M. Markerink-van Ittersum

Effects of two selective phosphodiesterase type 5 inhibitors, sildenafil and vardenafil, on object recognition memory and hippocampal cyclic GMP levels in the rat.

The present study investigated the effects of two cyclic GMP-specific phosphodiesterase enzyme type 5 inhibitors, sildenafil and vardenafil, on the memory performance in the object recognition task. Both compounds were given per orally (1, 3 and 10 mg/kg sildenafil; 0.1, 0.3, 1 and 3 mg/kg vardenafil) immediately after the exposure to two identical objects. The memory for the objects was tested 24 h later. Vehicle-treated rats spent equal times exploring a new and the familiar object demonstrating that they did not remember the familiar one. However, sildenafil improved the object discrimination performance of the rats with a high discrimination performance at a dose of 3 mg/kg. Rats treated with vardenafil also showed an improved object discrimination performance. Compared with sildenafil, vardenafil appeared to be even more potent in this respect since it already produced a high discrimination performance at a dose of 0.3 mg/kg. The effects of both compounds on cyclic GMP and cyclic AMP accumulation were studied in rat hippocampal slices incubated in vitro. Cyclic GMP levels were increased after incubation with the highest concentration of 100 microM vardenafil (together with 0.1 mM sodium nitroprusside), although no changes in cyclic GMP levels were detected after incubation with different concentrations of sildenafil. Both compounds had no effect on cyclic AMP levels. Additional cyclic GMP immunocytochemistry showed that incubation with vardenafil (in the presence of sodium nitroprusside) resulted in a concentration-dependent staining of cyclic GMP. Staining was predominantly found in neuronal fibres in the hippocampal CA2/CA3 region. It was already detected at a concentration of 0.1 microM vardenafil. Also positive fibres were detected after incubation with sildenafil but at a higher concentration of 10 microM. Taken together, these results suggest that inhibition of phosphodiesterase enzyme type 5 improves object recognition memory. This effect might be explained by increased levels of central cyclic GMP.

Distribution of nitric oxide synthase and nitric oxide-receptive, cyclic GMP-producing structures in the rat brain

The structures capable of synthesizing cyclic GMP in response to nitric oxide in the rat brain were compared relative to the anatomical localization of neuronal nitric oxide synthase. In order to do this, we used brain slices incubated in vitro, where cyclic GMP-synthesis was stimulated using sodium nitroprusside as a nitric oxide-donor compound, in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Nitric oxide-stimulated cyclic GMP synthesis was found in cells and fibers, but was especially prominent in varicose fibers throughout the rat brain. Fibers containing the nitric oxide-stimulated cyclic GMP production were present in virtually every area of the rat brain although there were large regional variations in the density of the fiber networks. When compared with the localization of nitric oxide synthase, it was observed that although nitric oxide-responsive and the nitric oxide-producing structures were found in similar locations in general this distribution was complementary. Only occasionally was nitric oxide-mediated cyclic GMP synthesis observed in structures which also contained nitric oxide synthase. We conclude that the nitric oxide-responsive soluble guanylyl cyclase and nitric oxide synthase are usually juxtaposed at very short distances in the rat brain. These findings very strongly support the proposed role of nitric oxide as an endogenous activator of the soluble guanylyl cyclase in the central nervous system and convincingly demonstrate the presence of the nitric oxide-cyclic GMP signal transduction pathway in virtually every area of the rat brain.

Localization of cGMP-dependent protein kinase type II in rat brain.

