High glucose concentrations stimulate renal papillary phosphatidylcholine biosynthesis.

The effects of a high glucose concentration (HGC) on renal phosphatidylcholine (PtdCho) biosynthesis were studied. In control rats, HGC increased papillary PtdCho biosynthesis. In chronic diabetic rats, an increase above that induced by diabetes was observed. Such glucose-responsive phospholipid pools were shown to be transient in adult control rats, while in acute diabetic and aged control and chronic diabetic rats they seem to be of slow breakdown or permanent. Deoxyglucose evokes the HGC effect only in the presence of 5 mM glucose. Neomycin, which blocks phospholipase C action, corrected the HGC effect in control and chronic diabetic rats, but not the increase due to diabetes. CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity was increased by both in vivo and simulated diabetes. Therefore, transient high extracellular glucose levels promote a reversible increase in papillary (32)P-PtdCho, while diabetes causes an irreversible increase resulting in PtdCho accumulation, possibly related to papillary necrosis.

Serotonin hypersensitivity in aorta of two kidney-two clip hypertensive rats: calcium contribution and prostanoids-nitric oxide interactions.

Previous studies from our own laboratory have shown that abdominal aorta rings from two kidney - two clip hypertensive rats (HT) develop hypersensitivity to serotonin (SER) which is related to a decreased nitric oxide (NO) availability and enhanced thromboxane A2 production. In the present study we investigated whether calcium and prostanoid-NO interactions are involved in these findings. To this purpose, the aortic responses to SER were analyzed in calcium-free medium and in calcium-depleted aorta placed in normal medium. Moreover, effects of ridogrel (RID, an antagonist of TxA 2/PGH2 receptors and inhibitor of thromboxane synthetase) were analysed by cumulative dose-response curves to SER in the presence and in the absence of the NO synthase inhibitor N(omega)-nitro-L-arginine (NOLA). Vascular responses to SER in vessels from HT rats were associated with increased intracellular calcium mobilization. In addition, hypersensitivity to SER in HT group respect to sham group (SH) disappeared in the presence of RID, NOLA and RID plus NOLA. RID decreases the maximum tension to SER and this effect was prevented by NOLA. This inhibition was of a greater magnitude in rings from sham rats (SH): 34 +/- 6% than in HT rats: 15 +/- 6% (p < 0.05). Besides, RID decreased the sensibility to SER in the presence of NOLA only in the HT group. In conclusion, the present study suggests that SER hypersensitivity observed in HT rats is related to a facilitated intracellular calcium mobilization and enhanced TxA2-endoperoxide response. Changes in membrane SER-gated calcium channels opening are observed only during the early hypertensive period. Besides, the lower depressor effect of RID on the maximal tension to SER in aorta rings from HT rats are related with a decreased NO availability in this model of renovascular hypertension.

Polyphosphoinositide synthesis in human neutrophils. Effects of a low metabolic energy state.

Phosphatidylinositol (PtdIns) synthesis and polyphosphoinositide (PPI) formation were measured as the incorporation of [32P]orthophosphate ([32P]Pi) or [3H]inositol into non-stimulated intact human neutrophil membrane phospholipids. The rate of PtdIns "de novo" synthesis appeared to be a slow mechanism when compared to the rapid incorporation of [32P]Pi into PPIs. Of the "de novo" synthesized [3H]PtdIns, 70% was further phosphorylated to PPI. Nevertheless, this PPI pool represented less than 0.01% of the total nmols of PPIs formed evaluated as [32P]Pi labeling, indicating that PPI formation mainly involves a no "de novo" synthesized phosphatidylinositol pool. When evaluated at short incubation times, oscillations in the formation of PPIs were detected. A rapid phase was characterized after 30 s of incubation with [32P]Pi Phosphorylation levels returned to an equilibrium state within a minute, and the second phase peaked at 5 min., returning to equilibrium at 15 min. The fluctuant kinetics though not the equilibrium level of PPI formation, could be abolished by neomycin. On the other hand, a selective inhibition of the rapid phase of PPI synthesis occurred in the presence of the tyrosine kinase inhibitor genistein. When the incorporations of [gamma-32P]-adenosine triphosphate (ATP) or [32P]Pi into human neutrophil particulate fraction membranes were evaluated, PPIs synthesis showed fluctuations independently of the precursor used. Noticeably, [32P]from [32P]Pi was incorporated more efficiently into PPIs than that from [gamma-32P]ATP, when evaluated in parallel using equal specific activities for both radiolabeled precursors and under non-ATP synthesizing conditions. Moreover, the incorporation of [32P]Pi into particulate fraction PPIs was not abolished by high concentrations of non-radiolabeled ATP, and metabolically inhibited PMNs showed high rates of PPI synthesis. These data suggest that PPI formation is not necessarily a futile cycle in PMNs.

