A Novel Phosphodiesterase 1 Inhibitor DSR-141562 Exhibits Efficacies in Animal Models for Positive, Negative, and Cognitive Symptoms Associated with Schizophrenia.

In our drug discovery program, we identified a novel orally available and brain-penetrant phosphodiesterase (PDE) 1 inhibitor, 3-methyl-7-(tetrahydro-2H-pyran-4-yl)-2-{[trans-4-(trifluoromethyl)cyclohexyl]-methoxy}imidazo[5,1-f][1,2,4]triazin-4(3H)-one (DSR-141562). In the present study, we characterized the preclinical profile of DSR-141562. This compound has preferential selectivity for predominantly brain-expressed PDE1B over other PDE1 family members, and high selectivity for the PDE1 family over other PDE families and 65 other tested biologic targets. Oral administration of DSR-141562 at 10 mg/kg slightly elevated the cGMP concentration, and it potently enhanced the increase of cGMP induced by a dopamine D1 receptor agonist in mouse brains. The cGMP level in monkey cerebrospinal fluid was also elevated after treatment with DSR-141562 at 30 and 100 mg/kg and could be used as a translational biomarker. Since PDE1B is believed to regulate dopaminergic and glutamatergic signal transduction, we evaluated the effects of this compound using schizophrenia-related behavioral assays. DSR-141562 at 3-30 mg/kg potently inhibited methamphetamine-induced locomotor hyperactivity in rats, while it had only minimal effects on the spontaneous locomotor activity. Furthermore, DSR-141562 at 1-100 mg/kg did not induce any signs of catalepsy in rats. DSR-141562 at 0.3-3 mg/kg reversed social interaction and novel object recognition deficits induced by repeated treatment with an N-methyl-D-aspartate receptor antagonist, phencyclidine, in mice and rats, respectively. In common marmosets, DSR-141562 at 3 and 30 mg/kg improved the performance in object retrieval with detour tasks. These results suggest that DSR-141562 is a therapeutic candidate for positive, negative, and cognitive symptoms in schizophrenia. SIGNIFICANCE STATEMENT: This is the first paper showing that a phosphodiesterase 1 inhibitor is efficacious in animal models for positive and negative symptoms associated with schizophrenia. Furthermore, we demonstrated that this compound improved cognitive function in the common marmoset, a nonhuman primate.

The Novel Phosphodiesterase 9A Inhibitor BI 409306 Increases Cyclic Guanosine Monophosphate Levels in the Brain, Promotes Synaptic Plasticity, and Enhances Memory Function in Rodents.

N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) is an established cellular model underlying learning and memory, and involves intracellular signaling mediated by the second messenger cyclic guanosine monophosphate (cGMP). As phosphodiesterase (PDE)9A selectively hydrolyses cGMP in areas of the brain related to cognition, PDE9A inhibitors may improve cognitive function by enhancing NMDA receptor-dependent LTP. This study aimed to pharmacologically characterize BI 409306, a novel PDE9A inhibitor, using in vitro assays and in vivo determination of cGMP levels in the brain. Further, the effects of BI 409306 on synaptic plasticity evaluated by LTP in ex vivo hippocampal slices and on cognitive performance in rodents were also investigated. In vitro assays demonstrated that BI 409306 is a potent and selective inhibitor of human and rat PDE9A with mean concentrations at half-maximal inhibition (IC50) of 65 and 168 nM. BI 409306 increased cGMP levels in rat prefrontal cortex and cerebrospinal fluid and attenuated a reduction in mouse striatum cGMP induced by the NMDA-receptor antagonist MK-801. In ex vivo rat brain slices, BI 409306 enhanced LTP induced by both weak and strong tetanic stimulation. Treatment of mice with BI 409306 reversed MK-801-induced working memory deficits in a T-maze spontaneous-alternation task and improved long-term memory in an object recognition task. These findings suggest that BI 409306 is a potent and selective inhibitor of PDE9A. BI 409306 shows target engagement by increasing cGMP levels in brain, facilitates synaptic plasticity as demonstrated by enhancement of hippocampal LTP, and improves episodic and working memory function in rodents. SIGNIFICANCE STATEMENT: This preclinical study demonstrates that BI 409306 is a potent and selective PDE9A inhibitor in rodents. Treatment with BI 409306 increased brain cGMP levels, promoted long-term potentiation, and improved episodic and working memory performance in rodents. These findings support a role for PDE9A in synaptic plasticity and cognition. The potential benefits of BI 409306 are currently being investigated in clinical trials.

