NOS-II is involved in early differentiation of murine cortical, retinal and ES cell-derived neurons—an immunocytochemical and functional approach

Nitric oxide (NO), a cell-derived highly diffusible and unstable gas is regarded to be involved in inter- and intracellular communication in the nervous system. Based on findings about the expression of the inducible NO synthase (NOS) isoform during development of early mouse olfactory as well as vestibulocochlear receptor neurons, we intended to prove a general role of this isoform for neuronal differentiation. Using immunohistochemical techniques, an exclusive expression of the inducible NOS-II isoform in early post-mitotic neurons of the developing mouse cortex and retina can be detected. In a pharmacological approach using cultures of the mouse cortex as well as embryonic stem cell-derived neural precursor cells, we investigated the functional role of NO on initial neuronal differentiation. Effects of NOS inhibitors and NO donors on the morphological differentiation were correlated with developmentally regulated calcium current densities, focusing on the effects of the specific NOS-II inhibitor GW 274150. Furthermore, involvement of the soluble guanylate cyclase (sGC)/cGMP signaling cascade was pharmacologically investigated. Our data indicate that while a specific block of NOS-II provokes a clear inhibition of neurite outgrowth formation as well as a decrease of calcium current densities, the inverse is true for exogenous NO donation. In line with lacking immunoreactivity for the sGC and cGMP there are only minor effects of compounds manipulating the sGC/cGMP pathway, suggesting the downstream sGC/cGMP pathway not to be essential in these early differentiation steps.     

Prenatal exposure to polybrominated diphenylether 99 enhances the function of the glutamate-nitric oxide-cGMP pathway in brain in vivo and in cultured neurons

Polybrominated diphenylethers (PBDEs) are widely used as flame retardants. Significant amounts of PBDEs are present in the milk of lactating women. The possible neurotoxic effects of PBDEs are not well known. Perinatal exposure to PBDEs affects both motor and cognitive functions by mechanisms that remain unclear. Some types of learning depend on N-methyl-D-aspartate receptor activation, which increases intracellular calcium that binds to calmodulin and activates nitric oxide synthase, increasing nitric oxide formation that activates guanylate cyclase, increasing cGMP formation. Part of this cGMP is released to the extracellular fluid. We studied whether prenatal exposure of rats to PBDE99 alters the function of this glutamate-nitric oxide-cGMP pathway in rat brain in vivo. At 10 weeks of age, rats treated with PBDE99 showed increased function of the glutamate-nitric oxide-cGMP pathway in brain in vivo, as assessed by microdialysis in freely moving rats. The increased function of the pathway was reproduced in primary cultures of cerebellar neurons prepared from rats prenatally exposed to PBDE99 as well as in neurons cultured from normal rats and treated in vitro with PBDE99. Increased calmodulin content and activation of soluble guanylate cyclase by nitric oxide contributed to the increased function of the pathway.     

The NO‐cGMP pathway in the rat locus coeruleus: electrophysiological, immunohistochemical and in situ hybridization studies

The effect of two nitric oxide (NO) donors, SIN-1 and DEA/NO, as well as of the inactive SIN-1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN-1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN-1 increased the firing rate. No effect could be recorded with the brain-inactive SIN-1 derivative molsidomin. The membrane permeable cGMP analogue 8-bromo-cGMP imitated the action of SIN-1. The hyperpolarizing effect of SIN-1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non-stimulated slices, whereas SIN-1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non-noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non-noradrenergic neurons ( Xu et al. 1994 ), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.     

Role of nitric oxide and cyclic GMP in glutamate-induced neuronal death

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors is neurotoxic, leading to neuronal degeneration and death. In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of NMDA receptors, leading to increased intracellular calcium which binds to calmodulin and activates neuronal nitric oxide synthase (NOS), increasing nitric oxide (NO) which in turn activates guanylate cyclase and increases cGMP. Inhibition of NOS prevents glutamate neurotoxicity, indicating that NO mediates glutamate-induced neuronal death in this system. NO generating agents such as SNAP also induce neuronal death. Compounds that can act as “scavengers” of NO such as Croman 6 (CR-6) prevent glutamate neurotoxicity. The role of cGMP in the mediation of glutamate neurotoxicity remain controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and NO neurotoxicity in cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and NO neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase increased extracellular but not intracellular cGMP and prevented glutamate neurotoxicity. Addition of cGMP to the medium also prevented glutamate neurotoxicity. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.     