In brain, signaling pathways initiated by atrial natriuretic peptide, or transmitters which stimulate nitric oxide synthesis, increase cGMP as their second messenger. One important class of target molecules for cGMP is cGMP-dependent protein kinases, and in the present study, biochemical and immunocytochemical analyses demonstrate the widespread distribution of type II cGMP-dependent protein kinase in rat brain, from the cerebral cortex to the brainstem and cerebellum. Also, colocalization of cGMP-dependent protein kinase type II with its activator, cGMP, was found in several brain regions examined after in vitro stimulation of brain slices with sodium nitroprusside. In western blots, cGMP-dependent protein kinase type II was observed in all brain regions examined, although cerebellar cortex and pituitary contained comparatively less of the kinase. Immunocytochemistry revealed cGMP-dependent protein kinase type II in certain neurons, and occasionally in putative oligodendrocytes and astrocytes, however, its most striking and predominant localization was in neuropil. Electron microscopy examination of neuropil in the medial habenula showed localization of the kinase in both axon terminals and dendrites. As a membrane-associated protein, cGMP-dependent protein kinase type II often appeared to be transported to cell processes to a greater extent than being retained in the cell body. Thus, immunocytochemical labeling of cGMP-dependent protein kinase type II often did not coincide with the localization of kinase mRNA previously observed by others using in situ hybridization. We conclude that in contrast to cGMP-dependent protein kinase type I, which has a very restricted localization to cerebellar Purkinje cells and a few other sites, cGMP-dependent protein kinase type II is a very ubiquitous brain protein kinase and thus a more likely candidate for relaying myriad cGMP effects in brain requiring protein phosphorylation.

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain.

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes.Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells.The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.

Behavioural, neurochemical and neuroanatomical effects of chronic postnatal N-nitro-l-arginine methyl ester treatment in neonatal and adult rats

In the present study we evaluated the consequences of interference with nitric oxide synthesis during development on brain function and behaviour in later life. Rat pups received a daily injection of the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg, s.c.) from postnatal day 0 to 24. At postnatal day 8 L-NAME-treated rats had enlarged and heavier stomachs, while body weights appeared to be reduced. The stomachs were not affected in size and weight anymore at postnatal day 24, whereas the body weights were still reduced by the L-NAME treatment, although they soon recovered after termination of the treatment. At four months-of-age, rats were tested in non-cognitive (open field) and cognitive (Morris water escape, two-way active avoidance) tasks. Open field behaviour of adult rats postnatally treated with L-NAME was not affected. In the water escape task there were no differences between the saline and L-NAME-treated rats in spatial discrimination learning and spatial reversal learning. Furthermore, postnatal L-NAME treatment did not have an effect on the acquisition of the two-way active avoidance task. Subsequently, we tested rat pups during the L-NAME treatment at postnatal day 19 through 24 in the open field and the two-way active avoidance task. L-NAME treatment appeared to increase the behavioural activity in the open field. There was no difference in behaviour in the active avoidance task between saline and L-NAME-treated rats. Biochemical and immunocytochemical studies showed that at postnatal day 8 the basal cyclic GMP level was reduced, while the cyclic GMP formation due to incubation with the nitric oxide donor sodium nitroprusside appeared to be increased in the hippocampus, striatum and frontal cortex of L-NAME-treated rats. Hence, nitric oxide synthase was inhibited whereas the soluble guanylyl cyclase activity may be increased in sensitivity. At postnatal day 24 basal cyclic GMP levels and nitric oxide-mediated cyclic GMP formation in the brain structures of L-NAME-treated rats had normal values again. Taken together, the findings of this study suggest that postnatal inhibition of nitric oxide synthase has profound neurochemical effects during development and may have short-lasting effects on non-cognitive behaviour, but it does not affect behaviour and brain function in later life.

Region-specific developmental patterns of atrial natriuretic factor- and nitric oxide-activated guanylyl cyclases in the postnatal frontal rat brain

In the rat central nervous system, cyclic GMP can be produced by two isoforms of guanylyl cyclase: a cytosolic isoform, which is activated by nitric oxide, and a membrane-bound isoform, activated by atrial natriuretic factor. We studied the development of guanylyl cyclase activity upon maturation of the rat forebrain from postnatal days 4 to 24, using a combined immunocytochemical and biochemical approach. Atrial natriuretic factor-activated particulate guanylyl cyclase activity was found to decrease in the frontal cortex, in the lateral septum and in the piriform cortex upon maturation. A transient expression of atrial natriuretic factor-sensitive guanylyl cyclase activity was observed at postnatal day 8 in the caudate putamen complex, whereas an increase was observed in the lateral olfactory tract from postnatal days 8 to 24. Biochemical and immunocytochemical studies using the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester, or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinaloxin-1-one, indicated high levels of endogenous nitric oxide release at postnatal days 4 and 8. This activity decreased strongly in all brain areas examined. From postnatal day 8 onwards, atrial natriuretic factor-responsive cyclic GMP-immunoreactive cells could be characterized as astrocytes, with the exception of those in the the lateral olfactory tract, where the myelinated fibers became cyclic GMP producing. Furthermore, our results on activation of both guanylyl cyclases at postnatal day 8 leads to the suggestion that both isoforms might be found in the same cells. This study shows that there are pronounced differences between various frontal brain areas in the development of the responsiveness of both the particulate and soluble isoforms of guanylyl cyclase, and lends further support to the hypothesis that natriuretic peptides have a role in neuronal growth and plasticity of the rat brain.

