Nitric oxide signaling modulates synaptic transmission during early postnatal development.

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.

The relationship between prostaglandin E receptor 1 and cyclooxygenase I expression in guinea pig bladder interstitial cells: proposition of a signal propagation system.

PURPOSE: We explored the structural relationship between enzymes producing prostaglandin (cyclooxygenase I) and 1 of the receptor families that respond to prostaglandin (prostaglandin E receptor 1) in the bladder muscle. MATERIALS AND METHODS: Nine male guinea pigs were sacrificed by cervical dislocation. Bladders were removed and fixed in 4% paraformaldehyde in phosphate buffered saline. Frozen sections (10 µm) were cut and stained with antibodies to prostaglandin E receptor type 1, cyclooxygenase I and vimentin. RESULTS: Prostaglandin E receptors 1 was identified on smooth muscle cells, and vimentin positive surface muscle and intramuscular interstitial cells. Muscle staining was less intense than on interstitial cells and had a punctuate appearance. Prostaglandin E receptor 1 expression on interstitial cells was highly localized. Discrete regions of intense staining were noted on interstitial cell processes. Cyclooxygenase I was also expressed in muscle interstitial cells. Cyclooxygenase I positive interstitial cells were more prevalent in the muscle bundles of the inner muscle than in the outer muscle layers. Cyclooxygenase I staining was noted on discrete regions of the cell or cell processes. Double staining with prostaglandin E receptor 1 and cyclooxygenase I suggested that cell regions expressing the former are different from those expressing the latter. CONCLUSIONS: The discovered arrangement of prostaglandin E receptor 1 and cyclooxygenase I may have the potential to facilitate the propagation of signals in the interstitial cell network. Such a signaling system may have a role in coordinating events, as in bladder pathology, facilitating the global coordinated changes associated with bladder wall remodeling.

M3 muscarinic receptor-like immunoreactivity in sham operated and obstructed guinea pig bladders.

PURPOSE: Type 3 muscarinic receptors, which are present in the bladder wall, are important for bladder function. However, their role in the context of the urothelium is not well defined. Understanding the role of type 3 muscarinic receptors has been limited by the lack of specific type 3 muscarinic receptor antibodies. Thus, we identified a specific type 3 muscarinic receptor antibody and investigated the site of type 3 muscarinic receptors in sham operated and obstructed guinea pig bladders. MATERIALS AND METHODS: The specificity of 4 commercially available type 3 muscarinic receptor antibodies was determined. Immunohistochemistry was then done in bladder tissue from sham operated and obstructed guinea pig bladders. RESULTS: One of the 4 antibodies examined had the needed specificity in terms of blocking peptide and Western blot characterization. Using this antibody type 3 muscarinic receptor immunoreactivity was associated with muscle cells, nerves and interstitial cells. Four types of interstitial cells were identified, including suburothelial, lamina propria, surface muscle and intramuscular interstitial cells. In the obstructed model the bladder wall was hypertrophied and there was nerve fiber loss. The number of lamina propria, surface muscle and intramuscular interstitial cells was increased but not the number of suburothelial interstitial cells. Also, surface muscle interstitial cells appeared to form clusters or nodes with type 3 muscarinic receptor immunoreactivity. CONCLUSIONS: Nerve loss and the up-regulation of interstitial cells with type 3 muscarinic receptor immunoreactivity may underlie major functional changes in the pathological bladder. This indicates that type 3 muscarinic receptor specific anticholinergic drugs may affect not only the detrusor muscle, as previously thought, but also interstitial cells and nerve fibers.

Prostaglandin receptor EP1 and EP2 site in guinea pig bladder urothelium and lamina propria.

PURPOSE: Urothelium has 2 main functions. It is a barrier to urine and has a sensory role. In response to stretch urothelium releases various substances that modulate afferent nerve activity. Recent data on the localization of cyclooxygenase type 1, the enzyme responsible for prostaglandin production, suggests that prostaglandin may have complex local action. MATERIALS AND METHODS: The bladders of 7 guinea pigs were stained for prostaglandin receptors type 1 and 2, and costained for vimentin and cyclooxygenase I. RESULTS: Prostaglandin receptor type 1 staining was seen in urothelial cells and in the suburothelium. Urothelial staining, which was often punctuate and weak, was detected in all urothelial cell layers, including suburothelial cells. In contrast, strong prostaglandin receptor type 2 staining was seen in the urothelium and in suburothelial cells. Cyclooxygenase I was absent in interstitial cells and umbrella cells with the highest concentration in the basal cell layer. CONCLUSIONS: Interstitial cells express prostaglandin receptor types 1 and 2, indicating that they can respond to prostaglandin. Umbrella cells do not express cyclooxygenase I. Cyclooxygenase I was present in basal urothelial cells, making them a possible site of prostaglandin synthesis. Thus, prostaglandin produced by urothelium may target prostaglandin receptor types 1 and 2 in the urothelium and suburothelium. Therefore prostaglandin is hypothesized to have a role in signal regulation in the bladder wall.

