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Experiments were carried out in rats to determine whether prostaglandin E2 (PGE2) synthesized in vivo by individual glomeruli could be measured in fluid obtained from early proximal tubule convolutions. A newly developed nonradioactive enzyme-linked immunoassay for PGE2, capable of measuring 0.1- to 0.25-pg quantities, made this approach feasible. In salt-deprived rats, recollection micropuncture samples were obtained from the earliest proximal convolution [tubular fluid-to-plasma insulin ratio (TF/PIn) 1.13] for measurement of PGE2, and then single-nephron glomerular filtration rate (SNGFR). PGE2 was also measured in serum ultrafiltrate, and theoretical maximum "filtered" PGE2 was calculated for each nephron. This was compared with the measured PGE2 from the same nephron. Indomethacin was then given to inhibit cyclooxygenase. We found that measured PGE2 in early proximal fluid (TF) was higher than could be attributed to glomerular filtration. Indomethacin markedly decreased measured PGE2 in early TF. Administration of probenecid, to block tubular transport of PGE2, did not alter the observations. In chronically salt-loaded rats early TF PGE2 was significantly less compared with the salt-deprived rats. Intrarenal infusion of a nonpressor dose of angiotensin II (ANG II) doubled PGE2 appearance in early TF samples. We conclude from these observations that PGE2 in early TF is derived in part from the glomerulus. In the intact rat, glomerular PGE2 synthesis is higher after salt deprivation than after salt loading, inhibited by indomethacin, and stimulated by ANG II. This new approach will allow evaluation of the role of in vivo glomerular PGE2 production in various pathophysiological conditions.

The interaction of inflammatory cells and glomerular prostaglandins (PG) may be important during glomerulonephritis. We therefore examined the influence of platelet-activating factor (PAF), (a mediator of inflammation released from leukocytes) and of phagocytosis of zymosan on arachidonic acid metabolism and on cell contractility in rat glomerular mesangial cells in culture. PAF increased PGE2 synthesis (determined by radioimmunoassay) within minutes (threshold: 10(-10)M; maximal effect: 10(-7)M). Serum-treated zymosan also stimulated PGE2, but with a slower onset. In cells prelabeled with [14C]arachidonic acid both PAF and serum-treated zymosan released 14C from phospholipids and increased free [14C]arachidonate. The ratio of 14C-release to PGE2 was, however, different with PAF and serum-treated zymosan, indicating different phospholipid pools. Under phase-contrast microscopy, PAF caused contraction of mesangial cells with a dose-response and time-course parallel to that for PGE2 synthesis. Serum-treated zymosan caused no contraction. The PAF-induced contraction was enhanced by PG synthesis inhibition and was attenuated by addition of PGE2, indicating a feedback mechanism. The mesangial contraction by PAF may be important in favoring deposition of immune complexes, while the PGE2 synthesis stimulated by PAF and by phagocytosis of zymosan may counteract the deleterious effects of PAF during induction of glomerulonephritis.

Pertussis toxin may inactivate N proteins linked to phospholipase C. We examined the effect of pretreatment with pertussis toxin at different concentrations and times on agonist-induced PGE2 synthesis in mesangial cells. Two to four hours with 10-50 ng/ml of pertussis toxin inhibited the response to angiotensin and platelet activating factor, but with a different sensitivity. This was associated with decreased [14C]arachidonic acid release in prelabeled cells. The response to A23187 was unaltered. At high concentrations (1 to 5 micrograms/ml) pertussis toxin increased basal PGE2 and the response to all agonists. Pertussis toxin pretreatment resulted in a dose-dependent ribosylation of a 40 kDa protein band. Thus, responses to different agonists have different sensitivity to pertussis toxin inhibition, which at high concentrations may even have opposite effects.