Nitric-oxide-induced cGMP synthesis in cholinergic neurons in the rat brain

Abstract. Nitric oxide (NO)-mediated cGMP synthesis is localized throughout the rat brain in close proximity to the NO-synthase-containing structures. However, characterization of the cGMP synthesizing structures in terms of co-localization with the classical neurotransmitter systems has not yet been reported. Here we present evidence, using double immunostaining for cGMP and the vesicular acetylcholine transporter, that virtually all of the cholinergic fibers in the cerebral cortex and the majority of the cholinergic fibers in the basal ganglia accumulate cGMP in response to a NO donor. In these areas, only few cGMP-containing fibers were observed not to be part of the cholinergic system. Co-localization between cGMP and the vesicular acetylcholine transporter was only observed to a minor degree in the ventral forebrain, the hippocampus, the reticular thalamic nucleus, and the nucleus ambiguus. No association of cGMP synthesis with the cholinergic system was observed to a similar extent in other brain areas. These results, in combination with literature data on the distribution of cholinergic receptors in the rat brain, suggest that NO has an anterograde and/or retrograde signaling function on subsets of cholinergic neurons.

The role of phosphodiesterase isoforms 2, 5, and 9 in the regulation of NO-dependent and NO-independent cGMP production in the rat cervical spinal cord

NO-responsive, cGMP-producing structures are abundantly present in the cervical spinal cord. NO-mediated cGMP synthesis has been implicated in nociceptive signaling and it has been demonstrated that cGMP has a role establishing synaptic connections in the spinal cord during development. As cGMP levels are controlled by the activity of soluble guanylyl cyclase (synthesis) and the phosphodiesterase (PDE) activity (breakdown), we studied the influence of PDE activity on NO-stimulated cGMP levels in the rat cervical spinal cord. cGMP-immunoreactivity (cGMP-IR) was localized in sections prepared from slices incubated in vitro. A number of reported PDE isoform-selective PDE inhibitors was studied in combination with diethylamineNONOate (DEANO) as a NO-donor including isobutyl-methylxanthine (IBMX) as a non-selective PDE inhibitor. We studied 8-methoxy-IBMX as a selective PDE1 inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitor, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. cGMP-IR structures (nerve fibers, axons, and terminals) were characterized using the following neurochemical markers: vesicular transporter molecules for acetylcholine, GABA, and glutamate (type 1 and type 2), parvalbumin, glutamate transporter molecule EAAT3, synaptophysin, substance P, calcitonin gene-related peptide, and isolectin B4. Most intense cGMP-IR was observed in the dorsal lamina. Ventral motor neurons were devoid of cGMP-IR. cGMP-IR was observed in GABAergic, and glutamatergic terminals in all gray matter laminae. cGMP-IR was abundantly colocalized with anti-vesicular glutamate transporter 2 (vGLUT2), however not with the anti-vesicular glutamate transporter 1 (vGLUT1), suggesting a functional difference between structures expressing vGLUT1 or vGLUT2. cGMP-IR did not colocalize with substance P- or calcitonin-gene related peptide-IR structures, however did partially colocalize with isolectin B4 in the dorsal horn. cGMP-IR in cholinergic structures was observed in dorsal root fibers entering the spinal cord, occasionally in laminae 1-3, in laminae 8 and 9 in isolated boutons and in the C-type terminals, and in small cells and varicosities in lamina 10. This latter observation suggests that the proprioceptive interneurons arising in lamina 10 are also NO-responsive. No region-specific nor a constant co-expression of cGMP-IR with various neuronal markers was observed after incubation of the slices with one of the selected PDE inhibitors. Expression of the mRNA of PDE2, 5, and 9 was observed in all lamina. The ventral motor neurons and the ependymal cells lining the central canal expressed all three PDE isoforms. Incubation of the slices in the presence of IBMX, DEANO in combination with BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase, provided evidence for endogenous NO synthesis in the slice preparations and enhanced cGMP-IR in all lamina. Under these conditions cGMP-IR colocalized with substance P in a subpopulation of substance P-IR fibers. It is concluded that NO functions as a retrograde neurotransmitter in the spinal cord but that also postsynaptic structures are NO-responsive by producing cGMP. cGMP-IR in a subpopulation of isolectin B4 positive fibers and boutons is indicative for a role of NO-cGMP signaling in nociceptive processing. cGMP levels in the spinal cord are controlled by the concerted action of a number of PDE isoforms, which can be present in the same cell.