Stretch independent regulation of prostaglandin E(2) production within the isolated guinea-pig lamina propria.

OBJECTIVE: To use an isolated preparation of the guinea-pig bladder lamina propria (LP) to investigate the effects of adenosine tri-phosphate (ATP) and nitric oxide (NO) on the release of prostaglandin E(2) (PGE(2)). MATERIALS AND METHODS: The bladders of female guinea-pigs (200-400 g) were isolated and opened to expose the urothelial surface. The LP was dissected free of the underlying detrusor muscle and cut into strips from the dome to base. Strips were then incubated in Krebs buffer at 37 degrees C. Each tissue piece was then exposed to the stable ATP analogue, BzATP, and a NO donor, diethylamine-NONOate (DEANO), and the effect on PGE(2) output into the supernatant determined using the Parameter(TM) PGE(2) enzyme immunoassay kit (R & D Systems, Abingdon, UK). Experiments were repeated in the presence of purinergic receptor and cyclooxygenase (COX) enzymes, COX I and COX II, antagonists. The cellular location of COX I, COX II and neuronal NO synthase (nNOS) within the bladder LP was also determined by immunohistochemistry. RESULTS: PGE(2) production was significantly increased by BzATP. Antagonist studies showed the purinergic stimulation involved both P(2)X and P(2)Y receptors. The BzATP response was inhibited by the COX inhibitor indomethacin (COX I >COX II) but not by DUP 697 (COX II >COX I). Thus, BzATP stimulation occurs because of COX I stimulation. NO had no effect on PGE(2) production over the initial 10 min of an exposure. However, PGE(2) output was increased 100 min after exposure to the NO donor. In the presence of NO, the BzATP stimulation was abolished. Immunohistochemistry was used to confirm the location of COX I to the basal and inner intermediate urothelial layers and to cells within the diffuse layer of LP interstitial cells. In addition, nNOS was also located in the basal urothelial layers whilst COX II was found in the interstitial cell layers. CONCLUSIONS: There is complex interaction between ATP and NO to modulate PGE(2) release from the bladder LP in the un-stretched preparation. Such interactions suggest a complex interrelationship of signals derived from this region of the bladder wall. The importance of these interactions in relation to the physiology of the LP remains to be determined.

Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins.

Recent results suggest that long-lasting potentiation at hippocampal synapses involves the rapid formation of clusters or puncta of presynaptic as well as postsynaptic proteins, both of which are blocked by antagonists of NMDA receptors and an inhibitor of actin polymerization. We have investigated whether the increase in puncta involves retrograde signaling through the NO-cGMP-cGK pathway and also examined the possible roles of two classes of molecules that regulate the actin cytoskeleton: Ena/VASP proteins and Rho GTPases. Our results suggest that NO, cGMP, cGK, actin, and Rho GTPases including RhoA play important roles in the potentiation and act directly in both the presynaptic and postsynaptic neurons, where they contribute to the increase in puncta of synaptic proteins. cGK phosphorylates synaptic VASP during the potentiation, whereas Rho GTPases act both in parallel and upstream of cGMP, in part by maintaining the synaptic localization of soluble guanylyl cyclase.

Thyroid hormone level positively regulates NOS and cGMP in the developing rat cerebellum.

