Modulation Of Activity Of Retinal Bipolar Cell By Brain Natriuretic Peptide

Natriuretic peptides (NPs; ANP, BNP and CNP) comprise a family of structurally related peptides, which are derived from three different genes and share a 17-amino acid internal ring. In addition to their peripheral involvement in diuresis and blood pressure regulation, NPs and their corresponding receptors (NPR-A, NPR-B and NPR-C) are found throughout the central nervous system (CNS) and may represent an important neuromodulatory system in the CNS. While NP system is known to be involved in modification of emotional behaviours, the sequelae of stress hormone release, foodtake, arousal and autonomic nervous system activation, little is known about the regulation by NPs of neurotransmitter receptors. Bipolar cells (BCs) are second-order neurons, which convey signal from photoreceptors to amacrine cells and ganglion cells in the retina. Two types of BCs, ON type BCs and OFF type BCs, generate ON and OFF signals through activation of distinct glutamate receptors. In the mammalian retina BCs are exclusively driven by either rods or cones. All rod BCs seem to be of ON type whereas cone BCs are of either ON type or OFF type. Here in this work, we focus on Rod-BCs. GABAa and GABAc receptors, glycine receptor and metabotropic glutamate receptor (mGluR) are localized on Rod-BCs.In this work, using the immunohistochemistry, whole-cell patch clamp recording and calcium imaging techniques, we report, for the first time, modulation of GABA receptor mediated currents by BNP on Rod-BCs.Firstly, using immunohistochemisty and confocal microscope techniques, we show the extensive expression of NPs and their receptors in rat retinal neuronal and glial elements. It was revealed that ANP-, BNP-and CNP-Immunoreactivity (IR) were seen in the OPL, IPL and INL. Moreover, the expression profiles for NPR-A and NPR-B are rather similar in that the distribution of these two receptors was observed in OPL, INL and INL. Interestingly, labeling for NPR-A and NPR-B was observed in all parts of both PKC-positive BCs, most of which may be of the Rod-BC, including the dendrites, somata, axons and axon terminals, and PKC-negative BCs, which may be of the OFF type. It is known that NPs can increase intracellular guanosine-3',5'-cyclic monophosphate (cGMP) concentraction via activation of particle guanylyl cyclase (pGC). Using patch clamp recording, it was shown that BNP induced cGMP-gated nonselected cation channel (CNC) mediated currents from Rod-BCs, as did by cGMP and 3-isobutyl-1-methylxanthine (IBMX).The fact that NPRs are localized in the Rod-BC suggests that NP/NPR system may be involved in information processing of the Rod-BCs. We first characterized GABA-induced currents (I_GABA) in Rod-BCs. The current response to 50μM muscimol, a GABA_A receptor agonist, showed an initial transient component (I_GABA(A)), which desensitized to a steady level with a time constant of 1．24 s. In contrast, the current induced by 200μM CACA (I_<sub>GABA(C)), a GABA_C receptor agonist, was rather sustained and did not show significant desensitization. Baclofen, a GABA_B receptor agonist, could not induce any discernable current from Rod-BCs. The average reversal potential was 1．2±2．4 mV and-1．4±3．2 mV for I4AA-and BIC-sensitive currents, respectively. The dose-response relationships of GABA_A and GABA_C currents yielded an EC₅₀ of 51．3±6．2μM and 6．0±0．6μM, respectively. These data indicated that GABA_A-and GABA_C-but not GABA_B-receptor are expressed on Rod-BC. Further experiments revealed that BNP greatly suppressed GABA_A currents, but not GABA_C currents, which was mediated by NPR-A since the effect could be blocked by anantin, a specific antagonist of NPR-A, and HS-142-1, a specific antagonist for pGC. Moreover, cANF, an agonist of NPR-C, did not mimic the BNP effect, suggesting no involvement of NPR-C. Focal application of 1mM GABA to the dendrite/soma or axon terminal could induce I_GABA of different amplitudes, with one induced at the axon terminal being larger (214．3±20．5 vs 108．9±25．5) . Interestingly, BNP suppressed the I_GABA induced at the terminal, but without effects on the I_<sub>GABA at the dendrite/soma.Intracellular mechanisms underlying the suppression of I_GABA by BNP were further studied. 8Br-cGMP, like BNP, suppressed GABA_A, but not GABA_C currents, and selective PKG inhibitor KT5823 greatly reduced the suppression effect of BNP on I_GABA, suggesting the involvement of the cGMP/PKG pathway. On the other hand, calcium imaging showed that NPs significantly increased [Ca²⁺]_i at the axon terminal of Rod-BC, but the change in [Ca²⁺]_i was much smaller at the soma/dendrite. No significant change in GABA responses was detected when extracellular Ca²⁺-free solution was delivered along with 10 mM EGTA, a calcium chelator, and application of 50 nM BNP persisted to reduce the GABA currents significantly. In contrast, intracellular Ca²⁺-free solution, buffered with 10 mM BAPTA, significantly increased the GABA current. During intracellular infusion of Ca²⁺-free solution, application of 50 nM BNP failed to suppress the GABA currents. These results strongly suggest that suppression of I_GABA by BNP was not due to the influx of extracellular calcium. During internal application of the inositol 1,4,5-trisphosphate receptor (IP₃R) antagonists, heparin and xestospongins-C (Xe-C), BNP persisted to suppress I_GABA. In contrast, following the application of ryanodine receptor (RyaR) modulators caffeine, ryanodine or ruthenium red, I_GABA was reduced, and the BNP caused suppression was no longer observed. Furthermore, the BNP effect was greatly reduced by the calmodulin (CaM) inhibitors W-7 and calmidazolium (CMZ). In sum, BNP increased cGMP levels via NPR-A thereby promote the release of calcium from ryanodine sensitive stores through the PKG pathway. The increased [Ca²⁺]_i levels leads to activation of CaM, resulting in the suppression of GABA_A receptors.We also investigated modulatory effects of BNP on glycine receptors on Rod-BCs. I_Gly induced from Rod-BCs was strychnine-sensitive, with an EC₅₀ of 40．5±5．2μM. BNP had no effect on I_Gly. It was further found that I_Gly could only be induced from the dendrite/soma. Since BNP could not increase [Ca²⁺]_i at the dendrite/soma, it was understandable why BNP did not change I_Gly.