Vanadate-induced inhibition of renin secretion is unrelated to inhibition Na,K-ATPase activity

There is evidence that three inhibitors of Na,K-ATPase activity--ouabain, K-free extracellular fluid, and vanadate--inhibit renin secretion by increasing Ca2+ concentration in juxtaglomerular cells, but in the case of vanadate, it is uncertain whether the increase in Ca2+ is due to a decrease in Ca2+ efflux (inhibition of Ca-ATPase activity, or inhibition of Na,K-ATPase activity, followed by an increase in intracellular Na+ and a decrease in Na-Ca exchange) or to an increase in Ca2+ influx through potential operated Ca channels (inhibition of electrogenic Na,K transport, followed by membrane depolarization and activation of Ca channels). In the present experiments, the rat renal cortical slice preparation was used to compare and contrast the effects of ouabain, of K-free fluid, and of vanadate on renin secretion, in the absence and presence of methoxyverapamil, a Ca channel blocker. Basal renin secretory rate averaged 7.7 +/- 0.3 GU/g/60 min, and secretory rate was reduced to nearly zero by 1 mM ouabain, by K-free fluid, by 0.5 mM vanadate, and by K-depolarization (increasing extracellular K+ to 60 mM). Although 0.5 microM methoxyverapamil completely blocked the inhibitory effect of K-depolarization, it failed to antagonize the inhibitory effects of ouabain, of K-free fluid, and of vanadate. A concentration of methoxyverapamil two hundred times higher (100 microM) completely blocked the inhibitory effects of vanadate, but still failed to antagonize the effects of ouabain and of K-free fluid. Collectively, these observations demonstrate that vanadate-induced inhibition of renin secretion cannot be attributed entirely to Na,K-ATPase inhibition, since in the presence of methoxyverapamil, the effect of vanadate differed from the effects of either ouabain (a specific Na,K-ATPase inhibitor) or K-free fluid. Moreover, it cannot be attributed entirely to a depolarization-induced influx of Ca2+ through potential-operated Ca channels, since methoxyverapamil antagonized K-depolarization-induced inhibition of renin secretion much more effectively than it antagonized vanadate-induced inhibition.     

Vanadium-induced Cl(-)-secretion in rabbit descending colon is mediated by prostaglandins.

Vanadium in the 4+ (vanadyl-ion) and 5+ (vanadate-ion) oxidation state stimulates furosemide-sensitive electrogenic Cl- secretion in isolated epithelia of rabbit descending colon. This effect is associated with an increased release of prostaglandin E2 from the tissue. Inhibitors of phospholipase A2 or cyclooxygenase abolish both vanadium-induced release of prostaglandin E2 and Cl- secretion. Neuronal mechanisms are not likely to be involved, as tetrodotoxin does not affect the vanadate induced Cl- secretion. Although vanadate is known to inhibit Na+,K(+)-ATPase activity, no inhibition of active Na+ transport was observed in intact colonic epithelia suggesting a rapid intracellular reduction of vanadate ions to vanadyl ions which have no inhibitory effect on the Na+,K(+)-ATPase. The present findings therefore indicate that vanadate stimulated colonic Cl- secretion involves intracellular conversion of vanadate to vanadyl and release of prostaglandin E2.     

Cyclosporine A inhibits prostaglandin E2 release, and has no effect on renin secretion, from rat renal cortical slices

These experiments were designed to test the hypothesis that cyclosporine A (CSA) inhibits renin secretion and stimulates renal prostaglandin E2 (PGE2) release in vitro. In rat renal cortical slices incubated at 37 degrees C in a buffered and oxygenated physiological saline solution containing 4 mM KCl, CSA concentrations ranging from 1 to 30 microM had no significant effect on renin secretion. Furthermore, partial depolarization of the cells, produced by increasing extracellular KCl concentration to 20 mM, failed to reveal any latent inhibitory or stimulatory effects of CSA on renin secretion. On the other hand, PGE2 release was significantly inhibited by CSA over the same range of concentrations. This inhibitory effect might be explained by the previous findings of others, that CSA inhibits phospholipase A2 activity, thereby decreasing arachidonic acid production, the rate-limiting step in PG synthesis. In conclusion, CSA inhibits PGE2 release but fails to affect renin secretion in vitro. These results suggest that the occasional effects of CSA on renin secretion in intact animals must be attributable to indirect and/or chronic effects.     

