Estrogen increases male rat aortic endothelial cell (RAEC) PGI2 release

Estrogen has been proposed as a negative risk factor for development of peripheral vascular disease yet mechanisms of this protection are not known. This study examines the hypothesis that estrogen stimulates rat aortic endothelial cell (RAEC) release of PGI₂. Male Sprague-Dawley rat abdominal aortic 1-mm rings were placed on 35 mm matrigel plates, and incubated for 1 week. The cells were transferred to a Primaria 60-mm dish and maintained from passage 3 in RAEC complete media and experiments performed between passages 4–10. Cells were incubated with Krebs-Henseleit buffer (pH 7.4) containing carrier or increasing concentrations of β-estradiol or testosterone for 60 min. The effluent was analyzed for eicosanoid release of 6-keto-PGF_1α (6-keto, PGI₂ metabolite), PGE₂ and thromboxane B₂ (TXB₂) by EIA (hormone stimulated — basal). Cells were analyzed for total protein by the Bradford method and for cyclooxygenase-1 (COX-1) and prostacyclin synthase (PS) content by Western blot analysis and densitometry. Testosterone did not alter RAEC 6-keto-PGF_1α release, whereas estrogen increased RAEC 6-keto-PGF_1α release in a dose-related manner. Estrogen preincubation (10 ng/ml) decreased COX-1 and PS content by 40% suggesting that the estrogen-induced increase in male RAEC PGI₂ release was not due to increased synthesis of COX-1 or PS. These data support the hypothesis that estrogen stimulation can increase endogenous male RAEC release of PGI₂.     

Prostacyclin contributes to inhibition of hypoxic pulmonary vasoconstriction by alkalosis

The mechanism by which extracellular alkalosis inhibits hypoxic pulmonary vasoconstriction is unknown. We investigated whether the inhibition was due to intrapulmonary production of a vasodilator prostaglandin such as prostacyclin (PGI₂). Hypoxic vasoconstriction in isolated salt-solution-perfused rat lungs was blunted by both hypocapnic and NaHCO₃_induced alkalosis (perfusate pH increased from 7.3 to 7.7). The NaHCO₃-induced alkalosis was accompanied by a significant increase in the perfusate level of 6-keto-prostaglandin F_1α (6-keto-PGF_1α), an hydrolysis product of PGI₁. Meclofenamate, an inhibitor of cyclooxygenase, counteracted both the blunting of hypoxic vasoconstriction and the increased level of 6-keto-PGF_1α. In intact anesthetized dogs, hypocapnic alkalosis (blood pH increased from 7.4 to 7.5) blunted hypoxic pulmonary vasoconstriction before but not after administration of meclofenamate. In separate cultures of bovine pulmonary artery endothelial and smooth muscle cells stimulated by bradykinin, the incubation medium levels of 6-keto-PGF_1α were increased by both hypocapnia and NaHCO₃-induced alkalosis (medium pH increased from 7.4 to 7.7). These results suggest that inhibition of hypoxic pulmonary vasoconstriction by alkalosis is mediated at least partly by PGI_2.     

Role of prostacyclin on steroidogenesis by frog interrenal gland

The role of prostacyclin (PGI₂) on amphibian adrenal steroidogenesis was studied in perifused interrenal fragments from adult male frogs. Exogenous PGI₂ (3×10⁻⁸ M to 3×10⁻⁵ M) and, in a lesser extent, 6-keto-PGF_1α increased both corticosterone and aldosterone production in a dose-related manner. Short pulses (20 min) of 0.88 μM PGI₂ administered at 90 min intervals within the same experiment did not induce any desensitization phenomenon. A prolonged administration (6 h) of PGI₂ gave rise to an important increase in steroid production followed by a decline of corticosteroidogenesis. Indomethacin (IDM, 5 μM) induced a marked reduction of the spontaneous secretion of corticosteroid which confirmed the involvement of endogenous PGs in the process of corticosteroid biosynthesis. The IDM-induced blockade of corticosterone and aldosterone secretion was totally reversed by administration of exogenous PGI₂ in our model. Angiotensin II (AII) induced a massive release of 6-keto-PGF_1α, the stable metabolite of PGI₂. The increase of 6-keto-PGF_1α preceded the stimulation of corticosterone and aldosterone secretions. In contrast, the administration of ACTH did not modify the release of 6-keto-PGF_1α. These results indicate that PGI₂ might be an important mediator of adrenal steroidogenesis in frog. They confirm that the corticosteroidogenic actions of ACTH and AII are mediated by different mechanisms.     

