Korean J Physiol Pharmacol.  2012 Jun;16(3):211-217. 10.4196/kjpp.2012.16.3.211.

Activation of the cGMP/Protein Kinase G Pathway by Nitric Oxide Can Decrease TRPV1 Activity in Cultured Rat Dorsal Root Ganglion Neurons

• Jin Y
• Kim J
• Kwak J

• KMID: 1768016
• DOI: http://doi.org/10.4196/kjpp.2012.16.3.211

Abstract

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents (Icap). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on Icap. Membrane-permeable cGMP analogs, 8-bromoguanosine 3', 5'-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on Icap. The PKG inhibitor KT5823 prevented the inhibition of Icap by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.

Keyword

Dorsal root ganglion neuron; Nitric oxide; Protein kinase G; Rat; TRPV1

MeSH Terms

Animals
Benzoates
Carbazoles
Cyclic GMP-Dependent Protein Kinases
Ganglia, Spinal
Guanosine
Guanylate Cyclase
Hemoglobins
Humans
Imidazoles
Neurons
Nitric Oxide
Nitroprusside
Penicillamine
Phosphotransferases
Proteins
Rats
Receptors, Cytoplasmic and Nuclear
Sensory Receptor Cells
Spinal Nerve Roots
Tissue Donors
Benzoates
Carbazoles
Cyclic GMP-Dependent Protein Kinases
Guanosine
Guanylate Cyclase
Hemoglobins
Imidazoles
Nitric Oxide
Nitroprusside
Penicillamine
Phosphotransferases
Proteins
Receptors, Cytoplasmic and Nuclear

Figure

• 

  Fig. 1 Effects of the NO donors, SNP and SNAP, on Icap in cultured DRG neurons. (A) Application of SNP (100 µM) with capsaicin (CAP, 0.3 µM) decreased Icap significantly (n=22). The representative trace shows Icap in the control and during application of SNP at a holding potential of -60 mV. Filled bars on the trace indicate the application of capsaicin for 20 s. The bar graph summarizes the effects of SNP on Icap. (B) SNAP (100 µM) co-applied with capsaicin also inhibited Icap significantly (n=13). The bar graph summarizes the effect of SNAP on Icap. (C) Dose-response curve for SNP on Icap inhibition. The curve was fitted using the Hill equation (IC50=0.67 µM). Data are expressed as a percentage of the control capsaicin response, represented by Icap in the absence of SNP, and are plotted as means±SEM. Numbers in parentheses indicate the number of cells tested. Asterisks indicate significant differences relative to the control, at **p<0.01.

• 

  Fig. 2 Effects of the NO scavengers, hemoglobin and carboxy-PTIO, on SNP inhibition of Icap. (A) The representative trace shows Icap during the control, application of SNP, and application of SNP in the presence of hemoglobin (Hb). Hemoglobin eliminated the inhibition of Icap caused by subsequent application of SNP. (B) The representative tracing shows Icap during the control, application of SNP, and application of SNP in the presence of carboxy-PTIO. Carboxy-PTIO also prevented the inhibition of Icap caused by subsequent application of SNP. Filled bars on the trace indicate the application of capsaicin for 20 s, and the open bar represents the application of the NO scavengers. (C) The bar graph summarizes the effects of SNP on Icap in the presence (filled bar) and absence (open bar) of the NO scavenger. Data are presented as means±SEM. Asterisks indicate significant differences at **p<0.01.

• 

  Fig. 3 Involvement of guanylyl cyclase and cGMP in the effects of SNP on Icap. (A) The representative trace shows Icap during the control situation, the application of 100 µM SNP, and the application of SNP in the presence of 10 µM ODQ. Pretreatment with ODQ partially blocked the inhibitory effect of SNP on Icap (n=6). (B) The representative trace shows Icap during the control situation, the application of 100 µM SNP, and the application of SNP in the presence of 1 mM IBMX, a phosphodiesterase inhibitor. The inhibitory effect of SNP was augmented by pretreatment with IBMX (n=9). Filled bars on the trace indicate the application of capsaicin for 20 s, and the open bar indicates the application of the inhibitor. (C) Each representative trace shows the effects of membrane-permeable analogs of cGMP, 8-Br-cGMP (100 µM) and 8pCPT-cGMP (100 µM), on the capsaicin responses. Both 8-Br-cGMP and 8-pCPT-cGMP mimicked the inhibitory effect of SNP on Icap. (D) The NO-independent guanylyl cyclase activator YC-1 (30 µM) decreased Icap. (E) Summary data show the effect of SNP on the amplitude of Icap before and after treatment with ODQ and IBMX. (F) The bar graph summarizes the effects of the membrane-permeable analogs of cGMP and YC-1 on Icap. Data are presented as means±SEM. Asterisks indicate significant differences at **p<0.01, vs. control.

• 

  Fig. 4 Effect of the protein kinase G inhibitor, KT5823 on the inhibitory effect of SNP on Icap. (A) KT5823 was applied for 1 min before SNP application. The representative trace shows Icap during the control situation, the application of 100 µM SNP, and the application of SNP in the presence of 1 µM KT5823. Pretreatment with KT5823 potently blocked SNP-induced Icap inhibition and even increased the amplitude of Icap (n=7). Each representative trace shows the effect of SNP in the absence and presence of KT5720 or BIM (10 µM). (B) The bar graph summarizes the effects of SNP on the Icap in the presence (filled bar) and absence (open bar) of the kinase inhibitors. Data are presented as means±SEM. Asterisks indicate significant differences at **p<0.01.

• 

  Fig. 5 S-nitrosylation of TRPV1 by 100 µM SNP. (A) The upper trace shows that SNP (100 µM) alone did not evoke a membrane current at -60 mV in DRG neurons. The lower trace shows that SNAP (100 µM) itself also failed to change the holding current. (B) Dithiothreitol (DTT, 10 mM) was pretreated for 1 min before application of SNP and capsaicin to reduce free sulhydryl groups on TRPV1. The representative trace and bar graph show the effect of SNP in the presence of DTT (n=4).

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