Thyroid hormones and nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling play a significant role in the structural development of the cerebellum, respectively. In the present study, the possible contribution of neuronal NO synthase (NOS) and cGMP in the thyroid hormone-induced structural changes was investigated in the cerebella of postnatal rats at different hormone levels. Animals were treated from postnatal day 4 until days 7, 14, and 21, by i.p. injection of 1 microg thyroxine (T(4))/10 g body weight/4 days, or p.o. with 100 microg 6-n-propyl-2-thyouracil (PTU)/10 g body weight/day. Control groups consisted of i.p. and p.o. vehicle controls and PTU/T(4)-treated animals. Measurement of serum fT(4), TSH as well as total T(3) and T(4) concentration of the cerebellar tissue indicated the changed thyroid status. nNOS extensively expressed in growing parallel fibers revealed by quantitative Western blot and layer analysis of immunohistochemically labeled coronal sections. Simultaneously, the cGMP concentration increased and the distribution of cGMP-immunoreactive (cGMP-IR) material in Purkinje cell perikarya and in the molecular layer expanded during cerebellar development. T(4) increased nNOS and cGMP level, and accelerated the development of nNOS-IR parallel fibers and cGMP-IR molecular layer. In contrast, PTU retarded the development by decreasing nNOS and cGMP concentration and slowing down layer formation. A single dose of T(4) could rescue the PTU-induced changes. Results suggest that the contribution of nNOS- NO/cGMP signaling in thyroid hormone regulated structural maturation of the cerebellum. The possible involvement of neurotrophins, calcium, and apoptotic events in this process was discussed.

On the origins of the sensory output from the bladder: the concept of afferent noise.

For many people a recurrent strong desire to void, sometimes with incontinence, diminishes their quality of life. At present there are few insights into what underlies these problems. The condition is described as the 'overactive bladder symptom complex' but this definition is proving to be unhelpful. It focuses on overt bladder contractions rather than the main problem, which is altered and heightened sensation. Also, current approaches that describe bladder sensations as episodic and leading to voiding as 'first and second sensation to void' might also be misleading if they are taken too literally and used to suggest mechanisms. Current research is beginning to focus on the mechanisms that generate afferent information from the bladder and how it can become altered. As these views develop it is crucial that we appreciate the diversity of the bladder afferent system and distinguish between afferent and sensory information; in this review we explore this underlying complexity. The central nervous system (CNS) receives vast amounts of information from the bladder, which arises from different locations, uses different fibre types and involves different methods. The CNS is continually being bombarded with 'afferent noise'. The challenge now is to understand the nature and components of this 'afferent noise' and which components are essential to sensation. The emerging picture is complex, but this complexity must not be negated or oversimplified. It must be embraced and incorporated it into thinking when designing experiments, analysing data, diagnosing patients and evaluating treatment.

Ubiquitin hydrolase (protein gene product 9.5) in the obstructed bladder: evidence for tissue remodelling involving a subset of interstitial cells.

OBJECTIVE: To examine the expression of ubiquitin hydrolase (UH), an enzyme which is part of the ubiquitin-proteasome system involved in the regulation of cell growth and differentiation, to gain an insight into the cell types and processes underlying the tissue remodelling that occur after bladder neck damage. MATERIALS AND METHODS: Three groups of male guinea pigs were used, comprising controls (not operated, four), sham (five) and obstructed (six). The bladder outlet was obstructed by implanting a silver ring around the urethra, which was left in situ for 2-4 weeks. Sham-operated guinea pigs had the same operative procedure but no ring was implanted. The bladders were removed and incubated in Krebs' solution at 36 degrees C, gassed with 95% O2 and 5% CO2, Tissues were then fixed in 4% depolymerized paraformaldehyde and processed for immunohistochemistry. We used antibodies raised against UH, cyclooxygenase type I and vimentin. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: Staining with an antibody to UH showed the presence of both sensory and motor nerves in control, sham and obstructed bladders. In the control bladders this was the predominant staining pattern. In the sham and obstructed bladders UH staining revealed additional positive cell types; cells associated with the outermost layers of the urothelium (the umbrella cells), in the lamina propria (the lamina propria interstitial cells (lp-ICs), on the surface of the muscle bundles (surface muscle, sm-ICs) and on the serosal surface (muscle coat, mc-ICs). All ICs stained with vimentin. The ICs within the muscle bundles (intramuscular, im-ICs) did not stain with UH. The number and density of the UH-positive cells was greater in the obstructed than in the sham bladders, suggesting a change in relation to the severity of damage to the bladder neck. CONCLUSION: The expression of UH implies the re-targeting of proteins marked for degradation in the proteasome. Increased expression of UH in the lp-ICs and sm-ICs shows that these cells are active in the early and late stages of the tissue remodelling resulting from obstruction. These results show a further subset of ICs that might be involved in the increased deposition of extracellular material and tissue remodelling.