Distribution and different activation of adenylate cyclase by naf and of guanylate cyclase by NaN3 in neuronal and glial cells separated from rat cerebral cortex

Distribution of adenylate cyclase and guanylate cyclase activities in neuronal perikarya and glial cells separated from rat brain, and cellular differences in activation between of adenylate cyclase by NaF and of guanylate cyclase by NaN3 have been studied. Adenylate cyclase activity was higher in the glial cells than in the neuronal fraction, while guanylate cyclase activity was equally detected in both cell fractions. Adenylate cyclase was mainly derived from the particulate fraction of both brain cell homogenates, whereas the major portion of guanylate cyclase activity was found in their soluble rather than in the particulate fractions. Although bulk-separated neurons and glial cells almost failed to change intracellular cyclic nucleotide levels in response to some putative neurotransmitters, activation of adenylate cyclase by NaF was found to be greater in neuronal than in glial cell fractions, and was observed more clearly in the soluble than in the particulate fractions. Sodium azide greatly increased guanylate cyclase in the particulate fraction, but did not affect it considerably in the soluble one. Addition of catalase to the reaction mixture together with NaN3 further stimulated guanylate cyclase both int he soluble and the particulate fractions. These results suggest that adenylate cyclase and guanylate cyclase without intimate coupling to the transmitter-receptor system, but with activation by NaF or NaN3, may be distributed ubiquitously in the cells separated from rat cerebral cortex.     

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture

Ca²⁺ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca²⁺-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca²⁺ entry could also down-regulate intracellular cGMP by activating a Ca²⁺/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 μM) and the Ca²⁺ ionophore A23187 (10 μM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca²⁺ and is prevented by the calmodulin inhibitor W7 (100 μM). Membrane depolarization, manipulations of the Na⁺ gradient, and intracellular Ca²⁺ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca²⁺ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia. J. Neurosci. Res. 49:333–341, 1997. © 1997 Wiley-Liss, Inc.     

Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway via neuron-glial communication in the peripheral nervous system

The NO-cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)-deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co-culture of neonatal DRG neurons with either Schwann cells pre-treated with an NO donor or a membrane-permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO-sGC-cGMP-mediated NGF and NT3 synthesis by Schwann cells protect neurons.     

Cyclic guanosine monophosphate signalling pathway plays a role in neural cell adhesion molecule-mediated neurite outgrowth and survival

The neural cell adhesion molecule (NCAM) plays a crucial role in neuronal development, regeneration, and synaptic plasticity associated with learning and memory consolidation. Homophilic binding of NCAM leads to neurite extension and neuroprotection in various types of primary neurons through activation of a complex network of signalling cascades, including fibroblast growth factor receptor, Src-family kinases, the mitogen-activated protein kinase pathway, protein kinase C, phosphatidylinositol-3 kinase, and an increase in intracellular Ca(2+). Here we present data indicating an involvement of cyclic GMP in NCAM-mediated neurite outgrowth in both hippocampal and dopaminergic neurons and in NCAM-mediated neuroprotection of dopaminergic neurons. In addition, evidence is presented suggesting that NCAM mediates activation of cGMP via synthesis of nitric oxide (NO) by NO synthase (NOS) and activation of soluble guanylyl cyclase by NO, leading to an increased synthesis of cGMP and activation by cGMP of protein kinase G.     

Phosphorylation of vasodilator-stimulated phosphoprotein: a consequence of nitric oxide- and cGMP-mediated signal transduction in brain capillary endothelial cells and astrocytes

