Cysteine-rich Protein 2, a Novel Substrate for cGMP Kinase I in Enteric Neurons and Intestinal Smooth Muscle

Huber, Andrea; Neuhuber, Winfried; Klugbauer, Norbert; Ruth, Peter; Allescher, Hans–Dieter

doi:10.1074/jbc.275.8.5504

Cited by 42 publications

(49 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…20), substance P (1:500; Santa Cruz Biotechnology), NeuN (1:1,000; Chemicon), N200 (1:1,000; Chemicon), and nNOS (1:500; Alexis Biochemicals, Grünberg, Germany). Binding sites were visualized with Alexa488-or Alexa568-conjugated species-specific secondary antibodies (Molecular Probes).…”

Section: Methodsmentioning

confidence: 99%

Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP-dependent protein kinase I

Tegeder

Turco²,

Schmidtko³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

cGMP-dependent protein kinase I (PKG-I) has been suggested to contribute to the facilitation of nociceptive transmission in the spinal cord presumably by acting as a downstream target of nitric oxide. However, PKG-I activators caused conflicting effects on nociceptive behavior. In the present study we used PKG-I ؊/؊ mice to further assess the role of PKG-I in nociception. PKG-I deficiency was associated with reduced nociceptive behavior in the formalin assay and zymosan-induced paw inflammation. However, acute thermal nociception in the hot-plate test was unaltered. After spinal delivery of the PKG inhibitor, Rp-8-Br-cGMPS, nociceptive behavior of PKG-I ؉/؉ mice was indistinguishable from that of PKG-I ؊/؊ mice. On the other hand, the PKG activator, 8-Br-cGMP (250 nmol intrathecally) caused mechanical allodynia only in PKG-I ؉/؉ mice, indicating that the presence of PKG-I was essential for this effect. Immunofluorescence studies of the spinal cord revealed additional morphological differences. In the dorsal horn of 3-to 4-week-old PKG-I ؊/؊ mice laminae I-III were smaller and contained fewer neurons than controls. Furthermore, the density of substance P-positive neurons and fibers was significantly reduced. The paucity of substance P in laminae I-III may contribute to the reduction of nociception in PKG-I ؊/؊ mice and suggests a role of PKG-I in substance P synthesis.spinal cord ͉ substance P ͉ nitric oxide ͉ pain T he second messenger cGMP is formed by activation of soluble and particulate guanylyl cyclases and has several targets, including cGMP-dependent protein kinase I (PKG-I) and PKG-II, of which PKG-I is expressed in the spinal cord (1, 2). Spinally delivered PKG inhibitors reduce formalin-induced nociceptive behavior in rats (3, 4), suggesting that PKG-I plays an important role in spinal nociceptive processing. It has been speculated that PKG-I mediates hyperalgesic effects of nitric oxide (NO) (5). This idea is supported by the observation that PKG-I inhibition causes a reduction of thermal hyperalgesia induced by injection of the NO donor, NOC-12 (6). Endogenous NO is produced by NO synthases, of which neuronal nitric oxide synthase (nNOS) is activated and up-regulated after N-methyl-D-aspartate receptor stimulation (7-10). NO probably acts as a retrograde messenger (11, 12) at nociceptive synapses, i.e., it is released from the postsynaptic neuron, diffuses back to the presynaptic neuron, and stimulates guanylyl cyclases. The latter step links NO to cGMP production and PKG-I activation. Because inhibition of NOS activity reduces nociception (13,14), the release of NO is thought to contribute to the development of hyperexcitability of nociceptive neurons under certain circumstances. Under the premise that PKG-I is a mediator of NO at nociceptive synapses, one would expect that PKG-I activation also causes hyperalgesia. However, effects of the spinally delivered PKG activator 8-Br-cGMP have been conflicting (15,16). (See Supporting Text, which is published as supporting information on the PNAS web site....

show abstract

Section: Methodsmentioning

confidence: 99%

Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP-dependent protein kinase I

Tegeder

Turco²,

Schmidtko³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Immunohistochemistry was performed on 10 m cryosections from WT and CRP2 Ϫ/Ϫ brains perfusion fixed with 2% paraformaldehyde. After preincubation with 10% normal donkey serum [in 1% BSA, 0.5% Triton X-100, and 0.05 M Tris-buffered saline (TBS)] and rinsing with TBS, serial slices were incubated with rabbit anti-CRP2 (Huber et al, 2000) (1:1000 in 1% BSA, 0.5% Triton X-100, 0.05 M TBS) overnight and tagged with Alexa 555-conjugated donkey anti-rabbit IgG (1:1000 in 1% BSA, 0.5% Triton X-100, 0.05 M TBS) after rinsing with TBS. CRP2 immunofluorescence was analyzed using a Bio-Rad (Hercules, CA) MRC1000 confocal laser scanning microscope equipped with a krypton-argon laser and attached to Nikon (Düsseldorf, Germany) Diaphot 300.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…The lumbar spinal cords were dissected, postfixed in the same fixative for 2.5 h, and cryoprotected in 30% sucrose overnight. Tissues were frozen in tissue-freezing medium on dry ice and cryostat sectioned at a thickness of 8 -16 m. Sections were permeabilized for 5 min in PBST (0.1% Triton X-100 in PBS), blocked for 1 h in blocking buffer (3% bovine serum albumin in PBST) containing 10% normal goat serum, and incubated overnight at 4°C with rabbit anti-CRP2 [1:500 (Huber et al, 2000)], rabbit anti-cGKI [1:100; recognizes the ␣ and ␤ isoform of cGKI (Pfeifer et al, 1998)], mouse anticalcitonin gene-related peptide (CGRP; 1:500; Sigma-Aldrich, Munich, Germany), mouse anti-nuclear neuronal protein (NeuN; 1:1000; Millipore, Billerica, MA), mouse anti-glial fibrillary acidic protein (GFAP; 1:1000; Millipore), rat anti-mouse CD11b (1:200; Serotec, Düsseldorf, Germany) dissolved in blocking buffer, or with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia isolectin B4 (IB4; 10 g/ ml; Sigma-Aldrich) dissolved in PBST. In double-labeling experiments, primary antibodies were consecutively incubated overnight.…”