M(3) muscarinic receptor expression on suburothelial interstitial cells.

OBJECTIVE: To identify the cells expressing the M(3) muscarinic receptor subtype in the lamina propria of the bladder. MATERIALS AND METHODS: The bladders from five normal guinea pigs were isolated and fixed in 4% paraformaldehyde. Tissues sections (10 microm) were then cut and stained with antibodies to the type 3 muscarinic receptor (M(3)), the interstitial cell marker vimentin and the nonspecific nerve marker PGP 9.5. The specificity of the antibody to the M(3) receptor was established using the complementary blocking peptide and Western blot analysis of human embryonic kidney (HEK) cells transfected to express the M(3) receptor protein. RESULTS The M(3) antibody pre-incubated with its blocking peptide showed no immunohistochemical staining. Investigating this antibody using HEK cells transfected to express the M(3) receptor protein and control HEK cells showed a single band in the transfected cells and no band in the control cells. M(3) receptor immunoreactivity (M(3)-IR) was detected primarily on a dense network of vimentin-positive (vim(+)) cells lying immediately below the urothelium, i.e. the suburothelial interstitial cells (Su-ICs). The M(3)-IR was punctate and appeared to be located on the cell surface. The diffuse network of cells in the remaining regions of the lamina propria showed no M(3)-IR. Few nerve fibres were associated with the M(3)-IR Su-ICs. The M(3)-IR Su-ICs were most numerous and prominent in the lateral wall. The number of M(3)-IR/vim(+) cells diminished towards the bladder base and were absent in the bladder urethral junction. In the base and urethral junction there were vim(+) cells that were not M(3)-IR. A population of umbrella cells in the lateral wall also showed weak punctate M(3)-IR. CONCLUSIONS Using a well-characterized M(3) antibody, these results show for the first time that the M(3) muscarinic receptor in the lamina propria is located specifically on the Su-ICs. The physiological role of these cells is unknown and consequently the significance of what appears to be a cholinergic signalling system is unclear. Previously published data showed that these cells respond to nitric oxide and atrial natriuretic peptide with an increase in cGMP and possibly prostaglandin. All of these observations, taken together, suggest that the Su-ICs receive multiple inputs and that they must be part of a complex signalling system in this region of the bladder wall.

Involvement of nitric oxide in depolarization-induced suppression of inhibition in hippocampal pyramidal cells during activation of cholinergic receptors.

Several types of neurons are able to regulate their synaptic inputs via releasing retrograde signal molecules, such as endocannabinoids or nitric oxide (NO). Here we show that, during activation of cholinergic receptors, retrograde signaling by NO controls CB1 cannabinoid receptor (CB1R)-dependent depolarization-induced suppression of inhibition (DSI). Spontaneously occurring IPSCs were recorded in CA1 pyramidal neurons in the presence of carbachol, and DSI was induced by a 1-s-long depolarization step. We found that, in addition to the inhibition of CB1Rs, blocking the NO signaling pathway at various points also disrupted DSI. Inhibitors of NO synthase (NOS) or NO-sensitive guanylyl cyclase (NO-sGC) diminished DSI, whereas a cGMP analog or an NO donor inhibited IPSCs and partially occluded DSI in a CB1R-dependent manner. Furthermore, an NO scavenger applied extracellularly or postsynaptically also decreased DSI, whereas L-arginine, the precursor for NO, prolonged it. DSI of electrically evoked IPSCs was also blocked by an inhibitor of NOS in the presence, but not in the absence, of carbachol. In line with our electrophysiological data, double immunohistochemical staining revealed an NO-donor-induced cGMP accumulation in CB1R-positive axon terminals. Using electron microscopy, we demonstrated the postsynaptic localization of neuronal NOS at symmetrical synapses formed by CB1R-positive axon terminals on pyramidal cell bodies, whereas NO-sGC was found in the presynaptic terminals. These electrophysiological and anatomical results in the hippocampus suggest that NO is involved in depolarization-induced CB1R-mediated suppression of IPSCs as a retrograde signal molecule and that operation of this cascade is conditional on cholinergic receptor activation.

Cyclic GMP in the pig vitreous and retina after experimental retinal detachment.