There is contradictory information on the relevance of nitric oxide (NO) and cGMP for the function of brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB). Therefore, NO/cGMP-mediated signal transduction was investigated in cell cultures of BCEC and of astrocytes (AC) inducing BBB properties in BCEC. Constitutive, Ca2+-activated isoforms of NO synthase (NOS) were found in BCEC (endothelial NOS: eNOS) and in AC (neuronal NOS: nNOS), leading to increased NO release after incubation with the Ca2+-ionophore A23187. Both cell types expressed inducible NOS (iNOS) after incubation with cytokines. Soluble guanylate cyclase (sGC) was detected in both cell types. NO-dependent cGMP formation were observed in BCEC and, less pronounced, in AC. Furthermore, both cell types formed cGMP independently of NO via stimulation of particulate guanylate cyclase (pGC). cGMP-dependent protein kinase (PKG) type Ibeta, but not type II, was expressed in BCEC and AC. In BCEC, vasodilator-stimulated phosphoprotein (VASP) was detected, an established substrate of PKG and associated with microfilaments and cell-cell contacts. Phosphorylation of VASP was intensified by increased intracellular cGMP concentrations. The results indicate that BCEC and, to a smaller degree, AC can form NO and cGMP in response to different stimuli. In BCEC, NO/cGMP-dependent phosphorylation of VASP is demonstrated, thus providing a possibility of influencing cell-cell contacts.     

Selective expression of the NOS II isoform during mouse vestibulocochlear receptorgenesis.

Based on in vitro studies, nitric oxide (NO) is reported to be involved in initial neuronal differentiation. In order to compare this finding with the situation in vivo, we have looked for the expression of the three NO synthase isoforms in the developing mouse vestibulocochlear system. From these isoforms only the inducible NOS II is expressed during inner ear development. Examination of a series of embryonic and early postnatal animals, up to postnatal day 6, reveals a maturation-dependent, monophasic expression of this isoform. Initial expression is observed by day 10 of gestation in nerve cells of the vestibolocochlear ganglion and on their fibres. By day 14 of gestation, these afferent fibres penetrate the epithelium of the prospective receptor fields making contact with early, differentiating immunoreactive cochlear hair cells and receptor cells of the macula and crista ampullaris. This receptor-cell-derived immunoreactivity vanished in differentiated sensory hair cells by postnatal day 6, when both the constitutive isoforms and subsequent activated members of the down stream second messenger cascade (guanylate cyclase/cGMP) of the adult mouse were not then detectable. The strict phasic expression of NOS-II, independent of the second messenger system mentioned above, implies that there is a unique role for the inducible NOS isoform in nerve cell differentiation, independent of the NO/guanylate cyclase/cGMP pathway.     

Mesencephalic trigeminal neurons are innervated by nitric oxide synthase-containing fibers and respond to nitric oxide

In the present study we found that mesencephalic trigeminal (Mes-V) neurons of the rat are innervated by nitrergic fibers and that nitric oxide (NO) modifies the electrophysiological properties of these cells. Mes-V neurons were surrounded by a network of fibers that contained neuronal nitric oxide synthase (nNOS); these fibers gave rise to terminal-, bouton-like structures which ended in Mes-V cells bodies. These cells, which did not display nNOS-like immunoreactivity were immunoreactive to a cGMP antibody. By performing intracellular recordings in the adult rat brain slice preparation, the effects of diethylenetriamine/NO adduct (DETA/NO) applications were examined. DETA/NO induced a depolarization that averaged 2.2 mV (range: 1–6 mV) in nine of 22 neurons. In 15 of 22 neurons (68% of the cells), there was a decrease in current threshold from 0.74 to 0.60 nA (19%; P<0.001). The excitatory effects of DETA/NO were abolished by ODQ, a blocker of soluble guanylate cyclase. Input resistance (R_in) decreased in 80% of the cells from a mean of 24.8 to 20.6 MΩ (17%; P<0.001) and the membrane time constant (τ_m) decreased from 7.5 to 5.6 ms (25%; P<0.05). The ‘sag’ seen in the membrane response of these cells to current pulses was augmented during DETA/NO application. These findings indicate that there is a nitrergic innervation of Mes-V neurons and that these sensory cells are target for NO that may act on them as an excitatory neuromodulator promoting the synthesis of intracellular cGMP.     

