Modulation by Brain Natriuretic Peptide of GABA Receptors on Rat Retinal ON-Type Bipolar Cells

Y.-C. Yu
2006 Journal of Neuroscience  
Natriuretic peptides (NPs) may work as neuromodulators through their associated receptors [NP receptors (NPRs)]. By immunocytochemistry, we showed that NPR-A and NPR-B were expressed abundantly on both ON-type and OFF-type bipolar cells (BCs) in rat retina, including the dendrites, somata, and axon terminals. Whole-cell recordings made from isolated ON-type BCs further showed that brain natriuretic peptide (BNP) suppressed GABA A receptor-, but not GABA C receptor-, mediated currents of the
more » ... currents of the BCs, which was blocked by the NPR-A antagonist anantin. The NPR-C agonist c-ANF [des(Gln 18 , Ser 19 , Gln 20 , Leu 21 , Gly 22 )ANF 4 -23 -NH 2 ] did not suppress GABA A currents. The BNP effect on GABA A currents was abolished with preincubation with the pGC-A/B antagonist HS-142-1 but mimicked by application of 8-bromoguanosine-3Ј,5Ј-cyclomonophosphate. These results suggest that elevated levels of intracellular cGMP caused by activation of NPR-A may mediate the BNP effect. Internal infusion of the cGMP-dependent protein kinase G (PKG) inhibitor KT5823 essentially blocked the BNP-induced reduction of GABA A currents. Moreover, calcium imaging showed that BNP caused a significant elevation of intracellular calcium that could be caused by increased calcium release from intracellular stores by PKG. The BNP effect was blocked by the ryanodine receptor modulators caffeine, ryanodine, and ruthenium red but not by the IP 3 receptor antagonists heparin and xestospongin-C. Furthermore, the BNP effect was abolished after application of the blocker of endoplasmic reticulum Ca 2ϩ -ATPase thapsigargin and greatly reduced by the calmodulin inhibitors W-7 and calmidazolium. We therefore conclude that the increased calcium release from ryanodine-sensitive calcium stores by BNP may be responsible for the BNP-caused GABA A response suppression in ON-type BCs through stimulating calmodulin. In the present work, we show the expression of NPR-A and NPR-B in rat BCs by immunocytochemistry and further demonstrate by patch-clamp techniques that BNP suppresses GABA A receptor-, but not GABA C receptor-, mediated currents through activation of NPR-A. Our results suggest that activation of the cGMP/PKG pathway, inducing an increase in calcium release from ryanodine-sensitive intracellular stores, may be responsible for modulation by BNP of GABA A responses through stimulating calmodulin (CaM). Materials and Methods Immunohistochemistry and confocal microscopy. The eyecups were prepared from adult male albino rats (Sprague Dawley) as described previously (Tian et al., 2003) . Animals were anesthetized deeply with 40 mg/ml sodium pentobarbital and killed by decapitation. Adequate care was taken to minimize pain and discomfort to animals in accordance with the National Institutes of Health guidelines for animal experimentation. The posterior eyecups were fixed in 0.1 M phosphate buffer (PB), pH 7.4, with 4% formaldehyde for 20 min at 4°C. After cryoprotected in 10, 20, and 30% (w/v) sucrose in 0.1 M PB for 2 h each at 4°C, the eyecups were sectioned vertically at 14 m thickness with a cryostat. The sections were then blocked and permeabilized with 6% donkey serum, 0.2% Triton X-100 in PBS overnight at 4°C and incubated with primary antibodies (rabbit polyclonal antibodies against NPR-A and NPR-B; Abcam, Cambridge, UK) at working dilutions of 1:200 and 1:400, respectively, for 3 d at 4°C in a medium containing 3% normal donkey serum, 0.2% Triton X-100, and 1% bovine serum albumin. After rinsing with PBS, the sections were incubated with the secondary antibodies to reveal binding sites. For double immunofluorescence labeling, one of the primary antibodies was used to immunolabel NPRs, and the labeling was revealed with Texas Red dye-conjugated donkey anti-rabbit IgG (1:100 dilution; Jackson ImmunoResearch, West Grove, PA). The mouse anti-PKC monoclonal antibody (1:4000 dilution; Sigma, St. Louis, MO), a specific marker of rat ON-type BCs, on the other hand, was used to label BCs, and the labeling was revealed with the donkey anti-mouse IgG tagged with fluorescein isothiocyanate (1:100 dilution; Jackson ImmunoResearch). Omission of one primary antibody yielded only the immunoreactivity for the remaining antibody, and omission of both abolished any immunolabeling. Staining by a mixture of the two secondary antibodies after incubating with one of the two secondary antibodies showed no crossreactivity of species-specific secondary antibodies. Immunohistochemical experiments were also performed on isolated BCs with different morphology. Retinal neurons were dissociated by enzymatic and mechanical treatments, following the procedure used for preparing isolated cells for patch-clamp recording (see below). Isolated BCs were placed on a slide in PBS for 30 -60 min at room temperature and fixed with 4% paraformaldehyde in 0.1 M fresh PB for 30 min, rinsed with PBS three times, and blocked for 1 h in PBS with 6% donkey serum plus 0.2% Triton X-100. The cells were then incubated with the primary antibodies for 2 h and further incubated with the secondary antibodies for 30 min at room temperature. The sections/cells were visualized with a Leica (Mannheim, Germany) SP2 confocal laser scanning microscope using a 40ϫ oil-immersion objective lens. Single optical sections were taken through the preparation and recorded digitally. To avoid any possible reconstruction stacking artifact, double labeling was precisely evaluated by sequential scanning on single-layer optical sections at intervals of 1.0 m. Western blot analysis. Rat retinal extracts were prepared following the procedure described in detail previously (Chen et al., 2004) , with minor modifications. The extract samples (2.0 mg/ml, 20 l) were loaded, subjected to 12% SDS-PAGE, and electroblotted onto polyvinylidene difluoride membranes using Mini-Protean 3 electrophoresis system and Mini Trans-Blot electrophoretic transfer system (Bio-Rad, Hercules, CA). The membranes were blocked in blocking buffer consisting of 20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 0.1% Tween 20, and 20% nonfat milk at room temperature for 2 h and then incubated with the antibody against NPR-A or NPR-B, at working dilutions of 1:500 and 1:1000, respectively, overnight at 4°C. The blots were washed, incubated with horseradish
doi:10.1523/jneurosci.3653-05.2006 pmid:16407567 fatcat:5o2scd4zxrewjdi5iojqmiicx4