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. 1999 May 25;96(11):6084-9.
doi: 10.1073/pnas.96.11.6084.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

J G Hoenderop  1 , A B Vaandrager, L Dijkink, A Smolenski, S Gambaryan, S M Lohmann, H R de Jonge, P H Willems, R J Bindels
Affiliations

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

J G Hoenderop et al. Proc Natl Acad Sci U S A. .

Abstract

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Ibeta or a membrane-bound cGK II/Ibeta chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

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Figures

Figure 1
Figure 1
Stimulation of 45Ca2+ uptake by ANP, SNP, and 8-Br-cGMP in freshly isolated CNT and CCD tubules. Freshly immunodissected CNT and CCD tubules were stimulated with ANP (100 nM), SNP (100 μM), and 8-Br-cGMP (100 μM) in the presence of tracer amount 45Ca2+ for 10 min at 37°C. Basal 45Ca2+ uptake is set at 100%, to which all values are related. Values are means ± SEM (n = 5). ∗, P < 0.05, significantly different from basal 45Ca2+ uptake.
Figure 2
Figure 2
Analysis of endogenous and reexpressed cGK I and cGK II in CNT/CCD cells. (A) Immunoblots of samples (20 μg protein each) from homogenates of either freshly immunodissected CNT/CCD tubules, monolayers of cells cultured from these tubules, or monolayers infected with adenoviral-cGK constructs (described in Materials and Methods) were labeled with antibodies against cGK I or cGK II (A). Shown in the right lane of each blot are standards (2 ng) of either purified bovine lung cGK I or recombinant rat intestine cGK II. The 86-kDa protein endogenously present in freshly isolated cells was identified as cGK II, and this was confirmed with immunoblots by using an additional antibody independently raised against cGK II (data not shown). (B) cGK II mRNA was detected by RT-PCR in freshly isolated and cultured CNT/CCD cells. Samples of RT-PCR-derived cGK II (420 bp) and glyceraldehyde-3-phosphate dehydrogenase (309 bp) cDNA were analyzed on an ethidium bromide-stained 2% (wt/vol) agarose gel. PCR was carried out either with (RT+) or without (RT−) reverse transcriptase. RNA (2 μg) was used as starting material for RT-PCRs.
Figure 3
Figure 3
Transcellular Ca2+ transport in monolayers of CNT/CCD cells expressing cGK II. Cultured monolayers were either infected (solid bars) or not infected (open bars) with adenovirus for expressing cGK II. Two days after infection, transcellular Ca2+ transport was measured in the absence (control) and presence of either 100 nM ANP (basolateral), 100 μM SNP (both sides), 100 μM 8-Br-cGMP (both sides), 100 μM 8-Br-cAMP (both sides), or 10 nM d-Arg-8-vasopressin (basolateral). Values are means ± SEM (n = 6). ∗, P < 0.05, significantly different from the control values.
Figure 4
Figure 4
Subcellular localization of cGK proteins expressed in CNT/CCD monolayers. Monolayers were infected with adenovirus for expressing either cGK Iβ (A), a chimer of the N terminus of cGK II linked to the N terminus of full-length cGK Iβ (B), cGK II (C), or a myristoylation-deficient (G2A) cGK II mutant (D). Two days after infection, immunolocalization of the cGK proteins was visualized by confocal laser-scanning microscopy. Membrane-associated (B and C) or cytosolic (A and D) localization was observed. Specificity of cGK immunostaining was confirmed by the absence of signal in noninfected monolayers (not shown). (Bar = 5 μm.)
Figure 5
Figure 5
cGMP-stimulated Ca2+ reabsorption in CNT/CCD monolayers expressing cGK proteins of different membrane-association properties. Monolayers were noninfected or infected with adenovirus for expressing either cGK II, a myristoylation-deficient cGK II mutant (G2A), cGK Iβ, or a chimer of the N terminus of cGK II fused to the N terminus of full-length cGK Iβ. Two days after infection, monolayers either were not stimulated (open bars) or stimulated with 100 nM basolateral ANP (shaded bars) or 100 μM 8-Br-cGMP added to both sides (solid bars). Values are means ± SEM of three experiments. ∗, P < 0.05, significantly different from control values.
Figure 6
Figure 6
Transcellular Na+ transport is not affected by cGK expression. Monolayers of CNT/CCD cells either were noninfected or infected with adenovirus for expressing cGK Iβ or cGK II. Two days after infection, the effect of 100 μM 8-Br-cGMP (both sides, for the period indicated in the figure) on the ISC was determined. Subsequently, transcellular Na+ transport was blocked by addition of 10 μM benzamil to the apical compartment. Representative traces of three experiments are shown.
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