Arachidonate metabolism in bovine gallbladder muscle

Incubation of [1-¹⁴C]arachidonic acid (AA) with homogenates of bovine gallbladder muscle generated a large amount of radioactive material having the chromatographic mobility of 6-keto-PGF_1α (stable product of PGI₂) and smaller amounts of products that comigrated with PGF_2α and PGE₂. Formation of these products was inhibited by the cyclooxygenase inhibitor indomethacin. The major radioactive product identified by thin-layer chromatographic mobility and by gas chromatography - mass spectrometric analysis was found to be 6-keto-PGF_1α. The quantitative metabolic pattern of [1-¹⁴C]PGH₂ was virtually identical to that of [1-¹⁴C]AA. Incubation of arachidonic acid with slices of bovine gallbladder muscle released labile anti-aggregatory material in the medium, which was inhibited by aspirin or 15-hydroperoxy-AA.These results indicate that bovine gallbladder muscle has a considerable enzymatic capacity to produce PGI₂ from arachidonic acid.     

Differential effects of acute thermal injury on rat splanchnic and renal blood flow and prostanoid release

This study examines the hypothesis that acute thermal injury decreases renal and splanchnic blood flow which correlates with altered endogenous vasodilator eicosanoid release. Anesthetized male Wistar rats were subjected to sham or a non-resuscitated 30% total body surface area burn. At 1, 2, 4, 8, and 24 h post-burn mean arterial pressure as well as superior mesenteric and renal artery in vivo blood flow were measured. The superior mesenteric and renal arteries were cannulated and perfused in vitro with their end organs with Krebs buffer (pH 7.4, 37°C). Renal and splanchnic 6-keto-PGF_1α (PGI₂), PGE₂, and thromboxane B₂ (TXB₂) release were measured by EIA at 15 min of perfusion. Renal and superior mesenteric artery blood flow decreased by 40% or more at 1 and 2 h post-burn despite mean arterial pressure remaining unchanged. The major eicosanoids released were PGI₂ from the splanchnic bed and PGI₂ and PGE₂ from the kidney. Splanchnic PGI₂ and TXB₂ release and renal TXB₂ increased 2–3 fold at 1 h post-burn but returned to the sham level at 2 h post-burn. By 24 h post-burn the vasodilator eicosanoids were increased in both the splanchnic and renal vascular beds. These data show that decreased renal and splanchnic blood flow was associated with increased endogenous release of the potent vasoconstrictor TXB₂. By 2 h post-burn, renal and splanchnic blood flow began returning toward the sham level as endogenous release of TXB₂ from both organs fell to sham levels. These data suggest that increased endogenous release of TXB₂ may contribute to the short-term decrease in renal and splanchnic blood flow in the immediate post-burn period and thus may contribute to ischemia of both vascular beds.     

Endogenous synthesis of prostacyclin was positively regulated during the maturation phase of cultured adipocytes

Prostacyclin alternatively called prostaglandin (PG) I₂ is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI₂ has not been determined comprehensively at different life stages of adipocytes. PGI₂ is rapidly hydrolyzed to the stable product, 6-keto-PGF_1α, in biological fluids. Therefore, the generation of PGI₂ can be quantified as the amount of 6-keto-PGF_1α. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF_1α. According to the typical calibration curve of our ELISA, 6-keto-PGF_1α can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF_2α. The resulting ELISA was applied to the quantification of 6-keto-PGF_1α generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF_1α during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI₂. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF_1α is useful for monitoring the endogenous levels of the unstable parent PGI₂ at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.     