Oxytocin induces penile erection when injected into the ventral tegmental area of male rats: role of nitric oxide and cyclic GMP

Oxytocin (80 ng) injected into the caudal mesencephalic ventral tegmental area (VTA) of male rats induces penile erection. Such an effect occurs together with an increase in nitric oxide (NO) production, as measured by the augmented concentration of NO(2)(-) and NO(3)(-) found in the dialysate obtained from this brain area by means of intracerebral microdialysis. Both effects are abolished by d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin (1 microg), an oxytocin receptor antagonist, by S-methyl-l-thiocitrulline acetate (20 microg), a neuronal NO synthase inhibitor, or by omega-conotoxin GVIA (50 ng), a N-type Ca(2+) channel blocker, all injected into the VTA 15 min before oxytocin. In contrast, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (40 microg), a guanylate cyclase inhibitor, given into the VTA 15 min before oxytocin, abolishes penile erection, but not the increase in NO production, while haemoglobin (40 microg), a NO scavenger, injected immediately before oxytocin reduces the increase in NO production, but not penile erection. 8-Bromo-cyclic guanosine monophosphate (0.5-10 microg) microinjected into the VTA induces penile erection with an inverted U-shaped dose-response curve; the maximal effective dose being 3 microg. Immunohistochemistry reveals that in the caudal VTA oxytocin-containing axons/fibres (originating from the paraventricular nucleus of the hypothalamus) contact cell bodies of mesolimbic dopaminergic (tyrosine hydroxylase-positive) neurons containing both NO synthase and guanylate cyclase. These results suggest that oxytocin injected into the VTA induces penile erection by activating NO synthase in the cell bodies of mesolimbic dopaminergic neurons. NO in turn activates guanylate cyclase present in these neurons, thereby increasing cyclic GMP concentration.     

Aging modulates nitric oxide synthesis and cGMP levels in hippocampus and cerebellum

The biological roles of nitric oxide (NO) and cGMP as inter- and intracellular messengers have been intensively investigated during the last decade. NO and cGMP both mediate physiological effects in the cardiovascular, endocrinological, and immunological systems as well as in central nervous system (CNS). In the CNS, activation of theN-methyl-d-aspartic acid (NMDA) type of glutamatergic receptor induces Ca²⁺-dependent NOS and NO release, which then activates soluble guanylate cyclase for the synthesis of cGMP. Both compounds appear to be important mediators in long-term potentiation and long-term depression, and thus may play important roles in the mechanisms of learning and memory. Aging and the accumulation of amyloid β (Aβ) peptides are important risk factors for the impairment of memory and development of dementia. In these studies, the mechanism of basal- and NMDA receptor-mediated cGMP formation in different parts of adult and aged brains was evaluated. The relative activity of the NO cascade was determined by assay of NOS and guanylate cyclase activities. In addition, the effect of the neurotoxic fragment 25–35 of Aβ (Aβ) peptide on basal and NMDA receptor-mediated NOS activity was investigated. The studies were carried out using slices of hippocampus, brain cortex, and cerebellum from 3- and 28-mo-old rats. Aging coincided with a decrease in the basal level of cGMP as a consequence of a more active degradation of cGMP by a phosphodiesterase in the aged brain as compared to the adult brain. Moreover, a loss of the NMDA receptor-stimulated enhancement of the cGMP level determined in the presence of cGMP-phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was observed in hippocampus and cerebellum of aged rats. However, this NMDA receptor response was preserved in aged brain cerebral cortex. A significant enhancement of the basal activity of NOS by about 175 and 160% in hippocampus and cerebellum, respectively, of aged brain may be involved in the alteration of the NMDA receptor response. The neurotoxic fragment of Aβ, peptide 25–35, decreased significantly the NMDA receptor-mediated calcium, and calmodulin-dependent NO synthesis that may then be responsible for disturbances of the NO and cGMP signaling pathway. We concluded that cGMP-dependent signal transduction in hippocampus and cerebellum may become insufficient in senescent brain and may have functional consequences in disturbances of learning and memory processes. Aβ peptide accumulated during brain aging and in Alzheimer disease may be an important factor in decreasing the NO-dependent signal transduction mediated by NMDA receptors.     

Nitric oxide synthesis and action in an invertebrate brain

Nitric oxide (NO) is synthesized in mammalian neurons by Ca²⁺/calmodulin activated NO synthase and functions as a signalling molecule by activating soluble guanylyl cyclases in target cells. We demonstrate here that both NO synthase and NO-activated guanylyl cyclase are present in the brain of the locustSchistocerca gregaria. Our observations indicate, for the first time, that the NO-cyclic GMP signalling pathway exists in invertebrate nervous systems.     

