cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.     

The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.     

Activation of cGMP-dependent protein kinase Ibeta inhibits interleukin 2 release and proliferation of T cell receptor-stimulated human peripheral T cells

Several major functions of type I cGMP-dependent protein kinase (cGK I) have been established in smooth muscle cells, platelets, endothelial cells, and cardiac myocytes. Here we demonstrate that cGK Ibeta is endogenously expressed in freshly purified human peripheral blood T lymphocytes and inhibits their proliferation and interleukin 2 release. Incubation of human T cells with the NO donor, sodium nitroprusside, or the membrane-permeant cGMP analogs PET-cGMP and 8-pCPT-cGMP, activated cGK I and produced (i) a distinct pattern of phosphorylation of vasodilator-stimulated phosphoprotein, (ii) stimulation of the mitogen-activated protein kinases ERK1/2 and p38 kinase, and, upon anti-CD3 stimulation, (iii) inhibition of interleukin 2 release and (iv) inhibition of cell proliferation. cGK I was lost during in vitro culturing of primary T cells and was not detectable in transformed T cell lines. The proliferation of these cGK I-deficient cells was not inhibited by even high cGMP concentrations indicating that cGK I, but not cGMP-regulated phosphodiesterases or channels, cAMP-dependent protein kinase, or other potential cGMP mediators, was responsible for inhibition of T cell proliferation. Consistent with this, overexpression of cGK Ibeta, but not an inactive cGK Ibeta mutant, restored cGMP-dependent inhibition of cell proliferation of Jurkat cells. Thus, the NO/cGMP/cGK signaling system is a negative regulator of T cell activation and proliferation and of potential significance for counteracting inflammatory or lymphoproliferative processes.     

Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.     

Evidence that nitric oxide-induced synthesis of cGMP occurs in a paracrine but not an autocrine fashion and that the site of its release can be regulated: studies in dorsal root ganglia in vivo and in vitro.

As nitric oxide is a gas, it cannot be stored and has to be synthesized as required. This suggests that it could be released wherever nitric oxide synthase (NOS) is activated and due to its unstable state will react with appropriate targets at this site of production. In both dissociated dorsal root ganglion (DRG) cultures and in acutely isolated, but intact, DRG, treatment with capsaicin or bradykinin caused cGMP synthesis, which could be blocked by NOS inhibitors. The cGMP was synthesized in cells different from those expressing the neuronal isoform of NOS (nNOS). In dissociated cultures many of the cells stimulated to produce cGMP were neurons, whereas in isolated ganglia they were always satellite glia cells. Surprisingly, the satellite glia cells surrounding the nNOS-containing neurons did not contain cGMP. Following nerve section in adult rats, many axotomized ganglion neurons expressed nNOS. Again in these axotomized ganglia, most cGMP was expressed in the satellite glia surrounding nNOS-negative neurons. However, an nNOS-selective inhibitor reduced the cGMP present in these axotomized ganglia, suggesting that the cGMP synthesized is stimulated by NO (nitrogen monoxide) produced by nNOS. In both dissociated cultures and axotomized ganglia, nNOS-containing processes were observed close to cGMP-positive cells. These observations lead to the suggestion that NO acts in a paracrine fashion when stimulating the synthesis of cGMP and may not be synthesized at all sites containing nNOS.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

SNV, a lipophilic superactive VIP analog, acts through cGMP to promote neuronal survival.

The current study explored whether the neuroprotective effects of vasoactive intestinal peptide (VIP) and its analog Stearyl-Nle17-VIP (SNV) were mediated through cGMP. SNV, was previously found to be 100-fold more potent than VIP in providing neuroprotection. Neuronal survival was assessed in rat cerebral cortical cultures. A cGMP antagonist (RP-8-pCPT-cGMPS, 10(-12)-10(-9) M) reduced the number of surviving neurons (40-60%), this decline was spared in the presence of SNV (10(-13)M). A cGMP agonist (Sp-8-pCPT-cGMPS, 10(-14)-10(-8)M) and SNV (10(-16)-10(-8)M) both provided significant neuroprotection against 10(-12) M of the cGMP antagonist. Immunoassays indicated that SNV induced increases in cGMP (two-threefold) in these cultures, whereas VIP was 1000-fold less potent. These results implicate cGMP as a second messenger for VIP/SNV-mediated effects on neuronal survival.     

E-cadherin expression in a particular subset of sensory neurons.

