Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1 and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206 -38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A 2 (cPLA 2 ) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA 2 is the key phospholipase A 2 downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 M) induced the translocation of full-length cPLA 2 from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA 2 in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA 2 and with the CaLB domain in a calcium-and lipid-specific manner with a K Ca of 1.54 M. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA 2 enzymatic activity as well as lowering the EC 50 of calcium for the enzyme from 191 to 31 nM. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA 2 , demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.
The glucocorticoid dexamethasone, but not the mineralocorticoid aldosterone, increased amiloride-sensitive Na'-H' exchange activity in rat proximal tubule brush border vesicles. Na' uptake, independent of amiloride, was not affected. The glucocorticoid decreased the Na' gradient-dependent phosphate uptake. Uptake in the absence of a Na' gradient was not inhibited. Dexamethasone did not affect the Na' gradient-dependent D-glucose uptake. METHODS AND MATERIALS Animals and Steroid Administration. Male Sprague-Dawley rats (295-345 g) were fed Purina rat chow pellets ad lib. Rats were adrenalectomized under light ether anesthesia. After surgery, normal saline was given to prevent volume depletion. Adrenalectomized animals were divided into three groups. One group received twice daily subcutaneous injections of 0.3 ml of 0.9% saline. The other two groups received twice daily subcutaneous injections (5 pLg/100 g of body weight) of dexamethasone or aldosterone in 0.9% saline, respectively. The quantity of dexamethasone administered daily as the glucocorticoid replacement was approximately equivalent to the amount of corticosterone secreted per day by the rat and was the dose that effected a return of Na+, K+, and water movements in the rat colon to control values after adrenalectomy (18). Likewise, the quantity of aldosterone administered daily was approximately equivalent to the amount ofaldosterone secreted per day by rats on a normal diet (18). Another set of rats underwent only bilateral flank incisions, without removal of adrenals, and subsequently were given tap water to drink. This set ofanimals was subdivided into two groups. One group, designated sham, received twice daily subcutaneous injections of 0.3 ml of 0.9% saline. The other group, designated sham with dexamethasone, received dexamethasone (60 ,g/100 g of body weight) on the same schedule. Transport studies were performed 2 days after surgery. Hormones were last administered 2 hr prior to sacrifice. Animals were killed between 10:00 and 10:30 a.m.At the time ofsacrifice, serum Na+ concentration was slightly higher in adrenalectomized rats given aldosterone relative to the other groups, but the value was not statistically different from that found in adrenalectomized animals (141.6 ± 1.0 vs. 139.2 ± 0.5 milliequivalents/liter; P > 0.05). Serum Pi was essentially similar in all groups, varying from 2.66 ± 0.04 to 2.90 + 0.08 mM, although sham animals that received high doses of dexamethasone had-a Pi concentration that was minimally lower statistically than that in sham animals (2.70 ± 0.05 vs. 2.90 + 0.08 mM; P < 0.05), and adrenalectomized rats given maintenance doses of glucocorticoid had a slightly lower serum Pi concentration than did the adrenalectomized animal (2.66 ± 0.04 vs. 2.81 ± 0.06 mM; P < 0.05). Serum Ca2+ ranged from 2.35 ± 0.04 to 2.56 ± 0.04 mM. Sham rats given high doses of dexamethasone had the highest serum Ca2+ level. This was significantly greater (P < 0.05) than the values found in the other groups, which did not significan...
The possibility has been explored of using a specific angiotensin ii antagonist, saralasin (P-113, 1-sar-8-ala-angiotensin ii) to recognize patients whose hypertension depends upon excessive angiotensin ii activity. Among 60 hypertensive patients, saralasin infusion reduced blood pressure in 16 "responders," but not in 44 "nonresponders." The "responders" had the following findings: elevated plasma renin activity in renal vein (or veins) or peripheral veins or both (16 of 16); reduced renal blood flow, shown by arteriography, isotopic studies or pyelography (15 or 16), or progressive azotemia (one of 16); and reduction in blood. These findings indicated that angiotensin ii probably caused hypertension in the "responders," One "nonresponder" had renal vein levels of plasma renin activity suggestive of angiotensinogenic hypertension. Since hypertension was invariably angiotensinogenic when it was reduced by saralasin and, with one possible exception, was never angiotensinogenic in "nonresponders," the antagonist appears to provide an efpressure to or toward normal after corrective operation (four of four) or propranolol therapy (eight of eight). fective means of recognizing angiotensinogenic hypertension.
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