Cyclic GMP-activated channel activity in renal epithelial cells (A6)

In this study, the presence of Na+-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patch-clamp analysis under cell-attached conditions indicated the presence of spontaneously active Na+-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an ∼3:1 Na+/K+permeability-selectivity ratio. The Na+-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na+channel activity. Spontaneous and vasopressin-induced Na+channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the αENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although αENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on αENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na+-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na+transport mechanisms in the mammalian proximal nephron.

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Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.90509.2008 ◽

2009 ◽

Vol 296 (1) ◽

pp. F87-F97 ◽

Cited By ~ 43

Author(s):

Malay K. Raychowdhury ◽

Arnolt J. Ramos ◽

Peng Zhang ◽

Margaret McLaughin ◽

Xiao-Qing Dai ◽

...

Keyword(s):

Epithelial Cells ◽

Signaling Pathway ◽

Primary Cilium ◽

Primary Cilia ◽

Sensory Function ◽

Vasopressin Receptor ◽

Channel Activity ◽

Renal Epithelial Cells ◽

Selective Channel ◽

Cation Selective Channel

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK1 renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R- gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK1 cells stained with anti-V2R antibodies and in LLC-PK1 cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 μM AVP ( trans) or forskolin ( cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

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