Inhibitory effect of insulin on prostacyclin production by isolated rat adipocytes

Physiologic concentrations of insulin completely inhibited the norepinephrine-induced increment in the production of 6-keto-prostaglandin (PG) F_1α, the stable derivative of prostacyclin (PGI₂), by isolated rat adipocytes. The inhibition of PGI₂ production by insulin in isolated rat adipocytes supports the view that the elevated plasma level of 6-keto-PGF_1α in rats with non-ketotic diabetes mellitus and diabetic ketoacidosis is derived at least in part from production of PGI₂ by the adipocyte cell mass.     

Pulmonary formation of prostacyclin in man

The pulmonary formation of prostacyclin (PGI₂), as reflected by the difference in concentration of pulmonary and systematic arterial radioimmunoassayed 6-keto-PGF_1α, was determined in six healthy waking subjects. The systematic arterial 6-keto-PGF_1α levels were low (50 pg/ml), and no evidence of pulmonary formation and release of the compound was noted. In other experiments systemic arterial 6-keto-PGF_1α levels were determined in patients prior to and during artificial ventilation, as well as during and after occlusion of the pulmonary circulation (extra-corporeal circulation, ECC). The arterial 6-keto-PGF_1α concentration prior to artificial ventillation was 17±4 pg/ml, i.e. within the range observed in the healthy subjects. During artificial ventilation the arterial levels of 6-keto-PGF_1α increased to 191±21 pg/ml, suggesting that pulmonary formation of PGI₂ was stimulated. In the patients subjected to ECC with occluded pulmonary circulation the arterial content of 6-keto-PGF_1α was stabilised at an elevated level (120−170 pg/ml). Following re-establishment of the pulmonary circulation the arterial concentrations of 6-keto-PGF_1α increased markedly, to 284±50 pg/ml. It is suggested that the basal pulmonary formation of PGI₂ in man is low or non-existent, and that enhanced formation of the compound in the lungs is a consequence of intervention with normal pulmonary ventilation or perfusion.     

Prostaglandin synthesis by fetal rat bone in vitro: Evidence for a role of prostacyclin

Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE₂, PGF_2α and 6-keto-PGF_1α, the metabolite of prostacyclin (PGI₂). Since PGI₂ had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI₂ stimulated release of previously incorporated ⁴⁵Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10⁻⁵ to 10⁻⁹M. Because of the short half life of PGI₂ in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI₂) which was found to be a stimulator of bone resorption at concentrations of 10⁻⁵ to 10⁻⁶M. These studies suggest that endogenous PGI₂ production may play a role in bone metabolism. Since vessels produce PGI₂ it is possible that PGI₂ release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis.     

Assay of prostacyclin synthesis in intact aorta by aqueous sampling

Conversion of 1-¹⁴C-arachidonic acid (AA) to 6-keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂) was assayed kinetically by employing an aqueous sampling technique. In this way, one can arrive at a kinetic view of PGI₂ synthesis from AA in intact tissue. The assay appears to be particularly suitable to tissues such as the aorta where PGI₂ constitutes the major metabolite of AA. The assay avoids the need for organic solvent extraction and relies on the essential absence of tissue binding of 6-keto-PGF_1α. The disappearance of AA can also be followed in this system but quantitation is complicated by avid tissue binding of the fatty acid. The assay, as described should be applicable to other vascular tissues and should greatly simplify kinetic analyses of prostacyclin synthesis.     