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway.

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 µM), or norbinaltorphimine (norBIN, 1 µM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Salvinorin A preserves cerebral pial artery autoregulation after forebrain ischemia via the PI3K/AKT/cGMP pathway

This study aimed to investigate the protective effect of salvinorin A on the cerebral pial artery after forebrain ischemia and explore related mechanisms. Thirty Sprague-Dawley rats received forebrain ischemia for 10 min. The dilation responses of the cerebral pial artery to hypercapnia and hypotension were assessed in rats before and 1 h after ischemia. The ischemia reperfusion (IR) control group received DMSO (1 µL/kg) immediately after ischemia. Two different doses of salvinorin A (10 and 20 µg/kg) were administered following the onset of reperfusion. The 5th, 6th, and 7th groups received salvinorin A (20 µg/kg) and LY294002 (10 µM), L-NAME (10 μM), or norbinaltorphimine (norBIN, 1 μM) after ischemia. The levels of cGMP in the cerebrospinal fluid (CSF) were also measured. The phosphorylation of AKT (p-AKT) was measured in the cerebral cortex by western blot at 24 h post-ischemia. Cell necrosis and apoptosis were examined by hematoxylin-eosin staining (HE) and TUNEL staining, respectively. The motor function of the rats was evaluated at 1, 2, and 5 days post-ischemia. The dilation responses of the cerebral pial artery were significantly impaired after ischemia and were preserved by salvinorin A treatment. In addition, salvinorin A significantly increased the levels of cGMP and p-AKT, suppressed cell necrosis and apoptosis of the cerebral cortex and improved the motor function of the rats. These effects were abolished by LY294002, L-NAME, and norBIN. Salvinorin A preserved cerebral pial artery autoregulation in response to hypercapnia and hypotension via the PI3K/AKT/cGMP pathway.

Reduced cGMP levels in CSF of AD patients correlate with severity of dementia and current depression.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder, primarily affecting memory. That disorder is thought to be a consequence of neuronal network disturbances and synapse loss. Decline in cognitive function is associated with a high burden of neuropsychiatric symptoms (NPSs) such as depression. The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are essential second messengers that play a crucial role in memory processing as well as synaptic plasticity and are potential therapeutic targets. Biomarkers that are able to monitor potential treatment effects and that reflect the underlying pathology are of crucial interest. METHODS: In this study, we measured cGMP and cAMP in cerebrospinal fluid (CSF) in a cohort of 133 subjects including 68 AD patients and 65 control subjects. To address the association with disease progression we correlated cognitive status with cyclic nucleotide levels. Because a high burden of NPSs is associated with decrease in cognitive function, we performed an exhaustive evaluation of AD-relevant marker combinations in a depressive subgroup. RESULTS: We show that cGMP, but not cAMP, levels in the CSF of AD patients are significantly reduced compared with the control group. Reduced cGMP levels in AD patients correlate with memory impairment based on Mini-Mental State Examination score (r = 0.17, p = 0.048) and tau as a marker of neurodegeneration (r = -0.28, p = 0.001). Moreover, we were able to show that AD patients suffering from current depression show reduced cGMP levels (p = 0.07) and exhibit a higher degree of cognitive impairment than non-depressed AD patients. CONCLUSION: These results provide further evidence for an involvement of cGMP in AD pathogenesis and accompanying co-morbidities, and may contribute to elucidating synaptic plasticity alterations during disease progression.