We found that the dorsal root ganglia (DRG) and trigeminal ganglia of mouse embryos express the E-cadherin cell-cell adhesion molecule and analyzed its expression profile. E-cadherin expression began around Embryonic Day 12 (E12) in these ganglia, thereafter increased, and persisted to the adult stage. This cadherin was expressed by 10 and 30% of DRG neurons in E17 and postnatal animals, respectively, as well as by satellite cells and some Schwann cells. E-cadherin-positive primary sensory fibers terminated only in a narrow region of the dorsal horn of the spinal cord, which was identified as part of lamina II by double-staining for E-cadherin and substance P or somatostatin. This E-cadherin expressing area of the spinal cord extended to part of the trigeminal nucleus in the medulla. These results showed that E-cadherin is expressed in a particular subset of primary sensory neurons which may have specific functional properties. We suggest that this adhesion molecule may play a role in the selective adhesion of sensory neuronal fibers.     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture

The soybean phytoestrogen, genistein (Gen), has anabolic effects on bone through mechanisms that remain to be elucidated. We examined the role of nitric oxide (NO) and its downstream effector guanylyl cyclase (GC) in mediating the effects of Gen on the proliferation and osteoblastic maturation of primary mouse bone marrow-derived mesenchymal stem cells (BMSCs). Gen (10(-8) approximately 10(-6) M) resulted in a dose-dependent increase in cell proliferation as measured by increased [3H]thymidine incorporation, and stimulated osteoblastic maturation as assessed by culture duration-dependent increments in alkaline phosphatase (ALP) activity, calcium deposition into extracellular matrix and Runx2/Cbfa1 gene expression in BMSCs cultures. Gen also resulted in a dose-dependent increase in NO synthase (NOS) activity, NO formation, and cGMP production in BMSCs cultures. The effects of Gen were mimicked by 17beta-estradiol (E2, 10(-8) M). Concurrent treatment with the estrogen receptor (ER) antagonist ICI182,780 (10(-7) M) or the NOS inhibitor L-NAME (3 x 10(-3) M) diminished the Gen (10(-6) M)-mediated increase in NOS activity, NO production, and cGMP content. In contrast, a soluble GC inhibitor 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ, 10(-6) M) selectively blocked the Gen (10(-6) M)-mediated increase in cGMP content but not in NO production and NOS activity. Moreover, inhibition of ER, NOS activity or cGMP blocked Gen-induced proliferation and osteoblastic differentiation of BMSCs and Runx2/Cbfa1 gene expression in culture. Gen has estrogen-like activity and stimulates the proliferation and osteoblastic differentiation of mouse BMSCs at least in part through NO/cGMP pathway.     

Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells

The cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) pathway regulates many cellular functions. The current study shows that 8-Br-cGMP stimulates the number of attached primary but not that of subcultured murine vascular smooth muscle cells (VSMCs). These effects of 8-Br-cGMP require the presence of cGKI. In agreement with previous studies, cGKI inhibited the number of cells in repeatedly passaged murine VSMCs. Activation of the cGMP/cGKI pathway in freshly isolated primary VSMCs slightly decreased apoptosis and strongly increased cell adhesion. The stimulation of cell adhesion by cGKI involves an inhibition of the RhoA/Rho kinase pathway and increased exposure of β₁ and β₃ integrins on the cell surface. Together, these results identify a novel proadhesive function of cGMP/cGKI signaling in primary VSMCs and suggest that the opposing effects of this pathway on VSMC number depend on the phenotypic context of the cells.     

Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study

Summary In rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

Cysteine-rich protein 2, a novel substrate for cGMP kinase I in enteric neurons and intestinal smooth muscle

Nitric oxide/cGMP/cGMP kinase I (cGKI) signaling causes relaxation of intestinal smooth muscle. In the gastrointestinal tract substrates of cGKI have not been identified yet. In the present study a protein interacting with cGKIbeta has been isolated from a rat intestinal cDNA library using the yeast two-hybrid system. The protein was identified as cysteine-rich protein 2 (CRP2), recently cloned from rat brain (Okano, I., Yamamoto, T., Kaji, A., Kimura, T., Mizuno, K., and Nakamura, T. (1993) FEBS Lett. 333, 51-55). Recombinant CRP2 is specifically phosphorylated by cGKs but not by cAMP kinase in vitro. Co-transfection of CRP2 and cGKIbeta into COS cells confirmed the phosphorylation of CRP2 in vivo. Cyclic GMP kinase I phosphorylated CRP2 at Ser-104, because the mutation to Ala completely prevented the in vivo phosphorylation. Immunohistochemical analysis using confocal laser scan microscopy showed a co-localization of CRP2 and cGKI in the inner part of the circular muscle layer, in the muscularis mucosae, and in specific neurons of the myenteric and submucosal plexus. The co-localization together with the specific phosphorylation of CRP2 by cGKI in vitro and in vivo suggests that CRP2 is a novel substrate of cGKI in neurons and smooth muscle of the small intestine.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Hanssons' enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mesencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.