Effect of the protein kinase inhibitors, staurosporine and K-252a, on PGI2 production by rat liver cells (the C-9 cell line)

Staurosporine and K-252a, known inhibitors of several protein kinases, stimulated PGI₂ production (measured as 6-keto-PGF_1α in rat liver cells (the C-9 cell line). Preincubation of the rat liver cells with staurosporine or K-252a enhanced the PGI₂ production stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), platelet activating factor (PAF) and the Ca²⁺-ionophore a-23187, but not the PGI₂ synthesis stimulated by exogeneous arachidonic acid. These results suggest that phosphorylation of some proteins or certain amino acids on a protein can regulate arachidonic acid metabolism probably in the pathway leading to deesterification of phospholipids.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI₂; PGD₂; PGF₂ and PGE₂); BaCl₂ or prostaglandin metabolites (15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α; 6-keto-PGF_1α and 6-keto-PGE₁ in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl₂, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF_2α was shifted to the right of that for PGF_2α itself; the curve for 6-keto-PGF_1α was displaced to the right of the curve for PGI₂ and that for 6-keto-PGE₁ to the left.It was also demonstrated that the uterine motility elicited by 10⁻⁵ M PGF_2α and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI₂; 6-keto-PGF_1α and BaCl₂, but not the contractions following 6-keto-PGE₁, which disappeared much earlier. The contractile tension after PGF_2α; 15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α and PGI₂, increased as time progressed whilst that evoked by 6-keto-PGF_2α or BaCl₂ fluctuated during the same period around more constant levels.The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI₂, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF_1α, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Prostacycin production by arterialized autogenous venous grafts in dogs

Arteries are capable of producing significantly larger quantities of protacyclin than are veins. To test the hypothesis, whether prostacyclin production by the vessel wall is related to blood pressure and flow, we measured the amounts of PGI₂ released and synthesized by venous segments transplanted for 6 weeks into the arterial circulation. These results were compared with the production of prostacyclin by normal veins and arteries. In 20 dogs a segment of jugular vein was interposed into the carotid system; a sham dissection was done on the opposite side. “Arterialized” vein grafts showed prominent intima lined by endothelium, medial smooth muscle cell proliferation and fibrotic proliferation in adventitia. Spontaneous and arachidonic acid- stimulated prostacyclin production (measured by radioimmunoassay for 6-keto-PGF_1α) was not significantly different between arterialized venous autografts and jugular veins. Significantly larger amounts of prostacyclin were synthesized by the carotid artery. Thus, histologic changes and rheologic effects occurring in vein grafts transposed to the arterial site do not affect prostacyclin production.     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI₂ as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to γ raidation results in an irreversible cessation of growth and enhanced production of PGI₂. The level of PGI₂ measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for γ radiation in the elevation of PGI₂ levels which is distinct from its effect on cell division. Result presented indicate that exposure to γ radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI₂ synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

The impermeability of the basic cell membrane to thromboxane-B2, prostacyclin and 6-keto-PGF1α

Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing ³H-labeled prostacyclin (PGI₂), thromboxane (TxB₂) or 6-keto-PGF_1α and ¹⁴C-labeled sucrose or thiourea. Following 1 to 30 min incubation with ¹⁴C-sucrose, ³H-TxB₂ or ³H-6-keto-PGF_1α, the ¹⁴C or ³H space of packed RBCs remained essentially constant, yielding mean values (±S.E.) for all time periods of 6.1 ± 0.3, 9.5 ± 0.5 and 6.5 ± 0.4%, respectively. After 1 min of incubation at 4° or 23°C at a pH of 7.4 or 8.5 with trace amounts (10⁻⁹M) of ³H-PGI₂ or in the presence of added PGI₂ (10⁻⁵M) or ethacrynic acid (1.6 × 10⁻⁴M), the apparent PGI₂ space of packed RBCs ranged from 16 to 27%, decreasing to about 7% by 30 min. When RBCs were resuspended in fresh ³H-PGI₂ every 5 min, their ³H content increased very slowly (apparent PGI₂ space <40% at 30 min) as compared to thiourea (distribution space > 80% within 5 min). Over 90% of this ³H activity was lost from the RBCs in less than 2 min during elution at 4° or 23°C. It is concluded that RBC membranes and thus, presumably, the basic cell membrane in general, is not fundamentally permeable to PGI₂, 6-keto-PGF_1α or TxB₂. Hence, the effective entry of these cyclooxygenase products into some cells or their passage across tight-junctional capillaries or epithelial membranes must require facilitated or active transport processes as was shown to be the case for E, F and A PGs. This implies that the distribution, pharmacological action and metabolism of these and presumably all related cyclooxygenase products are selective rather than unrestricted.     