Regional and age-dependent expression of the nitric oxide receptor, soluble guanylyl cyclase, in the human brain

Nitric oxide (NO), synthesized by neuronal NO synthase (NOS-I), plays essential physiological roles in the brain. The major molecular target for NO is soluble guanylyl cyclase (sGC), a heterodimeric hemoprotein composed of a larger alpha and a smaller beta subunit. Both subunits of sGC are needed to generate the second messenger cyclic GMP (cGMP). Here we show using subunit-specific antibodies and Western blot analysis that sGCalpha1 and sGCbeta1 protein subunits are present in all examined human brain regions. The relative distribution of the two subunits was similar and also correlated well with the known distribution of NOS-I. The highest expression levels of sGC were found in cortex, basal ganglia and the limbic system. These regions display the most prominent biochemical and histological changes during ageing. In cortex, a negative correlation between the amounts of sGC and age was found, while sex and post-mortem delay time did not affect sGC levels significantly. Our data suggest that sGCalpha1 and sGCbeta1 subunits are widely distributed in human brain, consistent with a major role in NO signaling. Moreover, the NO/cGMP pathway appears to be affected by ageing in the human brain.     

cGmp synthesis in cultured cerebellar neurons is stimulated by glutamate via a Ca2+-mediated, differentiation-dependent mechanism

Sensitivity of cyclic GMP synthesis to stimulation by excitatory amino acids, depolarizing agents, and divalent cation ionophores develops during the differentiation of cerebellar neurons in culture; in each case calcium influx appears responsible for activating guanylate cyclase. The developmental event being followed is not the appearance of either the soluble or the particulate form of the enzyme since both are present throughout. The possible role of a differentiation-dependent calcium-modulating protein is discussed.     

Immunohistochemical localization of nitric oxide synthase and soluble guanylyl cyclase in the ventral cochlear nucleus of the rat

The diffusible messenger nitric oxide (NO) is implicated in auditory processing. It acts in the brain largely through activation of soluble guanylyl cyclase (sGC), a heterodimer comprised of alpha and beta subunits. The authors used immunohistochemistry to study the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway in the cochlear nucleus of Sprague-Dawley rats. Central fibers of the cochlear nerve were stained for neuronal nitric oxide synthase (NOS-I) but not for sGCbeta. Within the ventral cochlear nucleus, a large fraction of principal cells were immunopositive for both NOS-I and sGCbeta; these cells could be seen at times receiving contacts from NOS-I-positive fibers. sGC staining of somatic cytoplasm extended into the distal dendritic tree. At variance with this pattern, NOS-I was concentrated mainly in somata. Double-labeling experiments showed that most of the principal neurons expressed both antigens. By contrast, in the granule cell domain, small cells that were immunopositive for NOS-I rarely corresponded to those that were immunopositive for sGC. To assess whether NOS-I and sGC immunoreactivities colocalize with their respective catalytic activities, the authors performed multiple labeling with L-citrulline (a by-product of the formation of NO from L-arginine) and cGMP, respectively. L-citrulline was restricted to NOS-I-positive elements, and the large majority of NOS-expressing neurons were positive for citrulline. Multiple labeling revealed that almost all sGC-positive neurons also accumulated cGMP both in the ventral cochlear nucleus and in the granule cell domain. These data suggest that NO is a signaling molecule in the cochlear nucleus, perhaps functioning in both a paracrine manner and an autocrine manner.     

Increasing the function of the glutamate‐nitric oxide‐cyclic guanosine monophosphate pathway increases the ability to learn a Y‐maze task