Isoproterenol-stimulated renin secretion in the rat: second messenger roles of Ca and cyclic AMP

These experiments were designed to elucidate which of two second messengers (cyclic 3',5' adenosine monophosphate [c-AMP]; intracellular calcium [Cai]) was more closely related to the renin secretory process. The rat renal cortical slice preparation was used. Agents which previously were shown to inhibit basal renin secretion by increasing Cai (ouabain, vanadate, angiotensin II, antidiuretic hormone, and 60 mM K) antagonized and/or blocked isoproterenol-stimulated secretion, which is thought to be mediated by adenylate cyclase activation and increased levels of c-AMP. The stimulatory effect of dibutyryl c-AMP was antagonized and/or blocked by the same agents which antagonized and/or blocked isoproterenol-stimulated secretion. Thus, the inhibitory effects of these agents on isoproterenol-stimulated secretion cannot be explained by a Ca-induced decrease in c-AMP production. Secretory rate was stimulated by a potent phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine). A combination of this and dibutyryl c-AMP produced even greater stimulation. Ouabain blocked the stimulatory effect of this combination. These results are not consistent with an invariant direct relationship between c-AMP and renin secretory rate, but are consistent with an inverse relationship between Ca; and renin secretion. Further, they are consistent with the hypothesis that in isoproterenol-stimulated renin secretion. c-AMP is the second and Cai the third or the final messenger.     

Comparison of the effects of calcium alpha-glycerophosphate and Ca2+ on prostaglandin synthesis in rabbit kidney medulla slices

The stimulatory effects of calcium alpha-glycerophosphate (CaG) and Ca2+ on the generation of medullary prostaglandin E2 have been compared. CaG (4mM) was significantly more effective than Ca2+ (4 mM) as a stimulus for prostaglandin E2 release. This effect of CaG was most pronounced in the presence of exogenous phospholipase A2 (0.1 unit/ml). These results suggest that CaG has a more powerful stimulatory effect on prostaglandin E2 formation than Ca2+ because of its high affinity for phospholipase A2.     

Synergistic Effect of Prostaglandin E2 and Ouabain on Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells

We recently reported that prostaglandin E2 (PGE2) stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain, an inhibitor of Na+,K+-ATPase, synergistically induced a gradual secretion of catecholamines from the cells. The effect on catecholamine release was specific for prostaglandin E1 (PGE1) and PGE2 among prostaglandins tested (E1 = E2 greater than F2 alpha greater than D2). The release evoked by PGE2 plus ouabain was greatly reduced in Na+-depleted medium and not observed in Ca2+-free medium. Here we examined the synergistic effect of PGE2 and ouabain on the release with specific reference to ion fluxes. Regardless of the presence of PGE2, ouabain stimulated the release in a dose-dependent manner with half-maximal stimulation at 1 microM, and omission of K+ from the medium, a condition which suppresses the Na+,K+-ATPase activity, also enhanced the release from chromaffin cells exposed to PGE2. Ouabain induced a continuous accumulation of 22Na+ and 45Ca2+, as well as secretion of catecholamines. Although PGE2 itself showed hardly any effects on these cellular responses, PGE2 potentiated all of them induced by ouabain. The time course of catecholamine release was correlated with that of accumulation of 45Ca2+ rather than with that of 22Na+. The release evoked by PGE2 and ouabain was inhibited in a dose-dependent manner by amiloride and the analogue ethylisopropylamiloride, inhibitors of the Na+,H+-antiport, but not by the Na+-channel inhibitor tetrodotoxin nor by the nicotinic receptor antagonist hexamethonium. Ethylisopropylamiloride at 1 microM inhibited PGE2-enhanced accumulation of 22Na+ and 45Ca2+ and release of catecholamine by 40, 83, and 71%, respectively. Activation of the Na+,H+-antiport by elevation of the extracellular pH from 6.6 to 8.0 increased the release of catecholamines linearly. Furthermore, PGE2 induced a sustained increase in intracellular pH by about 0.1 pH unit above the resting value, which was abolished by amiloride or in Na+-free medium. These results taken together indicate that PGE2 activates the Na+,H+-antiport by stimulating phosphoinositide metabolism and that the increase in intracellular Na+ by both inhibition of Na+,K+-ATPase and activation of Na+,H+-antiport may lead to the redistribution of Ca2+, which is the initial trigger of catecholamine release.     

Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices.

We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices. Exogenous phospholipase A2 [from Naja naja (Indian cobra) venom] and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner. At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2. Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4). Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2. These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.     

Role of Ca2+ and cyclic nucleotides in the control of prostaglandin E production in the rat anterior pituitary gland.

In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.     

Interleukin-1 beta- and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2.

Interleukin-1 beta and forskolin induce prostaglandin E2 release as well as 14-kDa group II phospholipase A2 gene expression and secretion of the enzyme from rat glomerular mesangial cells. We now report that pretreatment of mesangial cells with transforming growth factor-beta 2 prior to stimulation with interleukin-1 beta or forskolin inhibits the induced release of prostaglandin E2. At the same time the secretion of group II phospholipase A2, measured both as enzyme activity with sn-2-labeled phosphatidylethanolamine as substrate and as enzyme protein in immunoblot experiments, is dose-dependently inhibited by pretreatment of the cells with transforming growth factor-beta 2. Analyses of enzyme activity and enzyme protein levels in the cells indicated that this is not due to inhibition of enzyme secretion with a concomitant increase in cellular levels of the enzyme. Rather, pretreatment of the cells with transforming growth factor-beta 2 largely prevented both the interleukin-1 beta- and the forskolin-induced synthesis of phospholipase A2. This is the first report indicating an inhibition of group II phospholipase A2 gene expression by transforming growth factor-beta 2. In line with those results, transforming growth factor-beta 2 did not induce the synthesis and secretion of group II phospholipase A2. However, under conditions where the interleukin-1 beta-induced expression of group II phospholipase A2 is fully suppressed by transforming growth factor-beta 2, the growth factor itself stimulated prostaglandin E2 synthesis by a mechanism apparently not involving group II phospholipase A2. The immunochemical identification of the inducible and secretable phospholipase A2 from rat mesangial cells as a group II enzyme was confirmed by purification and N-terminal amino acid sequence determination.     

Influence of macrophage products on the release of plasminogen activator, collagenase, beta-glucuronidase and prostaglandin E2 by articular chondrocytes.

We describe the effects of products of mononuclear phagocytes on the secretory activity of chondrocytes. The primary confluent cultures of rabbit articular chondrocytes were exposed to standard medium alone or enriched with conditioned medium obtained from cultures of rabbit peritoneal macrophages, the mouse macrophage cell line P388D1 or human blood mononuclear cells. Four markers of release were assessed, the neutral proteinases plasminogen activator and collagenase, the acid hydrolase beta-glucuronidase and prostaglandin E2, and the kinetics of their changes were monitored. Chondrocytes that were cultured in standard medium secreted large amounts of plasminogen activator, some beta-glucuronidase, but no collagenase, and released only minor amounts of prostaglandin E2. The addition of conditioned medium from rabbit macrophages induced a rapid release of large quantities of prostaglandin E2 and an abundant secretion of collagenase, while abolishing or strongly decreasing plasminogen activator secretion. In addition, beta-glucuronidase secretion was markedly enhanced. The decrease in secretion of plasminogen activator appeared to reflect a diminished production, since no evidence was found for the generation of inhibitors or for an accelerated extracellular breakdown of the enzyme. Conditioned media of the mouse and human mononuclear cells influenced the secretory activities of rabbit articular chondrocytes in a similar way, suggesting that the factor (or factors) acting on chondrocytes is produced by a variety of macrophages, and that its action is not species-restricted. The time course and concentration-dependence of the effects observed indicate that the secretion of plasminogen activator and collagenase are influenced in a strictly reciprocal fashion by the macrophage products. The release of prostaglandin E2 paralleled that of collagenase.     