Prostaglandin E2 (PGE2) inhibits the action of the antidiuretic hormone (ADH) in isolated collecting tubules. A negative feedback loop has been postulated whereby ADH stimulates PGE2 synthesis. Furthermore, lysyl-bradykinin (LBK) inhibits the antidiuretic effect of ADH, probably via PGE2. Enhanced PGE2 synthesis has also been implicated as contributing to the inability to maximally concentrate urine during the neonatal period. We investigated PGE2 synthesis in microdissected cortical (CCT), medullary (MCT), and branched cortical (BCT) collecting tubules from adult and in corticomedullary collecting tubules (CT) from newborn rabbits. Isolated BCT produced significantly less PGE2 (12 +/- 2 pg X mm-1 X 20 min-1) than CCT (65 +/- 9) or MCT (76 +/- 8) from kidneys of adult rabbits. CT from newborn rabbits produced only 19 +/- 3 pg/mm, significantly less than either CCT or MCT from adults. A large variability in basal PGE2 production and hormonal response was observed from tubule to tubule. Under either basal conditions or in the presence of 2 microM arachidonic acid, LBK enhanced PGE2 synthesis in CCT and MCT from adults. ADH enhanced PGE2 production in MCT under basal conditions and in CCT in the presence of arachidonic acid. Neither LBK nor ADH stimulated PGE2 synthesis in neonatal CT. A23187 consistently stimulated PGE2 synthesis in CCT and MCT from adults and, to a lesser extent, in CT from newborn rabbits. Our results support the hypothesis that ADH and LBK enhance PGE2 synthesis in the collecting tubule. This response is, however, subject to large variations from tubule to tubule and depends on the in vitro incubation conditions.

The mechanism of arachidonic acid release and prostaglandin E2 (PGE2) synthesis was studied in cultured mesangial cells from rat kidneys. Both the ionophore A23187 and angiotensin II stimulated radioimmunoassayable PGE2 synthesis. The effect of angiotensin occurred within minutes, with half-maximal stimulation around 10(-9) M. In cells prelabeled with [14C]arachidonate, A23187 caused release of [14C]-arachidonate from all phospholipids. In contrast, angiotensin II caused preferential release of [14C]arachidonate from phosphatidylinositol, associated with a significant increase in 14C-labeled phosphatidic acid, mono- and diacylglyceride, and arachidonate. These results indicate that angiotensin preferentially, but not exclusively, stimulates a phosphatidylinositol-specific phospholipase C, whereas A23187 results in unspecific stimulation of phospholipases. The tight coupling between an angiotensin-responsive phospholipid-arachidonate pool and cyclooxygenase may be responsible for the specificity of the response to angiotensin.

The interaction of vasopressin with prostaglandins were examined in the toad bladder by determining water flows, cAMP levels, and cAMP-dependent protein kinase activity. Both water flow and activation of cAMP-kinase in response to vasopressin were enhanced after prostaglandin inhibition, consistent with inhibition of vasopressin-induced cAMP generation by endogenous prostaglandins. On the other hand exogeneous PGE stimulated cAMP generation. PGE1 (10(-7) M) alone did not increase water flow but activated kinase more than vasopressin only. Addition of PGE1 (10(-7) M) and vasopressin inhibited water flow as compared with vasopressin along but increased the kinase ratio above that with vasopressin only. PGE2 (10(-5) M) increased the cAMP content and kinase ratio even more than vasopressin but again resulted in no water flow. Addition of vasopressin and PGE2 (10(-5) M) increased water flow but did not alter cAMP content or the kinase ratio compared with PGE2 alone. Similar results were obtained with PGE1. Accordingly, prostaglandin dissociates cAMP levels and kinase ratio from the hydroosmotic response, suggesting that PGE2 inhibits steps distal to cAMP. Consistent with this, in bladders pretreated with naproxen or meclofenamate, PGE2 (10(-8) to 10(-6) M) inhibited the response to submaximal doses of cAMP (5 mM) or 8-bromo-cAMP (0.03 mM). Furthermore, pretreatment with naproxen significantly enhanced the response to cAMP (5 mM). These studies provide evidence for vasopressin-PGE interaction at the site of cAMP generation and also at a step(s) unrelated to cAMP generation.