Endogenous prostaglandins regulate rat renal phospholipid 'de novo' synthesis.

Rat renal papilla is the zone of the kidney enjoying the most active phospholipid metabolism and also the highest prostaglandin production. We studied the phospholipid biosynthesis and the relationship between phospholipid de novo synthesis and prostaglandin biosynthesis in rat renal papilla. Indomethacin inhibited the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and phosphatidic acid. Exogenous PGF2alpha and PGD2 restored biosynthetic activity in the presence of indomethacin and also increased the activity of the enzymes involved in the Kennedy pathway. The decrease in phospholipid biosynthesis maintained a linear relationship with the decrease in prostaglandin biosynthesis. Moreover, esculetin, which stimulates prostaglandin synthesis, brought about a significant increase in 32P incorporation to the three phospholipids studied. The evidence presented in this paper indicates that renal PGF2alpha and PGD2 modulate phospholipid de novo synthesis in rat renal papilla.

Compartmental study of rat renal prostaglandin synthesis during development.

The biosynthesis of prostaglandins (PGs) from the endogenous and exogenous precursor, arachidonic acid (AA), in renal papilla, medulla and cortex from neonatal to adult rats was investigated. Rat renal papilla and medulla incubated in the presence of [1-14C]AA released radioactive PGE2, PGF2 alpha and PGD2 which increased with age. No radioactive prostaglandins were found in the supernatants of renal cortex at any age studied. The amount of total prostaglandins released from the endogenous precursor also increased from 10 to 70 days of age, PGD2 being the prostaglandin that showed the most important rise. In the cortex, only PGE2 release increased with age. Cyclooxygenase (COX) activity was measured in papillary, medullary and cortical homogenates by using [1-14C]AA as substrate. Papillary and medullary COX activity increased after 10 days of age and continued to rise up to day 30 thereafter remaining unaltered until adulthood. Cortical COX activity was very low and decreased with age. These findings indicate the low capacity of the neonatal rat kidney to synthesize PGs.

Compartmental study of rat renal phospholipid metabolism.

Phospholipid content and metabolism were studied in rat renal papillary, medullary and cortical slices. The highest concentration of phospholipids was found in cortex and the lowest in papilla samples (ratio cortex/medulla, 1.3; cortex/papilla, 3.7). The profile of the various phospholipids was different depending on the zone. The most important difference was the relative concentrations of sphingomyelin (CerPCho) and phosphatidylinositol (PtdIns) with ratios for PtdIns/CerPCho of 5.0, 3.3 and 2.5 in papilla, medulla, and cortex, respectively. In the three zones, PtdIns showed the highest specific activity for [2-14C]glycerol and [1-14C]arachidonic acid incorporation. By contrast, a higher amount of [1-14C]palmitic acid was incorporated into phosphatidylcholine than into any other phospholipid. The various radioactive precursors were only poorly incorporated into phosphatidylethanolamine. No radioactivity was associated with phosphatidylserine. The papilla possesses the most active phospholipid metabolism of all the pathways studied.

Increase of phosphatidylinositol arachidonic acid incorporation induced by mepacrine.