Pharmacokinetic investigation of sildenafil using positron emission tomography and determination of its effect on cerebrospinal fluid cGMP levels.

Sildenafil (Viagra) is a selective inhibitor of phosphodiesterase type 5 (PDE5), which degrades cyclic guanosine monophosphate to the linear nucleotide. Sildenafil is acutely used in erectile dysfunction and chronically in pulmonary hypertension. Evidence in the last decade shows that sildenafil may have potential as a therapeutic option for Alzheimer's disease or other neurodegenerative disorders. The purpose of this work was to explore whether sildenafil crosses the blood-brain barrier. Pharmacokinetic properties of sildenafil in rodents were investigated using (11) C-radiolabeling followed by in vivo positron emission tomography (PET) and ex vivo tissue dissection and gamma counting. PET results in rats suggest penetration into the central nervous system. Ex vivo data in perfused animals suggest that trapping of [(11) C]sildenafil within the cerebral vascular endothelium limits accumulation in the central nervous system parenchyma. Peroral sildenafil administration to Macaca fascicularis and subsequent chemical analysis of plasma and cerebrospinal fluid (CSF) using liquid chromatography coupled with tandem mass spectrometry showed that drug content in the CSF was high enough to achieve PDE5 inhibition, which was also demonstrated by the significant increases in CSF cyclic guanosine monophosphate levels. Central actions of sildenafil include both relaxation of the cerebral vasculature and inhibition of PDE5 in neurons and glia. This central action of sildenafil may underlie its efficacy in neuroprotection models, and may justify the continued search for a PDE5 ligand suitable for PET imaging. Sildenafil interacts with phosphodiesterase type 5 (PDE5) expressed in the endothelium and/or smooth muscle cells of brain vessels and also crosses the blood-brain barrier to interact with PDE5 expressed in brain cells. At therapeutic doses, the concentration of sildenafil in the cerebrospinal fluid (CSF) is high enough to inhibit PDE5 in the neural cells (neurons and glia). In turn, the concentration of cGMP likely increases in parenchymal cells and, as shown in this report, in the CSF. Read the Editorial Highlight for this article on page 220. Cover Image for this issue: doi: 10.1111/jnc.13302.

Decreased levels of guanosine 3', 5'-monophosphate (cGMP) in cerebrospinal fluid (CSF) are associated with cognitive decline and amyloid pathology in Alzheimer's disease.

AIMS: Levels of the cyclic nucleotides guanosine 3', 5'-monophosphate (cGMP) or adenosine 3', 5'-monophosphate (cAMP) that play important roles in memory processes are not characterized in Alzheimer's disease (AD). The aim of this study was to analyse the levels of these nucleotides in cerebrospinal fluid (CSF) samples from patients diagnosed with clinical and prodromal stages of AD and study the expression level of the enzymes that hydrolyzed them [phosphodiesterases (PDEs)] in the brain of AD patients vs. METHODS: For cGMP and cAMP CSF analysis, the cohort (n = 79) included cognitively normal participants (subjective cognitive impairment), individuals with stable mild cognitive impairment or AD converters (sMCI and cMCI), and mild AD patients. A high throughput liquid chromatography-tandem mass spectrometry method was used. Interactions between CSF cGMP or cAMP with mini-mental state examination (MMSE) score, CSF Aß(1-42) and CSF p-tau were analysed. For PDE4, 5, 9 and 10 expression analysis, brains of AD patients vs. controls (n = 7 and n = 8) were used. RESULTS: cGMP, and not cAMP levels, were significantly lower in the CSF of patients diagnosed with mild AD when compared with nondemented controls. CSF levels of cGMP showed a significant association with MMSE-diagnosed clinical dementia and with CSF biomarker Aß42 in AD patients. Significant increase in PDE5 expression was detected in temporal cortex of AD patients compared with that of age-matched healthy control subjects. No changes in the expression of others PDEs were detected. CONCLUSIONS: These results support the potential involvement of cGMP in the pathological and clinical development of AD. The cGMP reduction in early stages of AD might participate in the aggravation of amyloid pathology and cognitive decline.

