Prostaglandin E2 production by renal inner medullary tissue slices: Effect of metabolic inhibitors

Increasing oxygen from 5 to 95% has previously been shown to increase prostaglandin (PG) production in renal inner medullary slices. The possible role of oxidative phosphorylation in this process was investigated. The oxidative phosphorylation inhibitors, dinitrophenol (DNP), oligomycin, and cyanide were evaluated for their effects on PGE₂ production and ATP levels. None of the inhibitors affected PGE₂ synthesis, although they lowered ATP levels at the concentrations tested. In contrast, incubation of inner medullary tissue slices with 0% oxygen resulted in decreases both in PGE₂ and ATP levels. This suggest that the effect of oxygen on prostaglandin synthesis may be due to substrate limiting effects rather an effect on oxidative phosphorylation.When 22 mM 2-deoxyglucose was added to the incubation medium or when glucose was ommitted, PGE₂ levels increased. Sodium fluoride, presumably acting as a glycolytic inhibitor, increased PGE₂ levels, with a maximal effect at 10mM. ATP levels were 37% of control values with 20 mM NaF. This indicates that glucose may inhibit prostaglandin synthesis.These results indicate that oxygen (substrate) availability can limit inner medullary PGE₂. In view of the low pO₂ in the inner medulla, especially during antidiuresis, oxygen can potentially regulate prostaglandin productin in this tissue.     

Effects of vasopressin and prostaglandin synthesis inhibition on cyclic AMP accumulation in the dog renal inner medulla

The interaction of arginine vasopressin (AVP) and endogenous prostaglandins on cAMP production was investigated in the dog. Cyclic AMP content of dog inner medullary tissue slices exposed to different concentrations of AVP in the presence and absence of various prostaglandin synthesis inhibitors was determined. If the slices were incubated in isotonic media with 95% O2 and 5% CO2 gas phase, inhibition of prostaglandin synthesis decreased cAMP accumulation. A significant correlation was found between the decrements in cAMP content and basal cAMP levels. AVP-induced increments in cAMP accumulation was, however, unaffected by prostaglandin synthesis inhibitors. If incubation was performed in a hypertonic medium and at low O2 concentration, basal cAMP content was significantly reduced and it was not altered by inhibition of prostaglandin synthesis. The cAMP response to AVP was practically identical in the presence and absence of prostaglandin synthesis inhibitors. In conscious dogs AVP and indomethacin in itself had no effect on urinary cAMP excretion, but there was a significant decrease if the two compounds were combined. These results fail to lend support the hypothesis that endogenous prostaglandins modulate AVP-induced cAMP accumulation in the inner medulla.     

Effect of acetaminophen on prostaglandin E2 and prostaglandin F2α synthesis in the renal inner medulla of rat

Effects of acetaminophen on the renal inner medullary production of prostaglandin E₂ and F_2α were compared with the well-known effects of aspirin on this process. Acetaminophen was found to elicit a dose-dependent inhibition of both prostaglandin E₂ and F_2α accumulation in media with a K_i of 100–200 μM. This inhibition could not be accounted for by increased accumulation of prostaglandins within slices. Acetaminophen inhibition was reversed by removal of acetaminophen during the incubation or by addition of arachidonic acid. Similar manipulations did not reverse aspirin or indomethacin-mediated inhibition of prostaglandin synthesis. Thin-layer and gas chromatographic analysis of acetaminophen following incubation with slices demonstrated that this material was identical to authentic acetaminophen. This, in addition to the lack of an effect of glutathione on inhibition, suggests that acetaminophen does not have to be metabolized to exert this inhibition. Arachidonic acid did not alter the metabolism or increase the efflux of acetaminophen. Lower levels of prostaglandin E₂ observed with 5 mM acetaminophen and 1 mM aspirin caused a corresponding decrease in cyclic AMP content. Removal of acetaminophen from the second incubation or addition of arachidonic acid caused increases in both prostaglandin E₂ and cyclic AMP. Aspirin inhibition of cyclic AMP content was not reversed by similar manipulations. In vivo inhibition of inner medullary prostaglandin E₂ and prostaglandin F_2α synthesis was observed 2 h after a 375 mg/kg, intraperitoneal injection of acetaminophen. These data suggest that acetaminophen, like aspirin, is capable of reducing tissue prostaglandin synthesis. However, the mechanisms by which these two analgesic and antipyretic agents elicit their inhibition of prostaglandin synthesis are quite different.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Influence of mitochondrial inhibitors on the respiration and energy-dependent uptake of iodide by thyroid slices

