Giulio Clari

Sequential phosphorylation of protein band 3 by Syk and Lyn tyrosine kinases in intact human erythrocytes: identification of primary and secondary phosphorylation sites

Treatment of intact human erythrocytes with pervanadate induces Tyr (Y)-phosphorylation of the transmembrane protein band 3; in parallel, the activity of the immunoprecipitated tyrosine kinases Syk and Lyn is increased. When erythrocytes are incubated with pervanadate together with PP1, a specific inhibitor of Src kinases, including Lyn, the Y-phosphorylation of band 3 is only partially reduced. Indeed, the PP1-resistant phosphorylation of band 3 precedes and is a prerequisite for its coimmunoprecipitation with Lyn, which interacts with the phosphoprotein via the SH2 domain of the enzyme, as proven by binding competition experiments. Upon recruitment to primarily phosphorylated band 3, Lyn catalyzes the secondary phosphorylation of the transmembrane protein. These data are consistent with the view that band 3 is phosphorylated in intact erythrocytes by both PP1-resistant (most likely Syk) and PP1-inhibited (most likely Lyn) tyrosine kinases according to a sequential phosphorylation process. Similar radiolabeled peptide maps are obtained by tryptic digestion of (32)P-band 3 isolated from either pervanadate-treated erythrocytes or red cell membranes incubated with exogenous Syk and Lyn. It has also been demonstrated by means of mass spectrometry that the primary phosphorylation of band 3 occurs at Y8 and Y21, while the secondary phosphorylation affects Y359 and Y904. (Blood. 2000;96:1550-1557)

Dietary manipulation of Δ-6-desaturase modifies phospholipid arachidonic acid levels and the urinary excretion of calcium and oxalate in the rat: Insight in calcium lithogenesis

An anomalous n-6 polyunsaturated fatty acid composition in plasma and erythrocyte membrane phospholipids, namely increased levels of arachidonic acid (AA), has been reported in calcium nephrolithiasis and has been proposed to play an important role in its pathogenesis. To confirm this, in rats we modified phospholipid AA levels by dietary manipulation of the delta-6-desaturase, the rate-limiting enzyme of the fatty acid biosynthetic pathway, and evaluated the effect on cellular and renal functions predisposing to lithogenesis. Increased AA levels led to conditions at risk for nephrolithiasis: higher oxalate flux and lower sodium cotransport in erythrocytes and a rise in urinary prostaglandin E2, calcium, sodium, and oxalate levels; reduced AA levels reversed these changes. In vitro, in human erythrocytes the incorporation of exogenous AA into membranes increased band 3 protein phosphorylation directly activating the Ser/Thr protein kinase CK1 and induced a parallel raise in band 3-mediated oxalate transport. These findings demonstrate the pivotal role of phospholipid AA in modulating erythrocyte and renal transport of calcium and oxalate.

Further insights about the beneficial effects of n-3 fatty acids in the early molecular events of renal fibrosis in vitro.

BACKGROUNDnAccumulating experimental and clinical evidence reveals beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) in kidney disease by modulating inflammation and fibrosis mechanisms that lead to renal failure.nnnMETHODSnEPA, DHA (n-3 PUFAs) and AA (n-6 PUFA) effects, compared to those of AngII, on renal fibrotic processes at the extracellular matrix (ECM) level were verified in human mesangial cells in vitro, by means of RT-PCR, mitogenic assay and Western-blot analysis.nnnRESULTSnUnlike AngII, EPA and DHA enhanced the expression of MMP2 and DN, a TGFbeta inhibitor, while decreasing mitogenic factors such as PDGF and bFGF, and cell proliferation. Moreover, n-3 PUFAs elicited Bax expression in AngII-treated cells and downregulated COX-2--an enzyme involved in the inflammatory cascade. The mechanism of action could implicate PPARgamma activation, as this transcription factor was shown to translocate to the nucleus upon n-3 PUFA treatment.nnnCONCLUSIONSnThese results complement our previous reports demonstrating that EPA and DHA prevent ECM accumulation and inflammation that typify the fibrotic process, providing new insights into the cellular and molecular mechanisms underlying their beneficial effects. We confirm that n-3 PUFAs could effectively counteract kidney fibrosis development providing a rationale for their use in clinical settings.

Serum Potassium, Thiazides, Aldosterone, and Mineralocorticoid Receptors

To the Editor:nnI read with interest the article by Alderman et al1 on the clinical significance of hypokalemia and hyperkalemia in the antihypertensive treatment and prevention of cardiovascular complications. The conclusion was that, in most patients, concerns about potassium levels should not influence the clinicians decision about initiating hypertension treatment with low-moderate doses of thiazides. Chlorthalidone reduces the risk of congestive heart failure, in comparison with lisinopril and amlodipine,1 but it also activates the renin-angiotensin-aldosterone system. We address the issue that, beyond plasma potassium levels, during thiazides, the activation of the renin-angiotensin-aldosterone system and, in particular, the stimulation of mineralocorticoid receptors (MRs) are key determinants in the inflammatory process and the progression of …