Section: Immunohistochemistrymentioning

confidence: 99%

“…Extracted proteins (30 g per lane) were separated by SDS-PAGE and transferred onto nitrocellulose membranes by electroblotting. After blocking of nonspecific binding sites with blocking buffer (PBS with 0.2% Tween and 5% low-fat milk), membranes were incubated overnight at 4°C with anti-CRP2 [1:1000 (Huber et al, 2000)] diluted in blocking buffer. Anti-extracellular signal-regulated kinase 2 (ERK-2; 1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated for 1 h and used as loading control.…”

Section: Western Blotmentioning

confidence: 99%

“…Slides were placed on the stage of an inverted wide-field fluorescence microscope equipped with fluorescence filters for FITC. Labeled rabbit antibodies [against CRP2 (Huber et al, 2000), cGKI (Pfeifer et al, 1998), or syntaxin 1A (Synaptic Systems, Göttingen, Germany)], propidium iodide, and wash solutions were added and removed robotically. After incubation for 15 min, phase-contrast and fluorescence images of the superficial dorsal horn of the spinal cord were acquired.…”

Section: Multiepitope Ligand Cartographymentioning

confidence: 99%

See 2 more Smart Citations

Cysteine-Rich Protein 2, a Novel Downstream Effector of cGMP/cGMP-Dependent Protein Kinase I-Mediated Persistent Inflammatory Pain

Schmidtko¹,

Gao²,

Sausbier³

et al. 2008

J. Neurosci.

View full text Add to dashboard Cite

The cGMP/cGMP-dependent protein kinase I (cGKI) signaling pathway plays an important role in spinal nociceptive processing. However, downstream targets of cGKI in this context have not been identified to date. Using a yeast two-hybrid screen, we isolated cysteinerich protein 2 (CRP2) as a novel cGKI interactor in the spinal cord. CRP2 is expressed in laminas I and II of the mouse spinal cord and is colocalized with cGKI, calcitonin gene-related peptide, and isolectin B4. Moreover, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and peripherin. CRP2 is phosphorylated in a cGMP-dependent manner, and its expression increases in the spinal cord and in DRGs after noxious stimulation of a hindpaw. To elucidate the functional role of CRP2 in nociception, we analyzed mice with a targeted deletion of CRP2. CRP2-deficient (CRP2 Ϫ/Ϫ ) mice demonstrate normal behavioral responses to acute nociception and after axonal injury of the sciatic nerve, but increased nociceptive behavior in models of inflammatory hyperalgesia compared with wild-type mice. Intrathecal administration of cGMP analogs increases the nociceptive behavior in wild-type but not in CRP2 Ϫ/Ϫ mice, indicating that the presence of CRP2 is important for cGMP-mediated nociception. These data suggest that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.

show abstract

cGMP-Dependent Protein Kinases (cGK)

Hofmann¹,

Wegener²

2013

Methods in Molecular Biology

View full text Add to dashboard Cite

cGMP-dependent protein kinases (cGK) are serine/threonine kinases that are widely distributed in eukaryotes. Two genes-prkg1 and prkg2-code for cGKs, namely, cGKI and cGKII. In mammals, two isozymes, cGKIα and cGKIβ, are generated from the prkg1 gene. The cGKI isozymes are prominent in all types of smooth muscle, platelets, and specific neuronal areas such as cerebellar Purkinje cells, hippocampal neurons, and the lateral amygdala. The cGKII prevails in the secretory epithelium of the small intestine, the juxtaglomerular cells, the adrenal cortex, the chondrocytes, and in the nucleus suprachiasmaticus. Both cGKs are major downstream effectors of many, but not all, signalling events of the NO/cGMP and the ANP/cGMP pathways. cGKI relaxes smooth muscle tone and prevents platelet aggregation, whereas cGKII inhibits renin secretion, chloride/water secretion in the small intestine, the resetting of the clock during early night, and endochondral bone growth. This chapter focuses on the involvement of cGKs in cardiovascular and non-cardiovascular processes including cell growth and metabolism.

show abstract

Cysteine-rich Protein 2, a Novel Substrate for cGMP Kinase I in Enteric Neurons and Intestinal Smooth Muscle

Cited by 42 publications

References 52 publications

Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP-dependent protein kinase I

Reduced inflammatory hyperalgesia with preservation of acute thermal nociception in mice lacking cGMP-dependent protein kinase I

Cysteine-Rich Protein 2, a Novel Downstream Effector of cGMP/cGMP-Dependent Protein Kinase I-Mediated Persistent Inflammatory Pain

cGMP-Dependent Protein Kinases (cGK)

Contact Info

Product

Resources

About