In view of the fact that mepacrine (Mp) is usually used as an inhibitor of the endogenous phospholipase A2, and since this enzyme produces the release of arachidonic acid (AA) from membrane phospholipids, we studied the effect of different concentrations of Mp on the mobilization of [1-14C]AA in rat renomedullary phospholipids. During the acylation period, 0.1 mM Mp did not produce any significant change in the incorporation of [1-14C]AA into phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and only a slight increase in phosphatidylinositol (PI). Higher concentrations of Mp (0.5 to 1.0 mM) produced a decrease of radioactivity in PE and PC with an increase in PI. Using prelabeled slices, a dose-dependent decrease in the 14C-radioactivity in PE and PC was observed, with a parallel increase in PI. This effect of Mp persisted even in the presence of a physiological activator of phospholipase A2, bradykinin (BK). No change in the net amount of phospholipids was observed at any of the Mp concentrations used. The results of this study show that Mp, at concentrations generally used to inhibit phospholipase A2, produced a transfer of arachidonic acid from PE and PC to PI, rather than a blockade in the release of AA from membrane phospholipids.

Bradykinin-increased phospholipid deacylation-reacylation in rat renal medulla is inhibited by dBc AMP.

The effect of bradykinin on the mobilization of arachidonic acid was analyzed separately by acylation and degradation. Acylating activity was determined by the incorporation of [14C]arachidonic acid into the phospholipids at different times. Different concentrations of bradykinin had no effect on the phospholipid acylating activities. The degradation of the phospholipids was performed on renal medullary slices prelabeled with [14C]arachidonic acid. Treatment with bradykinin produced an initial degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, with a concomitant increase in lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol within 5 min of incubation. Phosphatidylcholine-, phosphatidylethanolamine- and phosphatidylinositol increased thereafter and reached the control values after 10 min of incubation. After 30 min, incubation of prelabeled slices with bradykinin produced a significant concentration-dependent increase in the phospholipid-labeling by reutilization of [14C]arachidonic acid. The effect of bradykinin on the phospholipid-labeling was blocked by preincubation with increasing concentrations of dBc AMP. Mepacrine also blocked the bradykinin stimulation in phosphatidylcholine and phosphatidylethanolamine, but had no effect on bradykinin-induced changes in the phosphatidylinositol arachidonic acid moiety.

Bradykinin stimulates phospholipase C in rat renal medullary slices.

Rat renal medullary slices prelabeled with [14C]arachidonic acid generate [14C]diacylglycerol within 1 min of exposure to bradykinin action. Production of [14C]diacylglycerol is transient. 2 min after the addition of bradykinin, the levels of metabolite reach the maximum, but decrease thereafter. Simultaneously, bradykinin induces a parallel decrease of the radioactivity in phosphatidylinositol. No degradation of other phospholipids is observed, and triacylglycerol is not affected. The degradation of [14C]phosphatidylinositol to [14C]diacylglycerol indicated the presence of phospholipase C activity. Preincubation of prelabeled slices with 2 mM dibutyryl cyclic AMP prevents both the generation of diacylglycerol and the degradation of phosphatidylinositol. Neither mepacrine nor indomethacin block diacylglycerol production and phosphatidylinositol breakdown. We conclude that, when rat renal medullary slices are stimulated with bradykinin, phosphatidylinositol-specific phospholipase C is activated.

Sow (Sus scrofa) follicular fluid: prostaglandin content and effect on the motility of isolated oviducts.

