Precluded flow-mediated [Ca2 ]i responses even in the presence of the activated PKC and PKA cascades (Fig. 7B). The amplitudes of the flow-mediated [Ca2 ]i response were 33 2 nM in the control and 5 2 nM after treatment with forskolin, PMA, and HC-067047. Overall, we conclude that the coordinated stimulation of both PKC and PKA cascades additively increases the amplitude of the TRPV4mediated [Ca2 ]i response to flow and, importantly, augments the basal TRPV4 activity, resulting in a progressive increase in the resting [Ca2 ]i levels.DISCUSSION It has been recently demonstrated that the activity of the Ca2 -permeable TRPV4 channel is central for [Ca2 ]i elevations in distal nephron cells in response to dynamic changes in tubular fluid flow (11, 12, 16, 20). Adequate mechanosensitive [Ca2 ]i responses are important determinants of many physiological processes in late nephron segments, including flow-dependent K secretion (15, 25), regulatory volume decreases (26), etc. Furthermore, we and others have recently demonstrated that pharmacological stimulation of TRPV4 activity isJULY 12, 2013 VOLUME 288 NUMBERinstrumental for blunting renal cystogenesis in ARPKD models (18, 27). In this study, we defined two distinct intracellular signaling cascades separately controlling TRPV4 trafficking and functional activity in murine distal nephrons. We found that the PKC-dependent signaling pathway is responsible for augmented TRPV4 activation by elevated flow over the apical plasma membrane. In contrast, TRPV4 translocation to the apical plasma membrane is a PKA-dependent process. We have provided substantial experimental evidence that TRPV4 serves as a route of Ca2 influx into distal nephron cells in response to elevated luminal flow. First, we documented that silencing of TRPV4 expression in cultured collecting duct cells disrupts Ca2 responses to shear stress (10). Second, genetic ablation of TRPV4 in mice abolishes flow-induced [Ca2 ]i elevations in the connecting tubule and cortical collecting duct (12).Spartalizumab Consistently, in this study, we have demonstrated that pharmacological inhibition of TRPV4 with the highly selective antagonist HC-067047 precludes changes in [Ca2 ]i during elevations in flow (Fig.Abemaciclib 7).PMID:23892407 Finally, we and others found that the presence of extracellular Ca2 is mandatory for flow-induced [Ca2 ]i elevations in renal cells (10, 28). Therefore, we are confident that monitoring changes in [Ca2 ]i in freshly isolated split-opened murine distal nephrons is a reliable way to assess the rate of TRPV4 activation in native tissue by a physiologically relevant stimulus, i.e. elevated flow. In this study, we did not identify principal and intercalated cells. As we reported previously (12), principal cells exhibit a mildly increased amplitude of flow-mediated [Ca2 ]i responses compared with intercalated cells, and this correlates with higher levels of TRPV4 expression in the former. A similar amplitude of flow-induced increases in [Ca2 ]i in principal and intercalated cells was also reported in microperfused rabbit cortical collecting ducts (29, 30). The detailed analysis did not reveal noticeable heterogeneity in the rate of potentiation of flow-dependent [Ca2 ]i responses in individual cells during activation of PKC signaling with PMA (Fig. 1). Furthermore, we have also observed similar translocation of TRPV4 to the apical membrane in “low TRPV4-expressing” intercalated cells after treatment with forJOURNAL OF BIOLOGICAL CHEMISTRYRegulation of T.