ANP-mediated cGMP signaling and phosphodiesterase inhibition in the rat cervical spinal cord

Natriuretic peptides (NP) and the corresponding receptors are present in the rodent spinal cord. We have studied the structures which respond to atrial natriuretic peptide, brain natriuretic peptide, or C-type natriuretic peptide with an increased synthesis of cGMP. NP-responsive cGMP-producing structures were observed in laminae I-III, and X, and in addition in ependymal cells, astrocytes and a subpopulation of dorsal root ganglion cells. As the cGMP concentration is controlled by the rate of synthesis and the rate of breakdown by phosphodiesterases, we studied NP-responsive structures in spinal cord slices incubated in the presence of different phosphodiesterase inhibitors. We studied EHNA and BAY 60-7550 as selective PDE2 inhibitors, sildenafil as a selective PDE5 inhibitors, dipyridamole as a mixed type PDE5 and PDE10 inhibitor, rolipram as a PDE4 inhibitor, and SCH 81566 as a selective PDE9 inhibitor. Double immunostainings showed that cGMP-IR colocalized partial with the vesicular acetylcholine transporter molecule in lamina X, with Substance P in a subpopulation of neuronal fibers situated dorsolateral, and with a subpopulation of CGRP-IR dorsal root ganglion neurons. Colocalization of cGMP-IR was absent with parvalbumin, synaptophysin, and the vesicular transporter molecules for GABA and glutamate. It is concluded that NPs in the spinal cord are probably involved in integrating intersegmental sensory processing in the spinal cord although the greater part of the NP-responsive cGMP-producing fibers could not be characterized. PDE2, 5, and 9 are involved in regulating NP-stimulated cGMP levels in the spinal cord. NPs may have a role in regulating cerebrospinal fluid homeostasis.

NO-mediated cGMP synthesis in cultured cholinergic neurons from the basal forebrain of the fetal rat.

Previously, using brain slices, we reported NO-mediated cGMP synthesis in all cholinergic fibers in the rat neocortex. In order to answer the question whether this property of cholinergic fibers was present before or developed after birth, we investigated properties of NO-responsiveness of cultured cholinergic forebrain neurons. Basal forebrain neurons of E16 fetal rat were cultured. Under the conditions chosen and after one day of culturing, all cells had attained a cholinergic phenotype using choline acetyltransferase or the vesicular acetylcholine transporter molecule as markers. Between 95-99% of the cells also expressed neuronal NOS. In the presence of 1 mM IBMX, a non-selective phosphodiesterase (PDE) inhibitor, 10 microM of the NO donor diethylamine-NONOate (DEANO) increased cGMP synthesis in 80% of the cells. cGMP levels in the cultured forebrain neurons were also increased when cells were stimulated with DEANO in the presence of the selective PDE inhibitors BAY 60-7550 (PDE2), sildenafil (PDE5), or the mixed type inhibitor papaverine (PDE2,5,10). Subpopulations of cells from the basal forebrain expressed mRNA for PDE2, PDE5, and PDE9. Atropine increased cGMP levels in an NO-dependent manner in a small population of cultured forebrain cells in the presence of IBMX. In conclusion, cultured cholinergic basal forebrain neurons present a heterogeneous cell population in the magnitude of their response to NO. NO-responsiveness of the cultured cholinergic neurons is already detectable after one day of culturing and indicates that NO-sensitivity of the cholinergic neurons of the rat basal forebrain is present well before birth.