N‐methyl‐D‐aspartate (NMDA) receptors play a crucial role in learning. However, the molecular mechanisms by which NMDA receptors contribute to learning processes are not known in detail. Activation of NMDA receptors leads to increased calcium in the postsynaptic neuron. Calcium binds to calmodulin and activates neuronal nitric oxide synthase, increasing nitric oxide (NO), which activates soluble guanylate cyclase, increasing cGMP. Part of this cGMP is released to the extracellular space. Several reports indicate that impairment of this glutamate‐NO‐cGMP pathway reduces the ability to learn a Y‐maze conditional discrimination task by rats. The aim of this work was to assess whether enhancing the function of this pathway increases the ability to learn this task. Prenatal exposure to the polybrominated diphenylether PBDE‐99 during embryonic days 2–9 or 11–19 enhances the function of the glutamate‐NO‐cGMP pathway in cerebellum in vivo as assessed by microdialysis in freely moving rats. This was associated with an increase in the ability to learn the Y‐maze task. Rats prenatally exposed to PBDE need fewer trials than control rats to learn the Y‐maze task. These results show that the function of the glutamate‐NO‐cGMP modulates the ability of rats to learn the Y‐maze task, that the function of the pathway under physiological conditions is not optimal for learning, and that performance in the Y‐maze task may be improved by enhancing slightly the function of the pathway and cGMP formation. © 2009 Wiley‐Liss, Inc.     

Interstitial cells of Cajal contain signalling molecules for transduction of nitrergic stimulation in guinea pig caecum

Abstract  Nitric oxide (NO) is an inhibitory signalling molecule in the gastrointestinal (GI) tract that is released from neurons and from leucocytes during inflammation. NO stimulates soluble guanylate cyclase (sGC), elevates cyclic guanosine 3',5'-monophospate (cGMP), and subsequently activates cGMP-dependent protein kinase (PKG). Targets for NO in the guinea pig caecum were investigated by characterizing the cellular distribution of sGC, cGMP and PKG. Immunoreactivity for both isoforms of sGC, sGCα1 and sGCβ1, was observed in the interstitial cells of Cajal (ICC) and enteric neurons in the tunica muscularis . Double labelling with anti-Kit and anti-sGC antibodies showed sGCα1 and sGCβ1-like immunoreactivity (LI) in almost all intramuscular (IM) and myenteric ICC. Neuronal processes with neuronal NO synthase were closely apposed to ICC expressing sGC-LI. Cells with sGC-LI possessed ultrastructural features of ICC-IM: caveolae, close association with nerve bundles and contacts with smooth muscle cells (SMC). Sodium nitroprusside, added with the phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine and zaprinast), enhanced cGMP-LI in almost all ICC and in some enteric neurons. Nerve stimulation also increased cGMP-LI in ICC and enteric neurons. In contrast, no resolvable increase in cGMP-LI was observed in any cells when the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was present. ICC and SMC also expressed PKG type I-LI. These data show that ICC express the downstream signalling molecules necessary to transduce nitrergic signals and activate inhibitory pathways and thus are primary targets for NO released from neurons and other cells in the GI tract.     

Role of Nitric Oxide/Cyclic GMP Pathway in the Inhibitory Effect of GABA and Dopamine on Prolactin Release

The anterior pituitary gland is a site of nitric oxide (NO) production and action, suggesting a local regulatory function. We recently reported that NO inhibits in vitro prolactin release. The aim of the present study was to establish the mechanism of action of NO on prolactin release and to determine whether NO is involved in the inhibitory effect of GABA on prolactin release. Since NO exerts its action through cGMP by activating guanylate cyclase in different tissues, we examined the effect of sodium nitroprusside (NP), a NO releaser, on intrapituitary cGMP levels. Incubation of anterior pituitary glands with 0.5 mM NP 4-fold increased intrapituitary cGMP content, but decreased intrapituitary cAMP levels. In addition, we studied the effect of NP on prolactin release in the presence of LY 83583, an inhibitor of guanylate cyclase activity and 3-lsobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity. 10μM LY 83583 and 0.5 mM IBMX blocked the inhibitory effect of NP on prolactin release. (10^-3 M) 8Br-cGMP, an analogue of cGMP, mimicked the effect of NP on prolactin release. On the other hand, NO seems to be involved in the inhibitory effect of GABA on prolactin release since hemoglobin, a scavenger of NO, and N^w-nitro-L-arginine methyl ester, an inhibitor of NO synthase (NOS), blocked the pituitary response to GABA. Moreover, GABA (10 ⁶ M) stimulated NOS activity by almost 50%. GABA increased intrapituitary cGMP levels and decreased cAMP. Dopamine stimulated NOS activity weakly.
These observations suggest that NO, acting through the guanylate cyclase-cGMP pathway, inhibits prolactin secretion. In addition, NO may be involved in the inhibitory effect of GABA and dopamine on prolactin release.