The influence of mitochondrial inhibitors, including oligomycin, antimycin and rotenone, on the iodide and oxygen uptake and the nucleotide content of incubated sheep thyroid slices was investigated. Each inhibitor strongly suppressed both iodide and oxygen uptake, and decreased the nucleoside triphosphate content of the slices. In most cases the addition of glucose or mitochondrial substrates restored iodide uptake in inhibitor-treated slices. Inhibitor concentrations sufficient to inhibit iodide uptake strongly had only slight effects on the thyroidal Na(+)+K(+)-activated adenosine triphosphatase. It is concluded that the inhibitors produce their effects by the inhibition in vivo of mitochondrial oxidative phosphorylation. ATP synthesis appears to be essential for iodide uptake to occur, and the high-energy intermediates (or energized state) of oxidative phosphorylation cannot be used to energize the uptake process. To a limited extent glycolytic ATP synthesis can support iodide uptake, which is therefore not exclusively dependent on aerobic metabolism. The mechanism of energy-linked iodide uptake is discussed.     

Influence of substrate activation (hydrolysis of ATP by first steps of glycolysis and beta-oxidation) on the effect of enzyme deficiencies,inhibitors, substrate shortage and energy demand on oxidative phosphorylation

In intact tissues respiratory substrates (glucose, fatty acids) must be activated with the use of ATP before they may be oxidised and used for energy (ATP) production. This activation by product constitutes an example of a typical positive feedback. In the present paper, the influence of substrate activation on the effect of inborn enzyme deficiencies, inhibitors, lowered oxygen tension, respiratory fuel shortage and increased energy demand on respiration and ATP synthesis is studied with the aid of the dynamic computer model of oxidative phosphorylation in isolated mitochondria developed previously. Computer simulations demonstrate that, in the case where oxidative phosphorylation in the whole organism is partially inhibited, the necessity of substrate activation can have significant impact on the relationship between the activity of (particular steps of) oxidative phosphorylation (or the value of energy demand) and the respiration rate. Depending on the sensitivity of ATP usage to ATP concentration, substrate activation may either slightly enhance the effect of the decrease in the oxidative phosphorylation activity (increase in energy demand) or may lead to a non-stability and sudden collapse of the respiration rate and phosphorylation potential below (above) a certain threshold value of oxidative phosphorylation activity (energy demand). This theoretical finding suggests a possible causal relationship between the affinity of ATP usage to [ATP] and the tissue specificity of mitochondrial diseases.     

Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E1-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since from a series of trialkyltin chlorides, tri-n-propyltin, tri-n-butyltin and tri-n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri-n-octyltin affect neither of them; other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri-n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na⁺ and K⁺ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions.2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin.3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value.4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis.5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Glucose Metabolism Assessed with 2-Deoxyglucose and the Effect of Glutamate in Subdivisions of Rat Hippocampal Slices

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver. Inhibition of oxidative phosphorylation as the primary action.

1. In slices of rat liver, oligomycin inhibited the net transport of Na+ and K+ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions. 2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin. 3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value. 4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycinresistant ATP synthesis. 5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Osteoclast precursors display dynamic metabolic shifts toward accelerated glucose metabolism at an early stage of RANKL-stimulated osteoclast differentiation.

