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.     

Polychlorinated Biphenyls PCB 153 and PCB 126 Impair the Glutamate–Nitric Oxide–cGMP Pathway in Cerebellar Neurons in Culture by Different Mechanisms

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. Perinatal exposure to PCB 153 or PCB 126 impairs the glutamate–nitric oxide–cGMP pathway in cerebellum in vivo and learning ability in adult rats. The aims of this work were: (1) to assess whether long-term exposure of primary cultures of cerebellar neurons to PCB 153 or PCB 126 reproduces the impairment in the function of the glutamate–nitric oxide–cGMP pathway found in rat cerebellum in vivo; (2) to provide some insight on the steps of the pathway affected by these PCBs; (3) to assess whether the mechanisms of interference of the pathway are different for PCB 126 and PCB 153. Both PCB 153 and PCB 126 increase basal levels of cGMP by different mechanisms. PCB 126 increases the amount of soluble guanylate cyclase while PCB 153 does not. PCB 153 reduces the amount of calmodulin while PCB 126 does not. Also both PCBs impair the function of the glutamate–nitric oxide–cGMP pathway by different mechanisms, PCB 153 impairs nitric oxide-induced activation of soluble guanylate cyclase and increase in cGMP while PCB 126 does not. PCB 126 reduces NMDA-induced increase in calcium while PCB 153 does not. When PCB 153 and PCB 126 exhibit the same effect, PCB 126 was more potent than PCB 153, as occurs in vivo.     

Whole brain spheroid cultures as a model to study the development of nitric oxide synthase-guanylate cyclase signal transduction

Whole brain spheroids provide a suitable model to study neurodevelopment. In the literature a role for the nitric oxide (NO)-cyclic guanosine 3',5'-monophosphate (cGMP) signalling pathway during development has frequently been suggested. In this study we investigated whether functional cGMP pathways were present in differentiated spheroids. In 3-week-old spheroids soluble guanylate cyclase was stimulated with N-methyl D-aspartic acid or sodium nitroprusside (NO donor). The results showed that the NO synthase-cGMP pathway is present in the culture system. Soluble guanylate cyclase-dependent cGMP formation was found in NO synthase containing neurons, in neurons of the GABAergic, glutamatergic and cholinergic system, and in astroglia and oligodendroglia. Activation of particulate guanylate cyclase by atrial natriuretic peptide also triggered an increase in cGMP production. Particulate guanylate cyclase was found in astroglia and in microglia as well as in glutamic acid decarboxylase and calbindin containing structures and neuronal NO synthase containing neurons. Chronic inhibition of NO synthase during culture development had no effect on soluble or particulate guanylate cyclase functioning. Similarly, inhibition of soluble guanylate cyclase during culture development did not have any effect on NO synthase and particulate guanylate cyclase functioning. It is concluded that NO synthase and both soluble and particulate guanylate cyclase are present in whole brain spheroid cultures and that their activity can be influenced by several stimuli. The spheroid culture system constitutes a suitable model to study the NO-cGMP pathway during brain development in mammals.     

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.     

Chronic hyperammonemia impairs the glutamate–nitric oxide–cyclic GMP pathway in cerebellar neurons in culture and in the rat in vivo

The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 μm ), impair N-methyl-d -aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC₅₀ was 20 μm , and at 100 μm the protection was complete. The induction of the protective effect was not immediate, but took several hours. Treatment with 100 μm ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate–nitric oxide–cGMP (3′,5′-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide. To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate–nitric oxide–cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate–nitric oxide–cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.     

Restoration of learning ability in hyperammonemic rats by increasing extracellular cGMP in brain

Intellectual function is impaired in patients with hyperammonemia and hepatic encephalopathy. Chronic hyperammonemia with or without liver failure impairs the glutamate-nitric oxide-cGMP pathway function in brain in vivo and reduces extracellular cGMP in brain as well as the ability of rats to learn a Y maze conditional discrimination task. We hypothesized that the decrease in extracellular cGMP may be responsible for the impairment in learning ability and intellectual function and that pharmacological modulation of the levels of cGMP may restore learning ability. The aim of this work was to try to reverse the impairment in learning ability of hyperammonemic rats by pharmacologically increasing extracellular cGMP in brain. We assessed whether learning ability may be restored by increasing extracellular cGMP in brain by continuous intracerebral administration of: (1) zaprinast, an inhibitor of the phosphodiesterase that degrades cGMP or (2) cGMP. We carried out tests of conditional discrimination learning in a Y maze with control and hyperammonemic rats treated or not with zaprinast or cGMP. Learning ability was reduced in hyperammonemic rats, which needed more trials than control rats to learn the task. Continuous intracerebral administration of zaprinast or cGMP restored the ability of hyperammonemic rats to learn this task. Pharmacological modulation of extracellular cGMP levels in brain may be a useful therapeutic approach to improve learning and memory performance in individuals in whom cognitive abilities are impaired by different reasons, for example in patients with liver disease who present hyperammonemia and decreased intellectual function.     

