Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors

Abstract

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC's (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (alpha/beta) beta1 isoform of the beta subunit of NOGC (NOGCbeta1) was specifically focused. NOGCbeta1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCbeta1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCbeta1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 +/- 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 +/- 3.4 vs. 1.2 +/- 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCbeta1 abundance was reduced using specific siRNA to NOGCbeta1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCbeta1 (530.2 +/- 141.4 vs. 26.1 +/- 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCbeta1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 +/- 22 vs. 92 +/- 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCbeta1 and GC-A interact and that NOGCbeta1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.

Fig. 1

Panel A: siRNA against NOGCβ1 decreases basal and ANP-stimulated intracellular cGMP levels in HK-2.HK-2 cells (jumbled siRNA-white bars) and HK-2 cells transfected with siRNA directed against N-terminal part of NOGCβ1 (black bars) were treated with ANP for 45 min (ANP), L-NAME for 16 h (L-NAME) and both L-NAME and ANP and cGMP assay was carried out as described in “Materials and methods” section. Inset: Representative immunoblot with actin, GC-A, and NOGCβ1 specific polyclonal Abs showing abundance of GC-A, and NOGCβ1 levels (arrows with GC-A and NOGCβ1) in cells transiently transfected with jumbled siRNA or siRNA to NOGCβ1. * P < 0.05, ** P < 0.01; n = 4. Panel B: siRNA against NOGCβ1 decreases ANP-stimulated membrane guanylyl cyclase activity. HK-2 cells were transfected with siRNA duplex oligonucleotides and incubated in the presence or absence of 10⁻⁷ M ANP for 45 min. Membrane fractions were collected and membrane guanylyl cyclase activity was determined as described in “Materials and methods” section. Experiments are repeated twice. * P < 0.05; n = 4

Fig. 2

siRNA to NOGCβ1 blocks ANP-mediated inhibition of endogenous Na⁺/K/ATPase activity in HK-2 cells pre-treated with L-NAME. HK-2 cells (control) and HK-2 cells were pretreated with L-NAME (1 mM × 16 h) to inhibit endogenous NO formation. Cells were transfected with jumbled siRNA (2 μg) or siRNA to NOGCβ1 (2 μg) and treated with 10⁻⁷ M ANP (45 min) as indicated. * P < 0.05; n = 3

Fig. 3

NOGC β1 over expression increases basal and ANP-stimulated cGMP formation in HK-2 cells. HK-2 cells were transiently transfected with pcDNA3.1 vector (white bars) or NOGCβ1 expression plasmid (NOGCβ1, black bars) and incubated with IBMX (1 mM) and ANP (10⁻⁷ M × 45 min), L-NAME (1 mM × 16 h), ANP and L-NAME, or ANP and H-3048, an inhibitor of ANP (10⁻⁶ M × 45 min). At the end of treatment periods, the cells were harvested and cell lysates were subjected to cGMP assay as described in “Materials and methods” section; n = 4. Inset: Western analysis with Actin, GC-A, and NOGCβ1 specific polyclonal Abs showing GC-A abundance (arrow with GC-A), and NOGCβ1 levels (arrow with NOGCβ1) in cells transfected with vector (pcDNA3.1) or NOGCβ1 expression plasmid (NOGCβ1 o/e). n = 4; * P < 0.05, ** P < 0.01

Fig. 4

a YFP-tagged NOGCβ1 and GC-A localize to membrane in HK-2 cells. NOGCβ1: Confocal images of HK-2 cells transiently co-transfected with YFP-tagged NOGCβ1 and His-tagged GC-A (see “Materials and methods” section). Upper panels: control pIRES-YFP vector (yellow fluorescence): NOGCβ1: pIRES-NOGCβ1-YFP transfected cells (Green fluorescence); GC-A: Immunostaining with polyclonal Ab to His tag GC-A. (red fluorescence). Merged images of NOGCβ1 and GC-A. m membrane, n nucleus. Middle panel: NOGCβ1: HK-2 cells transfected with YFP-tagged NOGCβ1[YFP fluorescence (green)]. Caveolin: Immunostaining with polyclonal Ab to caveolin (red fluorescence); merged images of NOGCβ1 and caveolin. Lower panel: Caveolin: Immunostaining with polyclonal Ab to caveolin (red fluorescence); HIS GC-A: Immunostaining with polyclonal Ab to His tag GC-A. (blue fluorescence); merged images of HIS GC-A and caveolin. b NOGCβ1 interacts with GC-A. Human Kidney-2 (HK-2) cells were harvested and cell pellets lysed in RIPA buffer. Cell extracts were immunoprecipitated with control IgG (control) anti GC-A Ab (IP-GCA) and NOGCβ1 identified on Western analysis using a polyclonal anti NOGCβ1 Ab as described in “Materials and methods” section. Total lysate “Input” (Color figure online)