The present study provides information regarding the effects of the sow follicular fluid (FF) on the motility of isolated segments of swine and rabbit oviducts. In addition, the concentration of prostaglandins (PGs) F2 alpha, E2 and E1 in the follicular fluid of sow ovaries isolated at different stages of the sex cycle as well as the generation of the same PGs by walls of ovarian follicles in early and late proestrus, in estrus, in metestrus and in diestrus, were explored. The stimulatory contractile effect of proestrous FF in isolated segments of sow fimbria was antagonized by polyphloretin phosphate (PPP), a PG receptor blocker and by indomethacin, an inhibitor of PG synthesis. The positive inotropism evoked by the FF was mimiked by bradykinin and the influences of both interventions were similarly antagonized by PPP. It appears plausible that the inotropic effect of the preovulatory FF on the sow fimbria could be not only by PGs already present in the fluid, but also by the stimulation of the synthesis of tubal PGs by follicular fluid bradykinin. The FF also stimulated the ampullary tubal segments isolated from proestrous sows whereas the same volume of FF depressed significantly the isometric developed tension of rabbit ampulla. The total concentration of the three PGs in the FF from late proestrous follicles was significantly greater than that of the same PGs in the other two stages of the sex cycle (early proestrus and diestrus), whereas the concentration of each PG (PGE2, PGF2 alpha or PGE1), did not differ within any of the stages of the cycle. Furthermore, the total amount of the three PGs produced by the walls of follicles from late proestrous ovaries was also significantly greater than that generated by ovarian follicles from early proestrus, estrus, metestrus and diestrus. In summary the results document that the concentration of each one of the PGs measured (E2, E1 or F2 alpha) attained maximal values at the time of ovulation. The results regarding the effects of FF on the inotropic activity of fimbrial and ampullary segments of sow oviducts also suggest that the fluid might play a physiological role, favouring the capture and transfer of ova into the oviducts at the moment of ovulation.

Rat renal medulla possess high capacity to catabolize prostaglandins.

Prostaglandin E2 is converted to 15-keto-13,14 dihydro prostaglandin E2,15-keto-prostaglandin F2 alpha and 15-keto-13,14 dihydro prostaglandin F2 alpha, by supernatants from rat kidney medulla. The main pathway for prostaglandin E2 inactivation is the combined action of 15 hydroxy dehydrogenase and delta 13 reductase enzymes. 9-Keto-reductase route constitutes a minor pathway. Prostaglandin F2 alpha is converted into 15-keto-prostaglandin F2 alpha, 15-keto-13, 14 dihydro prostaglandin F2 alpha and 15-keto-dihydro prostaglandin E2. Enzyme activities are time and substrate-concentration dependent. In the presence of an excess of substrate, rat renal medulla inactivates 40 and 56 times more prostaglandin E2 and prostaglandin F2 alpha, respectively, than the amount which is released under basal conditions. These results are in contrast to the generally accepted concept that the kidney cortex is the sole site of renal prostaglandin catabolism, and suggest, for the first time, that rat renal medulla may be a key site for the modulation of prostaglandin levels in the kidney.

Phospholipase C activity in rat kidney. Effect of deoxycholate on phosphatidylinositol turnover.

Rat renal cortical and medullary slices incorporate [14C]arachidonate into phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and triacylglycerols. The percent distribution of [14C]arachidonate among the various phospholipids is similar in renal cortex and medulla, although the total amount of radioactively labeled phospholipids is higher in the renal medulla. Subsequent incubation of prelabeled slices in the presence of deoxycholate induces a loss of radioactivity from [14C]phosphatidylinositol, with a concomitant increase in 1,2-[14C]diacylglycerol. Neutral lipids are not affected. The degradation of phosphatidylinositol to [14C]diacylglycerol indicates the presence of phospholipase C activity. Renal medulla seems to be more sensitive to deoxycholate than the renal cortex. Deoxycholate also induces slightly the disappearance of some 14C radioactivity from phosphatidylethanolamine and phosphatidylcholine, which might reflect activation of phospholipase A2. The activity of the phospholipase C could constitute the first step in the sequence of reactions that leads to the release of arachidonic acid.

Direct evidence favouring the notion that erythropoietin alters iron transport across the isolated intestinal tract of the rat.

A simple and reproducible method, using the isolated not everted intestine of the rat, for the study of iron transport is presented. Erythropoietin (ESF) enhanced significantly the passage of 59Fe across the intestine augmenting its movement at mucosal and serosal layers of the intestinal well.