Does the circadian clock make RPE-mediated ion transport “tick” via SLC12A2 (NKCC1)?

Figures & data

Figure 1. The circadian clock likely regulates ion transport by the ARPE-19 monolayers via NKCC1. a. Both ARPE-19 monolayers (ARPE-19M) and dispersed cell cultures (ARPE-19CC) expressed SLC12A2 mRNA. Amplification products were confirmed by sequencing. b. Confocal microscopy revealed that NKCC1 localized on the apical membrane of ARPE-19 monolayers (white arrows). c. The mRNA expression of SLC12A2 was rhythmic in ARPE-19 monolayers as determined by RT-PCR analysis. d. In RPE monolayers, two-way ANOVA revealed that apical supernatants had higher Na⁺, K⁺ and Cl⁻ concentrations than basolateral supernatants. Time significantly affected K⁺ concentrations and tended to affect Na⁺. e. Spearman’s correlation analysis of the expression of SLC12A2 mRNA and the calculated inter-compartmental ion concentration gradients or fluid difference (Api – BL). Values are plotted c, e. or represented as means ± SEM d. n = 3.

Table 1. Statistical analysis of rhythmicity in ARPE-19 supernatant components. Units for ion amplitudes are in mmol/l, for fluid amplitudes in ml. Data were fitted to the equation y = y₀ + c⋅cos[2π⋅(t-ϕ)/τ] by non-linear regression. Bold values represent p < .05.

Compartment	Apical				Basolateral
Component	Na^+	K^+	Cl^−	Fluid	Na^+	K^+	Cl^−	Fluid
Amplitude	3.52 ± 0.92	0.093 ± 0.025	3.50 ± 0.93	0.057 ± 0.017	2.22 ± 1.36	0.064 ± 0.03	2.07 ± 1.38	0.018 ± 0.016
Acrophase (h)	10.48 ± 1.05	10.39 ± 1.06	10.39 ± 1.06	22.66 ± 1.28	13.75 ± 2.18	12.87 ± 1.89	13.59 ± 2.41	6.36 ± 3.08
Period (h)	20.00 ± 2.21	20.00 ± 2.23	20.00 ± 2.22	26.50 ± 2.45	20.00 ± 4.20	20.00 ± 3.78	20.00 ± 4.58	20.00 ± 4.46
P-value	0.013	0.015	0.015	0.04	0.45	0.25	0.52	0.72