Comparison of umbilical vein models for measurement of relative prostacyclin and thromboxane production

There is growing evidence that blood vessels generate TXA₂ in addition to PGI₂. We examined effluents from continously perfused human umbilical vein and supernatants from umbilical vein rings for TXB₂ and 6-keto-PGF_1α measurements (stable metabolites of TXA₂ and PGI₂, respectively). TXB₂ and 6-keto-PGF_1α were identified in all samples. 6-keto-PGF_1α to TXB₂ ratio was higher in intact vein effluents than in the venous ring supernatants (112:1 and 28:1, respectively, P<0.01). Arachidonate stimulation increased 6-keto-PGF_1α and TXB₂ levels similarly in the intact vein effluent. In contrast, stimulation of the venous rings resulted in a relatively larger increase in TXB₂ than in 6-keto-PGF_1α. This caused 6-keto-PGF_1α to TXB₂ ratio to decline (p<0.01). The identity of TXB₂ was confirmed in several different ways. These data suggest that 1) human umbilical veins produce TXA₂ in addition to PGI₂, 2) TXA₂ release is more by venous rings than by the intact vein probably reflecting contribution from non-endothelial layers, and 3) arachidonate stimulation causes relatively greater release of TXA₂ than of PGI₂ from the venous rings, whereas release of PGI₂ and TXA₂ is similar from the intact vein.     

Effect of dipyridamole of prostaglandin generation by human platelets and vessel walls

These experiments were conducted to determine the effects of dipyridemole on human platelet aggregation, platelet thromboxane A₂ (TXA₂) and human vessel wall prostacyclin (PGI₂) generation. Dipyridamole in varying concentrations (5 to 50 μg/ml) had no direct effect on ADP-induced platelet aggregation in vitro, but it potentiated PGI₂-induced platelet aggregation inhibition at these concentrations. Dipyridamole also inhibited arachidonic acid-induced platelet TXA₂ generation at these concentrations. In continuously perfused umbilical vein segments, dipyridamole treatment resulted in stimulation of PGI₂ release determined by bioassay and by measurement of its stable metabolite 6-keto-PGF_1α. Minimum concentration of dipyridamole causing PGI₂ release was 50 μg/ml. These in vitro studies suggest that anti-thrombotic effects of dipyridamole in man are mediated mainly by potentiation of PGI₂ activity and to some extent by TXA₂ suppression. Stimulation of PGI₂ release by human vessels may not be seen in usual therapeutic concentrations.     

The human placental anti-aggregating factor is neither prostacyclin, nor a prostacyclin metabolite

Using PGH₂ as substrate, we have previously demonstrated that human placenta synthetizes mainly PGE₂, TxB₂ and PGD₂(1,2). Other reports have shown that placental tissue generates a substance which inhibits ADP-induced platelet aggregation and which was supposed to be PGI₂ (3). The present study indicates that the stability of that substance is different from the stability of prostacyclin (released by umbilical artery pieces). By GC-MS and multiple ion-monitoring, we have shown the presence of 6 keto-PGF_1α (the stable metabolite of PGI₂) in the umbilical artery incubation medium, while no trace of 6-keto-PGF_1α could be found in the placental medium. No conversion of AA to 6-keto-PGF_1α by placental microsomes was observed, even in the presence of antioxidants. The placenta possesses, in addition to the known 15-OH-PGDH and Δ-13 reductase activities, a weak 9 OH pGDH which is specific for PGF_2α (and not PGI₂ nor 6-keto-PGF_1α). GC-MS analysis is showed that the expected metabolites of PGI₂ through those three enzymes were not found in the placental medium, indicating that neither PGI₂ synthesis nor metabolism could be demonstrated in the placenta.     