Arginine blocks gap junctions between retinal horizontal cells

Functions of nitric oxide are of common interest among a variety of tissues, since it activates soluble guanylate cyclase to produce cGMP. Here we report that intracellular application of L-arginine, the precursor of nitric oxide, blocked gap junctions between horizontal cells of the turtle retina. The input resistances of the cells were greatly increased and the cells were thereby easily polarized by current injections through microelectrodes. This procedure enables us to plot precise I-V curves and the reversal potential of light responses was estimated at around 0 mV. These results were quite similar to those obtained by intracellular application of cGMP, suggesting that the L-arginine:nitric oxide:cGMP pathway is present in retinal horizontal cells.     

Age-related Changes in the NMDA Receptor/Nitric Oxide/cGMP Pathway in the Hippocampus and Cerebellum of Freely Moving Rats Subjected to Transcerebral Microdialysis

The N-methyl-d -aspartate (NMDA) receptor/nitric oxide synthase/guanylate cyclase pathway was studied during aging by monitoring extracellular cGMP in the rat hippocampus and cerebellum during in vivo microdialysis. In the hippocampus the basal cGMP efflux decreased by 50% from 3 to 12 months of age, whereas it remained constant with age in the cerebellum. Locally perfused NMDA (1 mM) evoked remarkable cGMP responses in 3-month-old rats; in the hippocampus the cGMP production was already dramatically reduced at 12 months, whereas in the cerebellum a similar impairment occurred much later (24 months). The nitric oxide donor S-nitroso-N-penicillamine (1 mM) elicited cGMP responses which slightly decreased from 3 to 12–24 months in the hippocampus, while no significant decrement with age could be seen in the cerebellum. Local perfusion of the phosphodiesterase inhibitor 3-isobutyl-1 methylxanthine (IBMX, 1 mM) produced large increases in hippocampal cGMP levels. The response decreased at 12 and 24 months, apparently in parallel with the fall in the basal level of cGMP. No significant differences across ages were observed following IBMX infusion in the cerebellum. The decreases in basal outflow and in the NMDA-evoked cGMP response seen in the aged hippocampus were not compensated for by supplying l -arginine. Infusion of d -serine (1 mM) enhanced (150–200%) extracellular cGMP in the cerebellum with no age-related differences. The activity in vitro, of hippocampal nitric oxide synthase at 24 months was 33% lower than at 3 months, whereas the cerebellar enzyme did not show any age-related decay. Aging seems therefore to affect differentially the NMDA receptor/nitric oxide synthase/cGMP pathway in the rat hippocampus versus the cerebellum. In the hippocampus the early fall in the NMDA-evoked cGMP response seems to originate from deficits in NMDA receptor function and nitric oxide synthase and guanylate cyclase activities; in the cerebellum, the decreased response to NMDA in the old animals seems essentially to be due to impairment of NMDA receptor function.     

The Nitric Oxide/Cyclic GMP Messenger System in Olfactory Pathways of the Locust Brain

Nitric oxide is generated by a Ca²⁺/calmodulin-stimulated nitric oxide synthase and activates soluble guanylyl cyclase. Using NADPH diaphorase (NADPHd) staining as a marker for the enzyme nitric oxide synthase and an antiserum against cGMP, we investigated the cellular organization of nitric oxide donor and target cells in olfactory pathways of the brain of the locust ( Schistocerca gregaria ). A small subset of neuronal and glial cells expressed cGMP immunoreactivity after incubation of tissue in a nitric oxide donor. Nitric oxide-induced increases in cGMP immunoreactivity were quantified in a tissue preparation of the antennal lobe and in primary mushroom body cell cultures. The mushroom body neuropil is a potential target of a transcellular nitric oxide/ cGMP messenger system since it is innervated by extrinsic NADPHd-positive neurons. The mushroom body-intrinsic Kenyon cells do not stain for NADPHd but can be induced to express cGMP immunoreactivity. The colocalization of NADPHd and cGMP immunoreactivity in a cluster of interneurons of the antennal lobe, the principal olfactory neuropil of the insect brain, suggests a role of the nitric oxide/cGMP system in olfactory sensory processing. Colocalization of NADPHd staining and cGMP immunoreactivity was also found in certain glial cells. The cellular organization of the nitric oxide/cGMP system in neurons and glia raises the possibility that nitric oxide acts not only as an intercellular but also as an intracellular messenger molecule in the insect brain.     