PURPOSE: Earlier studies have revealed a decreased level of cGMP in vitreous fluid obtained from patients with a retinal detachment. To further investigate this phenomenon, we developed an experimental retinal detachment model in pigs. METHODS: Experimental unilateral retinal detachments were induced in pig eyes by subretinal injection of 0.25% sodium hyaluronate. Fourteen days later the vitreous and retinas were analyzed for cGMP expression. Following enucleation, the retinas were incubated in the presence of a nonselective phosphodiesterase inhibitor (IBMX), and the particulate guanylyl cyclase stimulator atrial natriuretic peptide (ANP) or the soluble guanylyl cyclase stimulator sodium nitroprusside (SNP). cGMP was visualized in retinal wholemounts by immunochemistry combined with a computer based stereology system. cGMP levels in vitreous were determined by ELISA. RESULTS: The mean vitreous cGMP level in pig eyes with a retinal detachment (1.45 pmol/ml) was significantly lower compared to the mean level of cGMP in healthy pig eyes (4.61 pmol/ml; p=0.028 was considered significant). In the inner retina, ANP as well as SNP induced cGMP immunoreactivity in both detached and healthy retinas. After incubation with ANP, cGMP could also be detected in the outer nuclear layer of the detached retina, whereas this was not the case in the normal retina. CONCLUSIONS: Experimental retinal detachment in the pig eye leads to a decrease of cGMP levels in vitreous similar to that observed in clinical studies. This model may be helpful to analyze the mechanisms involved in cGMP dynamics following retinal detachment.

Regional differences in sensory innervation and suburothelial interstitial cells in the bladder neck and urethra.

OBJECTIVE: To identify and characterize possible structural specialisations in the wall of the lower urinary tract (LUT) in the region of the bladder urethral junction (BUJ), with the specific objective of identifying regional variations in sensory nerve fibres and interstitial cells (ICs). MATERIALS AND METHODS: The bladder base and urethra was removed from five male guinea pigs killed by cervical dislocation. Tissue pieces were incubated in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), fixed in 4% paraformaldehyde and processed for immunohistochemistry. The nonspecific marker vimentin and the general neuronal marker protein gene product (PGP) 9.5 were used to identify ICs and nerve fibres, respectively. Specific antibody binding was visualized using the appropriate secondary antibodies. RESULTS: The wall of the LUT in the region immediately between the bladder base and the urethra, the BUJ, differed in its cellular composition relative to the adjacent areas. PGP-positive (PGP(+)) nerve fibres, presumptive afferent fibres, lay within the urothelium running between the epithelial cells. There were two general nerve patterns: branching fibres with no varicosities, and complex fibres with varicosities. Fibre collaterals with varicosities exited the urothelium and occupied the space under the urothelium adjacent to the layer of suburothelial ICs. The latter, lamina propria and around the muscle bundles were identified using vimentin (vim(+)). In the base a few vim(+) cells were also PGP(+). In the region of the BUJ there was a decrease in the amount of smooth muscle. In this region, below the lamina propria, there was an area densely populated with vim(+)/PGP(+) ICs. Nerve fibres ran between the cells in this region. CONCLUSION: These structural specialisations within the urothelium and deeper layers of the BUJ suggest that they might be associated with specific functions. The localized highly branched network of the putative afferent nerves suggests the presence of a local axonal reflexes involving possible cross-talk between the urothelium and suburothelial layer. The function of the specialized region of ICs is not known and must await further information on the functional properties of this novel cell type. These observations show further the cellular heterogeneity of the cells in the LUT and the complexity of the structures. One of the major current challenges in functional urology is to understand the relationships between these novel structures and overall bladder and urethral function.

Two autocrine pathways to regulate cyclic GMP synthesis in cultured human retinal pigment epithelial cells.

AIM: To investigate the role of two separate enzymatic pathways [soluble (sGC) vs. particulate (pGC) guanylyl cyclase] in the synthesis of cyclic GMP (cGMP) in cultured human retinal pigment epithelial (RPE) cells. METHODS: cGMP accumulation was evaluated by quantitative analysis of cGMP immunoreactivity. RPE cells were also stained for inducible nitric oxide synthase (iNOS), ANP and beta(1)- and alpha(2)-subunits of sGC. RESULTS: We showed nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and iNOS immunoreactivity in RPE cells. Incubation of the cells in the presence of 1 mM IBMX to inhibit phosphodiesterase activity, and the simultaneous inhibition of NOS activity with L-NAME suggested the involvement of sGC in maintaining a low level of cGMP in the RPE cells. The involvement of sGC was further supported by detection of the beta(1)- and alpha(2)-subunits of sGC. Incubation of the cells in the presence of atrial natriuretic peptide (ANP) to stimulate pGC strongly increased cGMP immunoreactivity. We also demonstrated the presence of ANP in all RPE cells. CONCLUSION: Cultured human RPE cells are capable of producing cGMP after stimulation of sGC or pGC. The presence of iNOS and ANP in all cells suggests two different autocrine pathways of stimulating cGMP production in these cells. The possible role of cGMP in the regulation iNOS gene expression and in the regulation of ANP is discussed.

