Role of the calcium/calmodulin‐dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters¹

Abstract

The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium-dependent dicarboxylate transporter are modulated by the calcium/calmodulin-dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H-PAH was used as marker substance of the anion transporter, and 14C-glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10(-7) M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 10(-5) M. In contrast to PAH transport, tubular 14C-glutarate uptake was not affected by KN93 (10(-5) M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca(2+)-ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca(2+)-ionophore could be abolished by KN93, but not by Rp-cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

Molecular and Cellular Physiology of Renal Organic Cation and Anion Transport

“…The extent to which this regulatory response to PKC activation occurs in intact tubules is not clear in light of the observation that exposure of isolated rabbit S2 segments to phorbol ester appears to have no effect on the rate of Na ϩ -dependent glutarate transport (359). In addition, exposure to PD98059 (MAPK inhibitor), genistein (tyrosine kinase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), or KN93 (CaMII inhibitor) has no effect on basolateral glutarate uptake in intact tubules (118,119).…”

“…Inhibition of CKII activity has been shown to decrease PAH uptake into isolated rabbit RPT (118). Activation of PKA with forskolin has also been shown to inhibit PAH uptake in isolated rabbit RPT (133).…”

Calmodulin-Mediated Regulation of Bicarbonate and Lactate Transports in Rat Jejunum

“…Following calcium binding calmodulin becomes loaded and can interact with one of its many target proteins in the cell, either directly or by phosphorylation-mediated processes. Among the members of Ca 2+ /CaM -dependent protein kinases, the ubiquitous CaMKII is known to modulate the activity of several transport mechanisms [1,[5][6][7]. Results of the enzymatic assays reported here show that both basolateral and brush border membranes of jejunal enterocytes contain CaMKII, even if the kinase seems to be mostly located in the cytoplasm; it has been reported that also in other cell types the subcellular localization of the enzyme is predominantly in cytoplasmic organules [23].…”

“…Multifunctional Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous enzyme involved in carbohydrate, lipid and amino acid metabolism, neuro-transmitter synthesis and release, and regulation of ion channels, cytoskeleton and gene expression [3,4]. Several studies have demonstrated that various epithelial transporters, which allow the movements of solutes across the apical and basolateral plasma membranes, are regulated via a mechanism involving CaMKII [1,[5][6][7].…”

Regulation of Taurocholate Transport in Freshly Isolated Proximal Tubular Cells of the Rat Kidney by Protein Kinases

“…As a first approach to identify individual protein kinases involved in the regulation of tubular taurocholate transport, the effect of specific activators or inhibitors of a series of protein kinases (PKA, PKG, PKC, PI3 kinase, CaM kinase II, MAP kinases, tyrosine kinases) on 3 H-taurocholate uptake into proximal tubular cells were studied. These protein kinases were selected because they have been shown previously to be involved in the regulation of transmembrane transporters in the proximal tubular cells of the kidney [15,16,17,18,19,20,21,22,23]. To obtain results most closely related to natural conditions the studies were performed on proximal tubular cells which were freshly isolated from rat kidneys.…”