Neurons exposed to ammonia reproduce the differential alteration in nitric oxide modulation of guanylate cyclase in the cerebellum and cortex of patients with liver cirrhosis

The activation of soluble guanylate cyclase by nitric oxide is increased in the frontal cortex but is reduced in the cerebellum of patients who died with liver cirrhosis. The aims of this work were to assess whether hyperammonemia is responsible for the region-selective alterations in guanylate cyclase modulation in liver cirrhosis and to assess whether the alteration occurs in neurons or in astrocytes. The activation of guanylate cyclase by nitric oxide was lower in cerebellar neurons exposed to ammonia (1.5-fold) than in control neurons (3.3-fold). The activation of guanylate cyclase by nitric oxide was higher in cortical neurons exposed to ammonia (8.7-fold) than in control neurons (5.5-fold). The activation was not affected in cerebellar or cortical astrocytes. These findings indicate that hyperammonemia is responsible for the differential alterations in the modulation of soluble guanylate cyclase in cerebellum and cerebral cortex of cirrhotic patients. Moreover, the alterations occur specifically in neurons and not in astrocytes.     

Chronic exposure to aluminium decreases NADPH-diaphorase positive neurons in the rat cerebral cortex

Aluminium (Al) exposure is neurotoxic and is considered a possible etiological factor for many neurodegenerative disorders. Since it is known that Al impairs the glutamate-nitric oxide-cGMP pathway in neurons, this study was carried out to monitor the expression of NADPH-d in some central nervous system areas of rats after chronic administration of Al in drinking water. We tested three different nervous areas known to contain NADPH-diaphorase positive neurons: two cortical area (somatosensory cerebral cortex and cerebral cortex), a deep brain area (dorsolateral periaqueductal gray matter) and a spinal area (lumbar enlargement of the spinal cord). Our data showed that Al significantly decreased NADPH-d positive neurons in the cerebral cortex and the NADPH-d staining of many granular neurons in the cerebellum. We also found that Al did not cause neuron loss or apoptosis in the cerebral cortex. These findings suggest that the cortical nitroxidergic neurons and granule cells were a specific target of Al neurotoxicity.     

Nitric oxide mediated formation of cyclic GMP in the olfactory system.

Olfactory cilia preparation from rats contain considerable activity of soluble guanylate cyclase as indicated by the formation of cyclic GMP (cGMP) upon application of nitroprusside, a nitric oxide generating agent. Stimulation of olfactory cilia with high doses of odorants elicited a delayed and sustained elevation of the cGMP-concentration. The odorant-induced cGMP-response was abolished by L-NG-nitro-arginine, a selective inhibitor of nitric oxide synthesis, as well as by haemoglobin which efficiently binds and inactivates nitric oxide. These observations suggest that the NO/cGMP cascade may plan an important role in signal processing of the olfactory system.     

Developmental exposure to polychlorinated biphenyls PCB153 or PCB126 impairs learning ability in young but not in adult rats

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in the food chain and in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. The underlying mechanisms remain unclear. Some PCBs are endocrine disrupters. The aim of this work was to assess whether exposure of rats to PCB126 (dioxin-like) or PCB153 (non-dioxin-like) during pregnancy and lactation affects the ability of the pups to learn a Y maze conditional discrimination task and/or the function of the glutamate–nitric oxide (NO)–cGMP pathway in brain in vivo when the rats are young (3 months) or adult (7–8 months). After finishing the learning experiments, the function of the pathway was analysed in the same rats by in vivo brain microdialysis. The results obtained show that perinatal exposure to PCB153 or PCB126: (1) impairs learning ability in young but not in adult rats, (2) impairs the glutamate–NO–cGMP pathway function in cerebellum in vivo in young but not in adult rats and (3) affect these parameters in males and females similarly. PCB126 is around 10 000-fold more potent than PCB153. In control rats the function of the glutamate–NO–cGMP pathway and learning ability are lower in adult than in young rats. These age-related differences are not present in rats exposed to PCBs. The impairment of the glutamate–NO–cGMP pathway function induced at young age by developmental exposure to the PCBs could be one of the mechanisms contributing to the cognitive impairment found in children whose mothers ingested PCB-contaminated food during pregnancy and lactation.     