Plasma prostaglandin levels and circulating fuel levels in rats with diabetic ketoacidosis: Effects of cyclooxygenase inhibitors and of alpha and beta adrenergic blockade

We studied the effects of two structurally unrelated inhibitors of the fatty acid cyclooxygenase and of alpha and beta adrenergic blockade on the elevated plasma levels of 13,14-dihydro-15-keto-prostaglandin (PG)E₂, 6-keto-PGF_1α and thromboxane(TX)B₂, the stable derivatives of PGE₂, PGI₂ (prostacyclin) and TXA₂, respectively, in rats with streptozotocin-induced diabetic ketoacidosis (DKA). Meclofenamic acid and indomethacin each produced a significant decrease in the elevated plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂. Phentolamine significantly reduced the plasma level of TXB₂ but had no effect on the elevated circulating levels of glucose, free fatty acids, total ketones, 13,14,-dihydro-15-keto-PGE₂ or 6-keto-PGF_1α. Propranolol significantly reduced the elevated circulating levels of glucose, free fatty acids and total ketones but had no effect on the levels of the three prostaglandin derivatives. The ability of meclofenamic acid and indomethacin to reduce the plasma levels of 13,14-dihydro-15-keto-PGE₂, 6-keto-PGF_1α and TXB₂ confirms that the plasma levels of these three derivatives are elevated in rats with DKA. Since abnormalities in the production of PGI₂ and perhaps other cyclooxygenase derivatives may contribute to the pathogenesis of certain important hemodynamic and gastrointestinal features of DKA, cyclooxygenase inhibitors may play a role in the management of selected patients with this disorder. Alpha adrenergic activity is essential for the maintenance of the elevated plasma TXB₂ level in rats with DKA. The fall in the plasma TXB₂ level during alpha adrenergic blockade appears to reflect inhibition of platelet aggregation and platelet TXA₂ production, but other sources of the elevated plasma TXB₂ level in DKA are not excluded. Beta adrenergic activity contributes to the maintenance of elevated circulating levels of glucose, free fatty acids and total ketones in experimental DKA but not to the elevated plasma levels of the prostaglandin derivatives.     

Formation of 6-keto-PGF1α by collecting tubule cells isolated from rabbit renal papillae

Homogeneous populations of collecting tubule epithelial cells have been isolated from rabbit renal papillae by a sequence of procedures involving: (a) dissociation of the tissue by mincing and treatment with trypsin; (b) destruction of contaminating non-collecting tubule cells by differential lysis in hypotonic media and (c) collection and washing by repeated centrifugation. The isolated cells have been characterized as being derived from the collecting tubules on the basis of anatomical source, size and histological staining for both NADH diaphorase activity and cyclooxygenase antigenicity. The cells are judged to be viable by several criteria including their ability to exclude both trypsin and vital dyes, their capacity to metabolize glucose and leucine and their ability to retain distinctive morphology following 10–14 days in culture media. Homogenates of freshly isolated collecting tubule cells when incubated with [³H]-arachidonic acid yielded radioactive products identified by thin-layer chromatographic behavior in multiple solvent systems as 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂ and a monohydroxy acid, probably HHT. No lipoxygenase-like activity was detected. At arachidonate concentrations of 2 or less, the major product was 6-keto-PGF_1α; while at substrate concentrations of greater than 10 , PGE₂ was the major radioactive prostaglandin formed. Similar distributions of products were observed when homogenates of dissociated renal papillae enriched in medullary interstitial cells were incubated with arachidonic acid. Our results indicate that collecting tubule cells do contain significant prostacyclin synthetase activity and suggest that PGI₂ plays a role in the function of mammalian collecting tubules.