Abstract
Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.
Highlights
PAR1 and Proteinase-activated receptors 2 (PAR2) are expressed in most tissues, where their stimulation is coupled to the activation of phospholipase C (PLC)  and, secondarily, to that of various protein kinases C (PKCs), which often trigger ERK1,2 phosphorylation [2,3,4]
PAR2 may be involved in the control of kidney ion transport because 1) it is expressed in renal tubule epithelial cells [5], 2) in many epithelia, including distal colon, pancreatic duct, and airways, activation of PAR2 induces chloride secretion through activation of calcium-dependent channels [12,13,14,15,16], and 3) similar observations have been made in M1 cells, a mouse cell line derived from renal collecting duct principal cells [5]
Expression of PAR2 in the cortical thick ascending limb of Henle’s loop (cTAL)—Because PAR2 are classically coupled to activation of PLC and calcium signaling, we attempted to demonstrate functional expression of PAR2 in the cTAL by searching for changes in [Ca2ϩ]i induced by activation of PAR2
Summary
After blocking in TBS-Nonidet P-40 (50 mM Tris base, 150 mM NaCl, 0.2% Nonidet P-40) containing 10% low ride, 1 mM CaCl2, 50 mM Tris-HCl, pH 7.4) after treatment with fat milk, blots were successively incubated with anti-ERK antiinhibitors and PAR2 agonists, and total ATPase activity was body (p44/42 MAP kinase antibody, Cell Signaling Technology, determined in a solution containing 10 mM MgCl2, 1 mM 1/500 in TBS-Nonidet P-40 ϩ milk) and anti-rabbit IgG antiEDTA, 100 mM Tris-HCl, 10 mM Tris-ATP, 5 nCi/l body coupled to horseradish peroxidase With or without agonist peptide SLIGRL-NH2 (AP) (10 min at ERK was calculated, and in each experiment, these ratios in 37 °C), pools of 40 cTALs were incubated in bovine serum albu- different experimental conditions were expressed as the permin-free microdissection solution containing 1.5 mg/ml EZ- centage of the control group. MEK, MAP kinase/ERK kinase; PTK, protein tyrosine kinases; PI3K, phosphatidylinositol 3-kinase
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