Mature osteoclasts have an increased citric acid cycle and mitochondrial respiration to generate high ATP production and ultimately lead to bone resorption. However, changes in metabolic pathways during osteoclast differentiation have not been fully illustrated. We report that glycolysis and oxidative phosphorylation characterized by glucose and oxygen consumption as well as lactate production were increased during receptor activator of nuclear factor-kappaB ligand (RANKL)-induced osteoclastogenesis from RAW264.7 and bone marrow-derived macrophage cells. Cell proliferation and differentiation varied according to glucose concentrations (0 to 100 mM). Maximal cell growth occurred at 20 mM glucose concentration and differentiation occurred at 5 mM concentration. Despite the similar growth rates exhibited when cultured cells were exposed to either 5 mM or 40 mM glucose, their differentiation was markedly decreased in high glucose concentrations. This finding suggests the possibility that osteoclastogenesis could be regulated by changes in metabolic substrate concentrations. To further address the effect of metabolic shift on osteoclastogenesis, we exposed cultured cells to pyruvate, which is capable of promoting mitochondrial respiration. Treatment of pyruvate synergistically increased osteoclastogenesis through the activation of RANKL-stimulated signals (ERK and JNK). We also found that osteoclastogenesis was retarded by blocking ATP production with either the inhibitors of mitochondrial complexes, such as rotenone and antimycin A, or the inhibitor of ATP synthase, oligomycin. Taken together, these results indicate that glucose metabolism during osteoclast differentiation is accelerated and that a metabolic shift towards mitochondrial respiration allows high ATP production and induces enhanced osteoclast differentiation.     

Effects of various inhibitors of oxidative phosphorylation on energy metabolism, macromolecular synthesis and cyclic AMP production in isolated rat thymocytes. A regulating role for the cellular energy state in macromolecular synthesis and cyclic AMP production

Inhibitors of oxidative phosphorylation such as several triorganotin compounds, oligomycin, 2,4-dinitrophenol and carbonylcyanide p-trifluoromethoxyphenylhydrazone suppress energy metabolism of isolated rat thymocytes as indicated by a reduction of ATP levels, an increase in glucose consumption and by a marked accumulation of lactate. Also these compounds effectively inhibit the incorporation of DNA, RNA and protein precursors into acid-precipitable material of thymocytes. Moreover, the prostaglandin E₁-induced elevation of cAMP is markedly reduced by these inhibitors. A correlation is observed between the effects on energy metabolism, macromolecular synthesis and cAMP production, since (i) from a series of trialkyltin chlorides, tri-n-propyltin, tri-n-butyltin and tri-n-hexyltin are very effective inhibitors of these functions, while trimethyltin and tri-n-octyltin affect neither of them; (ii) other inhibitors of oxidative phosphorylation, each of them with quite different mechanisms of action, also inhibit macromolecular synthesis and cAMP production. The finding that a rise in intracellular ATP concentrations leads to a reversion of the tri-n-butyltin-induced inhibition of cAMP production and uridine incorporation, indicates a regulating role for the cellular energy state in these aspects of cellular function.     

Effect of oxygen and solute on PGE and PGF production by rat kidney slices

Increasing oxygen from 5% to 95% resulted in an increased production of both PGE's and PGF's. The release of prostaglandins from slices of rat kidney cortex and outer and inner medulla was measured. Prostaglandin production was observed predominantly in the inner medulla, was close to the lower limit of detection in the outer medulla, and was undetectable in the cortex. Increasing oxygen concentration resulted in a threefold increase in inner medullary prostaglandin production. Synthesis at 95% O₂ was less at 2100 mOsm than at 300 mOsm, while synthesis at 5% O₂ was not affected by high solute concentration. The implications of these results with respect to kidney function are discussed.     

Effects of prostaglandins on rat renal adenylate cyclase-cyclic AMP systems.

