Okadaic acid 1 microM, a potent inhibitor of protein phosphatases, mimicked the hormone effects on the antiport and abolished the different time-course due to hormone concentration, suggesting a role of kinases and phosphatases in the signal transduction. The effect of all activators was abolished by amiloride analog, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), confirming the specificity of these effects.

Insulin modulation of Na/H antiport in rat red blood cells.

Red blood cells have been shown to possess specific insulin receptors, with characteristics similar to the receptors of typical target cells. The present work was carried out to study the modulation of amiloride sensitive Na/H antiport by insulin on rat red blood cells. The activity of the Na/H antiport was determined by a new technique which involves the measurement of the fluorescence of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) loaded rbc's as function of their intracellular pH (pHi). Our results show that the antiport in red blood cells displays the same behaviour as in other cells and it is inhibited by amiloride. Insulin stimulates the antiport with a dose-dependence similar to other typical insulin effects: a maximum at 10 nM and a smaller effect at higher and lower hormone concentrations. Insulin effect on the antiport was completely abolished by amiloride (0.1 mM) and significantly inhibited by ouabain (1 mM) showing, also in red blood cells, the strict dependence of the Na/H antiport on the functioning of the Na pump.

Muscarinic stimulation of SK-N-BE(2) human neuroblastoma cells elicits phosphoinositide and phosphatidylcholine hydrolysis: relationship to diacylglycerol and phosphatidic acid accumulation.

Muscarinic stimulation of the human neuroblastoma cell line SK-N-BE(2) elicits hydrolysis of phosphoinositides and phosphatidylcholine (PtdCho) and produces a rapid and sustained elevation of diacylglycerol (DG) mass. PtdIns(4,5)P2 cleavage by phospholipase C (PLC) occurred immediately after carbachol (CCh) addition, and phosphoinositide hydrolysis was then sustained for at least 5 min. Cell stimulation, after extensive PtdCho labelling by long-term [3H]choline administration, resulted in an enhanced release of [3H]phosphocholine (PCho) into the external medium; enhanced [3H]PCho release, which occurred with a 15 s delay with respect to CCh addition, was particularly pronounced within the first minute of stimulation and proved to be caused by PtdCho-specific PLC activation. In fact, when cells were exposed to [3H]choline for a short period, to extensively label the intracellular PCho pool but not PtdCho, stimulation did not result in an enhanced release of [3H]PCho into the medium. PtdCho-specific phospholipase D (PLD) activation was documented by the accumulation of [3H]phosphatidylethanol in cells prelabelled with [3H]myristic acid and stimulated in the presence of 1% (v/v) ethanol; this metabolic pathway, however, proved to be a minor one leading to generation of phosphatidic acid (PtdOH) during cell stimulation, whereas DG production by the sequential action of PtdCho-specific PLD and PtdOH phosphohydrolase was not observed. Studies on cells which were double-labelled with [3H]myristic acid and [14C]arachidonic acid indicated that within 15 s of stimulation DG is uniquely derived from PtdIns(4,5)P2, whereas PtdCho is the major source at later times. Evidence is provided that rapid and selective conversion of phosphoinositide-derived DG into PtdOH may play an important role in determining the temporal accumulation profile of DG from the above-mentioned sources.

Calcium ion independent membrane leakage induced by phospholipase-like myotoxins.

The two snake venom myotoxins ammodytin L and myotoxin II, purified respectively from Vipera ammodytes ammodytes and Bothrops asper, have phospholipase-like structures but lack an Asp-49 in the active site and are without normal phospholipase activity. The interaction of these proteins with different types of liposomes indicated that the myotoxins were able to provoke rapid and extensive release of the aqueous content of liposomes. Leakage was measured by two different methods: fluorescence dequenching of liposome-entrapped carboxyfluorescein and ESR measurement of intravesicular TEM-POcholine reduction by external ascorbate. The process was independent of Ca2+ and took place without any detectable phospholipid hydrolysis. Nonmyotoxic phospholipases tested under the same conditions were unable to induce liposome leakage, which could be detected only when Ca2+ was added to the medium and with the concomitant hydrolysis of phospholipids. The kinetics of Ca(2+)-dependent and Ca(2+)-independent leakage were completely different, indicating two different mechanisms of interaction with the lipid bilayer. Studies using diphenylhexatriene as a probe of lipid membrane organization indicated that the myotoxins gave rise to a profound perturbation of the arrangement of the lipid chains in the membrane interior, whereas interaction of Naja naja phospholipase A2 with the membrane surface did not affect lipid organization. On the basis of these results we suggest that a new type of cytolytic reaction mechanism is responsible for the effects of phospholipase-like myotoxins in vivo.

Insulin effect on isolated rat hepatocytes: diacylglycerol-phosphatidic acid interrelationship.

It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC). This consideration prompted us to investigate the insulin effect on the mechanism leading to the accumulation of diacylglycerol (DAG) and phosphatidic acid (PA) in rat hepatocytes. Basically, insulin induces: (i) a significant increase of both [3H]glycerol and fatty acid labelling of DAG; (ii) a significant increase of PA labelling preceding DAG labelling and paralleled by a decrease of phosphatidylcholine (PC) labelling. These observations, which suggest an insulin-dependent involvement of a phospholipase D, are strengthened by the increase of PC-derived phosphatidylethanol in presence of ethanol. Finally, the observation that the PA levels do not return to basal suggests that other mechanisms different from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated.