Differential regulation of TRPC4 in the vasopressin magnocellular system by water deprivation and hepatic cirrhosis in the rat

Abstract

Transient receptor potential canonical subtype 4 (TRPC4) is expressed in the magnocellular paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. In this study, the regulation of TRPC4 expression was investigated in water deprivation and hepatic cirrhosis. We used laser capture microdissection technique for precise dissection of pure AVP cell population in the PVN and SON followed by quantitative real-time RT-PCR, and immunodetection techniques by Western blot analysis and immunofluorescence. Bile duct ligation elevated TRPC4 transcripts in the SON but not PVN with correlated changes in the protein expression in these regions, as well as increased colocalization with AVP in the SON, with no changes in the PVN. Water deprivation resulted in increased TRPC4 mRNA expression in the PVN, while it decreased channel expression levels in the SON. In both of these regions, protein expression measured from tissue punches were unaltered following water deprivation, with no changes in the number of TRPC4-positive cells. Thus, TRPC4 expression is differentially regulated in physiological and pathophysiological models of vasopressin release.

Keywords: TRPC4; bile duct ligation; osmolality; vasopressin; water deprivation.

Fig. 1.

TRPC4 mRNA expression by real-time RT-PCR from 7–10 laser microdissected vasopressin cells from the PVN (A) and SON (B) in BDL vs. sham. The data depict means ± SE mRNA levels as calculated by the 2^−ΔΔCT method. *P < 0.05 compared with sham ligation. n = 5 or 6 for each group. Western blot quantification and representative immunoblots of TRPC4 protein abundance in the brain punches from PVN (C and D) and SON (E and F) of BDL vs. sham. S and B in the immunoblots represent sham and BDL, respectively. The data are presented as means ± SE estimated by relative intensity by normalizing with GAPDH. n = 6 for each group.

Fig. 2.

TRPC4 mRNA expression by real-time RT-PCR from 7–10 laser-microdissected vasopressin cells from the paraventricular nucleus (PVN; A) and supraoptic nucleus (SON; B) in 48-h WD vs. controls. The data depict means ± SE mRNA levels as calculated by the 2^−ΔΔCT method. *P < 0.05 compared with sham ligation. n = 7 or 8 for each group. Western blot quantification and representative immunoblots of TRPC4 protein abundance in the brain punches from PVN (C and D) and SON (E and F) of 48 h WD vs. controls. C and W in the immunoblots represent control and water deprivation, respectively. The data are presented as means ± SE estimated by relative intensity by normalizing with GAPDH. n = 6 for each group.

Fig. 3.

Specificity of TRPC4 antibody in the magnocellular hypothalamic regions. Specific TRPC4 staining in the SON (A) and PVN (B) is completely abolished by use of TRPC4 blocking peptide in the SON (C) and also in the PVN (D). Scale bars represent 100 μm for these images. E: Western blots show complete elimination of TRPC4-immunoreactive bands using TRPC4-blocking peptide.

Fig. 4.

Confocal ×60 pseudocolored images of AVP and TRPC4 colocalization in the SON of sham (A–C) vs. BDL (D–F) and in the PVN of sham (G–I) vs. BDL (J–L). Red immunofluorescence represents AVP staining (A, D, G, and J) while green immunofluorescence represents TRPC4 staining (B, E, H, and K). Merged images of AVP and TRPC4 are represented by panels C, F, I, and L. Scale bar = 10 μm for all images.

Fig. 5.

Quantitative estimates of AVP, TRPC4, and colocalization in the PVN (A) SON (B) from sham and BDL. *P < 0.05 compared with sham ligation; n = 6 for each group.

Fig. 6.

Confocal ×60 pseudocolored images of AVP and TRPC4 colocalization in the PVN of control (A–C) vs. 48 h WD (D–F) and in the SON of control (G–I) vs. 48 h WD (J–L). Red immunofluorescence represents AVP staining (A, D, G, and J) while green immunofluorescence represents TRPC4 staining (B, E, H, and K). Merged images of AVP and TRPC4 are represented by panels C and F. Scale bar = 10 μm for all images.

Fig. 7.

Quantitative estimates of AVP, TRPC4, and colocalization in the PVN (A) and SON (B) from control vs. 48 h WD. *P < 0.05 compared with water deprivation; n = 6 for each group.