Studies on ouabain-complexed (Na+ +K+)-ATPase carried out with vanadate

Vanadate is able to promote the binding of ouabain to (Na+ +K+)-ATPase and it is shown that vanadate is trapped in the enzyme-ouabain complex. Also ouabain-bound enzyme, the formation of which was facilitated by (Mg2+ +Na+ +ATP) or (Mg2+ +Pi), is accessible to vanadate when washed free of competing ligands used for the promotion of ouabain binding. For vanadate binding to (Na+ +K+)-ATPase and to enzyme-ouabain complexes a divalent cation (Mg2+ or Mn2+) is indispensable, indicating that the cation does not remain attached to the ouabain-bound enzyme. K+ further increases vanadate binding in the absence of ouabain, but seems to have no additional role in case of vanadate binding to enzyme-ouabain complexes. Mn2+ is more efficient than Mg2+ in promoting binding of vanadate and ouabain to (Na+ +K+)-ATPase. That K+ in combination with Mn2+, in analogy with the effect in combination with Mg2+, increases the equilibrium binding level of vanadate and decreases that of ouabain does not seem to favour the hypothesis of selection of a special E2-subconformation by Mn2+. The vanadate-trapped enzyme-ouabain complex was examined for simultaneous nucleotide binding which could demonstrate a two-substrate mechanism per functional unit of the enzyme. The acceleration by (Na+ +ATP) of ouabain release from the (Mg2+ +Pi)-facilitated enzyme-ouabain complex does not, as anticipated, support such a mechanism. On the other hand, the deceleration of vanadate release as well as of ouabain release from a (Mg2+ +vanadate)-promoted complex could be consistent with a two-substrate mechanism working out-of-phase.     

Interleukin-1 beta and transforming growth factor-beta 2 enhance cytosolic high-molecular-mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells.

Interleukin-1 beta induces gene expression and secretion of group-II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin-1 beta-induced synthesis of group-II phospholipase A2 is prevented by transforming growth factor-beta 2, whereas transforming growth factor-beta 2 potentiated the interleukin-1 beta-evoked prostaglandin E2 production. Transforming growth factor-beta 2 itself did not induce synthesis of group-II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin-1 beta and transforming growth factor-beta 2 on a cytosolic phospholipase A2 activity and prostaglandin E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono-Q anion-exchange column and a Superose 12 gel-filtration column, the cytosolic phospholipase A2 activity was assigned to a high-molecular-mass phospholipase A2. Measured with 1-stearoyl-2-[1-14C]arachidonoylglycero-phosphocholine as substrate, both interleukin-1 beta and transforming growth factor-beta 2 enhanced the high-molecular-mass phospholipase A2 activity. The stimulation of rat mesangial cells with interleukin-1 beta and transforming growth factor-beta 2 was time- and dose-dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10 ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin-1 beta and transforming growth factor-beta 2 enhanced the cytosolic phospholipase A2 activity 2.2 +/- 0.6-fold and 2.5 +/- 0.6-fold, respectively. These results strongly suggest that an enhanced cytosolic high-molecular-mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor-beta 2. Whether interleukin-1 beta induced group-II phospholipase A2 and/or interleukin-1 beta-enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2.

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI2 by cortical microsomes. In the present studies PGI2 was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins in vivo. The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10(7 M to 10(-5) M. At similar concentrations 6-keto-prostaglandin F1alpha was not active on these slices. Thus, it is proposed that PGI2 exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

Inhibition of in vitro renin release by low extracellular K

Renin release from rat renal cortical slices was measured as a function of the concentration of K in the incubation medium. Release was completely abolished within a 30-minute period of exposure to nominally K-free or to 1.0 mM K media. Release tended to increase with increasing K, over the range 2 – 6 mM, although release was near maximal at 2.0 mM K. Omission of Ca from the incubation medium prevented the full inhibitory effect of nominally K-free medium. It is suggested that Ca accumulation, perhaps via Na-Ca exchange, is required to produce the inhibitory effect of low K medium on renin release in vitro.     