Application of structure-based drug design and parallel chemistry to identify selective, brain penetrant, in vivo active phosphodiesterase 9A inhibitors.

Phosphodiesterase 9A inhibitors have shown activity in preclinical models of cognition with potential application as novel therapies for treating Alzheimer's disease. Our clinical candidate, PF-04447943 (2), demonstrated acceptable CNS permeability in rats with modest asymmetry between central and peripheral compartments (free brain/free plasma = 0.32; CSF/free plasma = 0.19) yet had physicochemical properties outside the range associated with traditional CNS drugs. To address the potential risk of restricted CNS penetration with 2 in human clinical trials, we sought to identify a preclinical candidate with no asymmetry in rat brain penetration and that could advance into development. Merging the medicinal chemistry strategies of structure-based design with parallel chemistry, a novel series of PDE9A inhibitors was identified that showed improved selectivity over PDE1C. Optimization afforded preclinical candidate 19 that demonstrated free brain/free plasma ≥ 1 in rat and reduced microsomal clearance along with the ability to increase cyclic guanosine monophosphosphate levels in rat CSF.

CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis.

BACKGROUND: The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD). METHODOLOGY/PRINCIPAL FINDINGS: Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (-70%) and cGMP (-55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99). CONCLUSIONS/SIGNIFICANCE: We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control.

Simultaneous LC-MS/MS analysis of the biomarkers cAMP and cGMP in plasma, CSF and brain tissue.

The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are important second messengers. They are useful biomarkers to indicate biological activity of drugs such as phosphodiesterase (PDE) inhibitors which block the degradation of these nucleotides. Here, we established a fast and sensitive method for the simultaneous analysis of cAMP and cGMP by LC-MS/MS with broad applicability. The limit of detection is 50 pM. Linearity is given in a range of 0.5-500 nM for both nucleotides, with a high intra- and inter-assay precision and accuracy and an analysis time of 3.5 min. We validated the suitability of the method by pharmacological modulation of cAMP or cGMP concentrations in mice with the PDE4 inhibitor rolipram and the PDE5 inhibitor zaprinast. Rolipram significantly increased cAMP concentrations in plasma, CSF and brain tissue. Zaprinast increased cGMP concentrations in plasma but not in brain tissue, which is in accordance with its blood brain barrier permeability. In conclusion, the LC-MS/MS method described here could be a valuable analytical tool for investigating pharmacodynamic effects of PDE inhibitors and to monitor disease-related changes of cAMP and cGMP in the periphery as well as in the central nervous system.

[Clinical and laboratory assessment of indicators of oxidative status in the cerebrospinal fluid of patients with ischemic stroke].

The objective of the present study was to estimate parameters of oxidative status in the cerebrospinal fluid in the course of ischemic stroke and in the prediction of recovery of neurological functions. Concentration of superoxide dismutase (SOD) as a marker of antioxidant adaptation, the secondary lipid peroxidation products reacting with thiobarbituric acid (PRTBA), cyclic guanosine monophosphate (cGMP) as an indirect product of NO generation, and N-acetylneuraminic acid (NANA) as a marker of destruction of neuronal membranes were studied. One hundred and fifty patients with hemispheric ischemic stroke admitted to a hospital during the first 12 h after stroke were examined. It has been shown that the development of cerebral infarction is accompanied by increased concentrations of oxidative stress markers. Progressive ischemic stroke was characterized by the significantly prolonged increase in PRTBA, cGMP, NANA to the third day from the first symptoms of disease while regressive course was accompanied by the lack of higher production of TBKRP, cGMP, NANA to the third day of disease. The decrease in concentrations of factors of brain damage (PRTBA, cGMP, NANA) in the cerebrospinal fluid, along with the increasing role of processes of antioxidant adaptation, expressed in the growth of SOD concentrations, can be considered as a criterion for the prediction of recovery of disturbed neurologic function to the 21th day of disease.