Dissociable effects of acetylcholinesterase inhibitors and phosphodiesterase type 5 inhibitors on object recognition memory: acquisition versus consolidation.

RATIONALE: Phosphodiesterase enzyme type 5 (PDE5) inhibitors and acetylcholinesterase (AChE) inhibitors have cognition-enhancing properties. However, it is not known whether these drug classes affect the same memory processes. OBJECTIVE: We investigated the memory-enhancing effects of the PDE5 inhibitor sildenafil and AChE inhibitors metrifonate and donepezil in the object recognition task to find out whether acquisition or consolidation processes were affected by these drugs. METHODS: The object recognition task measures whether rats remembered an object they have explored in a previous learning trial. All drugs were given orally 30 min before or immediately after learning to study acquisition and consolidation, respectively. RESULTS: Sildenafil given immediately after the first trial improved the memory performance after 24 h and resulted in an inverted U-shaped dose-effect curve with the peak dose at 3 mg/kg. When given before the first trial, sildenafil also improved the memory performance. However, the dose needed for the best performance under this condition was 10 mg/kg, suggesting that the dose-effect curve shifted to the right. This can be explained by the metabolic clearance of the high dose of sildenafil. Donepezil had no memory improving effect when given after the first trial. However, when given before the first trial, a gradually increasing dose-effect curve was found which had its maximum effect at the highest dose tested (1 mg/kg). Likewise, only when metrifonate (30 mg/kg) was given before the first trial did rats show an improved memory performance. CONCLUSION: Our data strongly suggest that PDE5 inhibitors improve processes of consolidation of object information, whereas AChE inhibitors improve processes of acquisition of object information.

Localization and characterization of cGMP-immunoreactive structures in rat brain slices after NO-dependent and NO-independent stimulation of soluble guanylyl cyclase.

Possible differences in the localization of the cGMP response were investigated in rat brain coronal slices after in vitro incubation and NO-dependent or NO-independent stimulation of soluble guanylyl cyclase (sGC). Dose-dependent stimulation of cGMP synthesis by the NO donors, sodium nitroprusside, S-nitrosoglutathione, 3-morpholinosydnonimine and diethylamino NONOate was studied in the somatoparietal cortex, the hippocampus and the thalamus. cGMP accumulation was evaluated using a radioimmunoassay and by measuring cGMP-immunofluorescence using image analysis. All four NO donors induced similar cGMP staining patterns in the somatoparietal cortex, the hippocampus and the thalamus. NO-mediated cGMP synthesis in the cortical areas colocalized predominantly with the acetylcholine transporter and occasionally with parvalbumin (GABAergic cells) or the neuronal glutamate transporter. Incubation of the slices in the combined presence of a NO donor and the NO-independent activators YC-1 or BAY 41-2272 strongly potentiated cGMP synthesis and induced abundant cGMP-immunoreactivity in cortical GABAergic and glutamatergic cells. These findings indicate that the mechanism of NO release from the NO donors used does not determine the location of the cGMP response. The results suggest that YC-1 and BAY 41-2272 trigger a NO-sensing mechanism in cells in which the sGC is otherwise not sensitive to NO.

GSA: behavioral, histological, electrophysiological and neurochemical effects.

