PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

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To understand the mechanism by which phorbol esters (PMA) stimulate c-jun transcription in human leukemic cell line U937, we have mutated specific enhancer sequences within the c-jun promoter. We find in the region of DNA from -132 to +170 containing Sp1, C-TF and AP-1 sequences that mutation of the AP-1 sequence alone is not sufficient to abrogate transcription, and mutation of the Sp1 sequence increases transcription 4-fold. Although mutation of the CTF site had no effect, CTF and AP-1 mutations together totally abrogate PMA-induced transcription. In comparison mutations of either of these sites alone or together in a construct containing -1639/+740 of the c-jun promoter had no effect on transcription. Because this data suggested the possibility of other upstream control regions, we sequenced the promoter from -142 to -1639. This sequence demonstrates a greater than 70% homology between human, and mouse c-jun promoters for the region from -142 to -441, and a second AP-1-like site in the -183 to -192 region. Mutation of this site did not influence transcription by PMA. By making constructs containing varying portions of the promoter, we have identified the region between -142 and -711 to be responsible for mediating PMA-induced c-jun transcription.

The voltage-dependent K (KV) channel in Daudi human B lymphoma cells was characterized by using patch-clamp techniques. Whole-cell voltage-clamp experiments demonstrated that cell membrane depolarization induced a transient (time-dependent) outward current followed by a steady-state (time-independent) component. The time-dependent current resembled behavior of the type n channel, such as use dependence and a unique blockade by tetraethylammonium (TEA). Both time-dependent and time-independent currents were blocked by quinine with a similar IC50 (14.2 mM and 12.6 mM). Treatment with antisense oligonucleotide of human Kv1.3 gene significantly reduced both currents by 80%. Single-channel experiments showed that only one type of KV channel was recorded with a unitary conductance of approximately 19 pS. Consistent with whole-cell recordings, the channel activity in cell-attached patches remained in response to prolonged depolarization, and the remaining channel activity was blocked by quinine, but not TEA. Channel activity was scarcely seen in cell-attached patches after antisense treatment. Whole-cell current-clamp data showed that TEA, which blocks only the time-dependent current, caused a slight decrease in the membrane potential. In contrast, quinine and antisense, which block both time-dependent and -independent currents, strongly reduced the membrane potential. These data together suggest that the KV channel in Daudi cells does not completely inactivate and that the remaining channel activity due to this incomplete inactivation appears to be primarily responsible for maintaining the membrane potential.

Okadaic acid, a protein phosphatase inhibitor, is a strong tumor promoter which activates protein phosphorylation. Because another activator of protein phosphorylation, phorbol esters, stimulates hematopoietic differentiation, we sought to determine whether okadaic acid could also induce the differentiation of the human leukemic cell line U937. Differentiation was assessed by measuring changes in the following: mRNA levels, cell growth, morphology, cell surface markers, and the ability to induce superoxide. We found that okadaic acid treatment of U937 cells induces immediate increases in total cellular levels of both c-jun and c-fos mRNAs. Nuclear run-on experiments demonstrate that initial increases are secondary to increases in transcription, whereas latter changes may be secondary to mRNA stabilization. Like phorbol esters, okadaic acid treatment also activates AP-1 enhancer activity and induces the phosphorylation of c-Jun protein. Approximately 6-12 hours after treatment with okadaic acid, mRNA levels of c-myc, p34cdc2, and p58GTA, two cell cycle regulated protein kinases, decrease. Okadaic acid inhibits the growth of U937 cells, induces changes in nuclear morphology, stimulates increases in Mac-1 and Leu 11 surface antigens, and induces these cells to produce superoxide. These changes, taken together, suggest that U937 cells have been induced by okadaic acid to differentiate towards a more mature cell type.

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