A nitric oxide (NO) synthase inhibitor accelerates amygdala kindling.

In response to NMDA receptor activation, hippocampal, striatal and cerebellar neurons synthesize nitric oxide (NO), which in turn elevates cGMP levels via guanylate cyclase. NO is increasingly being considered as a transsynaptic retrograde messenger, involved in neuronal plasticity. The effect of an inhibitor of NO synthase, L-NG-nitroarginine (NOArg), was studied on amygdala kindling and on kindled seizures in rats. NOArg increased kindling rate, particularly in its initial period, but did not modify seizure severity in previously kindled rats, although we have no definitive explanation for this effect. However, an enhanced post-synaptic excitability could be attributed to the blockade of the negative feed-back exerted by NO on the NMDA receptor.     

Natriuretic Peptides Stimulate Cyclic GMP Production in an Immortalized LHRH Neuronal Cell Line

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro . Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors—which represent GCs—we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.     

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.     

Nitric oxide directly inhibits ghrelin-activated neurons of the arcuate nucleus

The hypothalamic arcuate nucleus (Arc) is a target site for signals regulating energy homeostasis. The orexigenic hormone ghrelin directly activates neurons of the medial arcuate nucleus (ArcM) in rats. Nitric oxide (NO) is a neuromodulator implicated in the control of food intake and body weight. NO is produced by nitric oxide synthase (NOS) and induces the formation of cyclic guanosine monophosphate (cGMP) via a stimulation of soluble guanylate cyclase (sGC). Both enzymes NOS and sGC have been identified in the Arc. Using extracellular recordings we characterized the effects of NO signaling on ArcM neurons and their co-sensitivity to ghrelin. The artificial NO donor sodium nitroprusside (10(-4) M) reversibly inhibited 91% of all ArcM neurons by a direct postsynaptic mechanism. 52% of ArcM neurons were excited by ghrelin. In all but one of these neurons SNP caused inhibitory responses. The SNP-induced inhibitions were mediated by cGMP since they were blocked by the specific sGC inhibitor ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, 10(-4) M). Furthermore, the membrane permeating cGMP analogue 8-Br-cGMP (10(-4) M) mimicked the inhibitory responses of SNP. In immunohistological in vitro studies SNP induced a cGMP formation, which could also be blocked by ODQ. The current studies demonstrate that NO/cGMP signaling inhibits a large population of ArcM neurons including ghrelin-excited cells. Since an activation of the latter neurons is regarded as a correlate of negative energy balance, NO may represent an anorectic neuromodulator in the Arc and/or restrain the action of signals promoting energy intake. NO signaling in the Arc is also induced following inflammation suggesting a possible role of Arc-intrinsic NO in disease-related anorexia.     

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.     

Localization of natriuretic peptides and their activation of particulate guanylate cyclase and nitric oxide synthase in the retina

In the vertebrate retina, cyclic guanosine monophosphate (cGMP) mediates photoreceptor signal transduction and modulates ion channel and gap junction conductivity. Although most previous studies have focused on its synthesis by nitric oxide (NO)-sensitive soluble guanylate cyclase, cGMP is also synthesized by NO-insensitive particulate guanylate cyclases (pGC). Natriuretic peptides and their associated pGC-coupled receptors have been reported in retina, but few studies have localized these natriuretic peptides or pGCs to specific retinal cells or demonstrated that activation of pGCs by natriuretic peptides increases cGMP synthesis. In this study, we immunocytochemically localized atrial, brain, and C-type natriuretic peptide-like immunoreactivity (ANP-LI, BNP-LI, and CNP-LI, respectively) in turtle retina by using isoform specific antisera, and determined the ability of each natriuretic peptide isoform to increase cGMP-like immunoreactivity (cGMP-LI) in retinal cells. ANP-LI and CNP-LI were localized in sparsely distributed amacrine cells with thin, intermittently varicose processes in the inner plexiform layer. BNP-LI was localized to abundant somata in the inner nuclear and ganglion cell layers and in specific amacrine and horizontal cells. Stimulation of turtle eyecups with each of these natriuretic peptides increased cGMP-LI in multistratified amacrine cells by means of NO-independent mechanisms in the central retina, and in select amacrine and bipolar cells in the peripheral retina by a nitric oxide-dependent mechanism. These results indicate that natriuretic peptides can modulate the synthesis of cGMP in select retinal neurons by two distinct signal transduction pathways in a regionally specific manner.