Effects of medium calcium, and agents affecting cytoskeletal function, on cellular volume and morphology in liver tissue in vitro

The possible role of an exocytotic, vesicular mechanism in cellular volume regulation under iso-osmotic conditions has been studied in slices of rat liver. The effects of incubation conditions and agents affecting the actin cytoskeleton were examined for changes of water, ionic composition, and ultrastructure. Slices were pre-incubated at 1°C in an iso-osmotic buffered medium to induce swelling. Upon restoration to 37°C in the same medium, tissue lost water. The Na+-K+ adenosine triphosphatase (ATPase) inhibitor ouabain inhibited water extrusion of about 50%, an effect that was accompanied by the formation of characteristic vesicles in the cytoplasmic region between the Golgi apparatus and the bile canaliculi. Water extrusion in the presence of ouabain was partially inhibited by trifluoroperazine and completely inhibited when the medium was free of Ca2+. In the presence of ouabain, brefeldin A caused a small reduction of water extrusion, whereas phalloidin and cytochalasins A, D, or E caused a marked inhibition. In these conditions there was a marked increase in size and number of cytoplasmic vesicles and a more widespread distribution of them within the cells, lacking the more specific orientation to the Golgi and canalicular regions that was seen in the presence of ouabain alone. Water extrusion was inhibited by phalloidin and cytochalasins in the absence of ouabain. In conclusion, our results are consistent with the hypothesis that iso-osmotic expulsion of water from hepatocytes can proceed partly through an accumulation of water in cytoplasmic vesicles, followed by exocytosis. This mechanism does not depend on Na+-K+ ATPase activity.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Stimulation by melittin of adrenocorticotropin and beta-endorphin release from rat adenohypophysis in vitro

The effect of melittin on the release of adrenocorticotropin (ACTH) and beta-endorphin from the corticotropic cells of the rat adenohypophysis was examined in vitro. Anterior pituitary quarters were perifused or incubated in vitro and ACTH- (ACTH-IR) or beta-endorphin-like immunoreactivity (beta-End-IR) in the medium was measured by radioimmunoassays. Melittin stimulated ACTH-IR and beta-End-IR release. This effect was rapid in onset, reversible, and concentration-related (50-5000 ng/ml) and depended on the presence of calcium ions in the incubation medium. Melittin also elevated the tissue content of unesterified 3H-arachidonic acid that had previously been incorporated into lipids. Purported phospholipase A2 inhibitors, mepacrine (up to 1 mM), dexamethasone (0.5 mg/kg in vivo, 50 nM in vitro), or p-bromophenacylbromide (100 microM), did not decrease the melittin (500 ng/ml) - induced beta-End-IR release, although mepacrine and dexamethasone may have inhibited phospholipase A2 activity as indicated by an inhibition of melittin-evoked prostaglandin E2 formation. After stimulation by melittin (500 ng/ml), beta-End-IR release was not affected by the cyclooxygenase inhibitor indomethacin (up to 140 microM), whereas nordihydroguaiaretic acid (100 microM), a lipoxygenase inhibitor, or BW755C (250 microM), an inhibitor of both cyclooxygenase and lipoxygenase, abolished melittin-induced hormone secretion. We conclude that melittin generates a signal in the corticotropic cells of the rat adenohypophysis which induces hormone secretion by exocytosis. This signal may be unrelated to the activation by melittin of phospholipase A2.     

Effect of diarachidonin on prostaglandin E2 synthesis in rabbit kidney medulla slices.

The effect of diarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. The addition of diarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. At three concentrations (10, 50 and 100 microM), increases in prostaglandin E2 formation induced by exogenous diarachidonin were 2-fold greater than those induced by exogenous arachidonic acid. Diacylglycerol or phosphatidic acid from egg lecithin had little or no effect on prostaglandin E2 production. Moreover, EGTA failed to inhibit diarachidonin-stimulated prostaglandin E2 formation, indicating that the stimulatory effect of diarachidonin is not mediated through the activation of endogenous phospholipase A2 (including phosphatidic acid-specific phospholipase A2). These results are discussed in the light of our former hypothesis that arachidonic acid release from kidney medulla phospholipids might occur through the sequential action of a phospholipase C coupled to diacylglycerol and monoacylglycerol lipases [Fujimoto, Akamatsu, Hattori & Fujita (1984) Biochem. J. 218, 69-74].     

Cytokine- and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.     

Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus.

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.