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Protein kinase C‐dependent superoxide production by the renal medullary thick ascending limb in normal and high glucose environments
Author(s) -
Carmines Pamela K.,
Mayhan William G.,
Pollock Jennifer S.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a335-c
Subject(s) - protein kinase c , chemistry , medicine , superoxide , endocrinology , nad+ kinase , apocynin , chelerythrine , nad(p)h oxidase , oxidase test , biochemistry , enzyme , biology
Experiments were performed to determine the enzymatic source of glucose‐induced superoxide anion (O 2 ·− ) production by the medullary thick ascending limb (mTAL) and the involvement of protein kinase C (PKC) in this phenomenon. mTAL suspensions prepared from normal rat kidney were incubated 30 min in normal (5.5 mM; NG) or high (20 mM; HG) glucose media. O 2 ·− production (lucigenin chemiluminescence) by the mTALs averaged 484 ± 107 RLU/sec/mg protein ( n = 9) in NG media and was significantly greater in HG media (1146 ± 297 RLU/sec/mg; n = 8). Pre‐incubation in either 100 μM apocynin (APO; NAD(P)H oxidase inhibitor) or 250 μM L‐NAME (NO synthase inhibitor) did not alter O 2 ·− production in NG media, but prevented the stimulatory impact of HG (HG + APO, 204 ± 92 RLU/sec/mg; HG + L‐NAME, 490 ± 168 RLU/sec/mg; both P > 0.05 vs NG). PKC inhibition (30 μM chelerythrine) virtually abolished O 2 ·− production in both NG and HG media. PKC activation (1 μM PMA in NG media; 30 min) markedly increased O 2 ·− production (4712 ± 808 RLU/sec/mg), a response that was ~50% abated by pretreatment with either APO, L‐NAME, or APO + L‐NAME (2389 ± 333, 2392 ± 302, and 2806 ± 467 RLU/sec/mg, respectively). We conclude that both NAD(P)H oxidase and NO synthase contribute to glucose‐stimulated O 2 ·− production by the mTAL. PKC is essential for O 2 ·− production under basal and glucose‐stimulated conditions. Moreover, PKC‐stimulated O 2 ·− production by the mTAL involves, at least in part, NAD(P)H oxidase and NO synthase acting in a sequential manner. (Supported by DK63416)