Renal insufficient patients suffer from a variety of complications as direct and indirect consequence of accumulation of retention solutes. Guanidinosuccinic acid (GSA) is an important probable uremic toxin, increased in plasma, urine, cerebrospinal fluid and brain of patients with uremia and supposed to play a role in the pathogenesis of some neurological symptoms. GSA, an NMDA-receptor agonist and GABA-receptor antagonist, is suggested to act as an excitotoxin and shown to be convulsive. The effect of hippocampal (i.h.) GSA injection on behavior and hippocampal volume in mice is presented here. In addition, hippocampal cGMP concentration after systemic injection of GSA was measured. The effect of co-application of NMDA-receptor antagonist CGP37849 with GSA was tested, in vivo, after hippocampal GSA injection and, in vitro, on GSA evoked currents in spinal cord neurons. A significant dose-dependent effect of i.h. injection of GSA on cognitive performance, activity and social exploratory behavior was observed. There was a protective effect of CGP37849 on GSA induced behavioral alterations. Volume of hippocampal cornu ammonis region decreased significantly and dose-dependently after GSA injection. Systemic GSA injection increased cGMP concentration in hippocampal formation. It can be concluded that GSA is an important neurotoxin. As GSA is increased in patients with uremia, it probably contributes to their neurological symptoms. Knowledge of neurotoxic effects and mechanisms of action of GSA and other uremic retention solutes could help in the development of more efficient treatment of uremic patients. Animal models like the 'GSA mouse model' are useful tools for research in this context.

Expression of nitric oxide synthase and nitric oxide-sensitive guanylate cyclase in the crustacean cardiac ganglion.

The cardiac ganglion is a simple central pattern-generating network that controls the rhythmic contractions of the crustacean heart. Enzyme assays and Western blots show that whole heart homogenates from the crab Cancer productus contain high levels of nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of arginine to citrulline with concomitant production of the transmitter nitric oxide (NO). Crab heart NOS is calcium-dependent and has an apparent molecular weight of 110 kDa. In the cardiac ganglion, antibodies to NOS and citrulline indicate the presence of a NOS-like protein and NOS enzymatic activity in the four small pacemaker neurons and the five large motor neurons of the cardiac network. In addition, all cardiac neurons label positively with an antibody to cyclic guanosine monophosphate (cGMP). The NO donor sodium nitroprusside (SNP, 10 mM) stimulates additional cGMP production in the isolated ganglion. This increase is blocked by [(1)H](1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ, 50 microM), an inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). Taken together, our data indicate that NO- and cGMP-mediated signaling pathways are enriched in the cardiac system relative to other crab tissues and that the cardiac network may be a target for extrinsic and intrinsic neuromodulation via NO produced from the heart musculature and individual cardiac neurons, respectively. The crustacean cardiac ganglion is therefore a promising system for studying cellular and synaptic mechanisms of nitrergic neuromodulation in a simple pattern-generating network.

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

Effect of peripheral nerve injury on cGMP and nitric oxide synthase levels in rat dorsal root ganglia: time course and coexistence.

Using the indirect immunofluorescence method, the distribution of cyclic GMP (cGMP) and nitric oxide synthase (NOS) was investigated in lumbar 5 dorsal root ganglia (DRGs) of untreated rats 1, 3 and 7 days following sciatic nerve section (axotomy). Untreated and axotomized (7 days) rats were also studied after perfusion with the NO donor sodium nitroprusside (SNP). Moreover, rats were injected with carrageenan lambda into the unilateral hindpaw and studied after 6 h, 1 day or 2 days. An increase in the number of cGMP-positive satellite cell profiles was found in axotomized DRGs at 3 days with lower numbers after 7 days. In contrast, no change in cGMP-like immunoreactivity (LI) in satellite cell profiles was detected 1 day after axotomy or 6h, 1 day or 2 days after inflammation, as compared to controls. Axotomy induced a marked increase in the percentage of NOS-immunoreactive (IR) neuron profiles in the ipsilateral DRGs as follows: 3.0% at 1 day, 15% at 3 days and 25% at 7 days, whereas no significant change was found in the expression of NOS-LI in the inflamed DRGs as compared to untreated DRGs. Between 15 and 20% of all NOS-positive neuron profiles were surrounded by, or in partial contact with, cGMP-IR satellite cells in controls 1 and 3 days after axotomy, whereas the corresponding figure was around 5% after 7 days. After SNP perfusion 60-70% of all DRG neuron profiles were partly or totally associated with cGMP-positive satellite cell profiles, with no significant difference between untreated and axotomized ganglia. The nerve injury-induced, parallel upregulation of NOS in DRG neurons and cGMP in satellite cells in the initial phase after axotomy suggests an involvement of NO as a signalling molecule between neurons and satellite cells in DRGs, especially after peripheral nerve injury, perhaps exerting a survival effect as recently proposed by Thippeswamy and Morris (1997).