The effects of prostaglandin (PG) E1, E2, A1, F1alpha, F2alpha or D2 on the rat renal cortical, outer medullary and inner medullary adenylate cyclase-cyclic AMP systems were examined. While high concentrations (8X10-4M) of each prostaglandin stimulated adenylate cyclase activity in each area of the kidney, PGE1 was the only prostaglandin to stimulate at 10-7M. PGA's were the only prostaglandins tested besides PGE's which stimulated adenylate cyclase at less than 10-4M. This effect of PGA's was limited to the outer medulla. PGD2 was the least stimulatory. Observations with renal slices yielded qualitatively similar results. The PGE's were the most potent in each area with PGA's only stimulatory in the outer medulla. O2 deprivation (5% O2) lowered the slice cyclic AMP content in each area of the kidney. In the cortex and outer medulla, prostaglandin mediated increases in cyclic AMP content were either lower or absent at 5% O2 compared to 95% O2. However, in the inner medulla PGE stimulation was observed only at 5% O2 and not 95% O2. No other prostaglandins were found to increase inner medullary cyclic AMP content at 95% or 5% O2. These results illustrate that the adenylate cyclase-cyclic AMP system responds uniquely to prostaglandins in each area of the kidney. Consideration of these results along with correlative observations suggests that inner medullary produced PGE's may act as local modulators of inner medullary adenylate cyclase.     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles

It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Effect of protein synthesis inhibitors on leukocytic pyrogen-induced in vitro hypothalamic prostaglandin production

In order to study the antipyretic effect of inhibitors of protein synthesis, hypothalamic tissue was incubated in vitro under controlled conditions and the amount of prostaglandin E2 (PGE2) measured in the supernatant medium. Rabbit anterior hypothalamic tissue was incubated with purified human leukocytic pyrogen (LP) and after 60 minutes the supernatant fluid was assayed for PGE2 by radioimmunoassay. Control tissue incubated with Eagle's medium (MEM) released elevated levels of PGE2; however, the addition of polymyxin B (PmxB), a cationic antibiotic which blocks the activities of bacterial endotoxins, significantly reduced PGE2. In addition, endotoxin added to MEM induced from the brain tissue PGE2 production which could be reduced by the addition of PmxB. Thus, commercial culture media such as MEM may contain sufficient amounts of endotoxin to stimulate brain PGE2 production in vitro. Purified human LP incubated with hypothalamic tissue in the presence of PmxB induced PGE2 production in a dose-dependent fashion. This release could be reduced (p less than 0.001) by the presence of either cycloheximide or puromycin during incubation with LP. The addition of these inhibitors to unstimulated hypothalamic tissue incubations did not reduce background levels of PGE2. It is concluded that the antipyretic effect of protein synthesis inhibitors results in a specific decrease in LP-induced levels of PGE2.     

Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C.

Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.     

Characterization of the PGI2/IP system in cultured rat mesangial cells

Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E(2) and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E(2) receptor subtype EP(2) was not detected, EP(1,3,4) mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E(2) had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI(2) synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI(2)/IP system in cultured rat mesangial cells.     

Regulation of prostaglandin production by nitric oxide in rat smooth muscle myometrial cells.

Smooth muscle myometrial cells isolated by an enzymatic method from estrogenized rats were used after 7-10 days of culture. They were incubated for 24 h with two distinct competitive nitric oxide (NO) inhibitors: NG-monomethyl-L-arginine (L-NMMA: 300 microM) and L-nitro-arginine methylester (L-NAME: 600 microM, 5 mM and 10 mM). Afterwards, the supernatants were separated in order to measure nitrite production and prostaglandin PGE synthesis. In the present report, we demonstrate that myometrial cells from estrogenized rats are able to produce NO, since all the inhibitors significantly decrease the production of nitrites in the culture media. Furthermore, we report that both inhibitors inhibited PGE synthesis by myometrial cells. We also used a donor of NO in the incubation medium for 24 h, sodium nitroprusside (NP), obtaining an strong (P< 0.001) increase in both nitrite and PGE production. We conclude that myometrial cells can produce NO and that one possible role of the NO synthetized by this cells may be the modulation of PGE production.