Selective D1 agonism but not D2 antagonism is reflected in cAMP and cGMP levels in rat CSF.

Cyclic adenosine 3'5'-monophosphate (cAMP) and cyclic guanosine 3'5'-monophosphate (cGMP) serve as second messengers in several cellular pathways within the central nervous system. In various neurological and psychiatric disorders with known deficits in neurotransmission function, CSF levels of cAMP and/or cGMP in patients were studied. Very little information is currently available on cAMP and cGMP levels in CSF of animals. Moreover, this is the first study on the effects of pharmacological treatment on cAMP and cGMP levels in rat CSF. Effect of systemic treatment with a D1 receptor agonist SKF82958 and a D2 receptor antagonist haloperidol on cAMP and cGMP levels, as well as baseline cAMP and cGMP levels in CSF of rats was determined. A significantly increased cAMP and cGMP level in cisternal CSF of rats systemically treated with the D1 receptor agonist SKF82958 was observed, while when treated with the D2 antagonist haloperidol, no effect on cAMP and only a slight decrease of cGMP was observed after treatment with the highest dose. Determining cAMP and/or cGMP in CSF of experimental animals can serve as a useful tool to study neural processes affected by disease and treatment.

Decreased cerebrospinal fluid cGMP levels in patients with amyotrophic lateral sclerosis.

The role of cyclic guanosine 5' monophosphate (cGMP) in neurodegeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is still controversial. The aim of this study was to measure levels of cGMP in cerebrospinal fluid (CSF) of patients with ALS, and to investigate whether there is a relationship between CSF cGMP levels and clinical parameters of the disease. The study involved 30 ALS and 20 control group patients. The CSF cGMP was measured by the enzyme-linked immunosorbent assay. The results showed that levels of CSF cGMP were significantly decreased in the group of ALS patients compared to controls and did not depend on clinical state of ALS patients, type of ALS onset, or the duration of the disease. Decreased levels of CSF cGMP observed in this study may suggest the role of cGMP in neurodegeneration in ALS. The CSF cGMP cannot be a marker of the disease activity.

Regulation of rat pial arteriolar smooth muscle relaxation in vivo through multidrug resistance protein 5-mediated cGMP efflux.

Multidrug resistance protein 5 (MRP5) has been linked to cGMP cellular export in peripheral vascular smooth muscle cells (VSMCs) and is widely expressed in brain vascular tissue. In the present study, we examined whether knockdown of MRP5 in pial arterioles [via antisense oligodeoxynucleotide (ODN) applications] affected nitric oxide (NO)/cGMP-induced dilations. The antisense or (as a control) missense ODN was applied to the cortical surface approximately 24 h before study via closed cranial windows. The efficacy of the antisense vs. missense ODN in eliciting selective reductions in MRP5 expression was confirmed by analysis of MRP5 mRNA in pial tissue. Unexpectedly, in initial studies, a significantly lower maximal pial arteriolar diameter increase in the presence of the NO donor S-nitrosoacetylpenicillamine (SNAP) was seen in the antisense vs. missense ODN-treated rats (35 vs. 48% diameter increase, respectively). It was suspected that this related to a reduced vascular smooth muscle cell sensitivity to cGMP due to prolonged exposure to increased intracellular cGMP levels elevated by overnight restriction of cGMP efflux. That postulate was supported by a finding of a diminished vasodilating response to the cGMP-dependent protein kinase-activating cGMP analog 8-p-chlorophenylthio-cGMP in antisense vs. missense ODN-treated rats. To prevent desensitization, additional rats were studied in the presence of chronic NOS inhibition via Nomega-nitro-L-arginine. In the NO synthase (NOS)-inhibited rats, the maximal SNAP response was much higher in the antisense (62% increase) vs. the missense ODN (40% increase) group. A similar result was obtained when monitoring responses to the soluble guanylyl cyclase-activating drugs YC-1 and BAY 41-2272. Moreover, in the presence of NOS inhibition, the normal SNAP-induced rise in periarachnoid cerebrospinal fluid cGMP levels, which reflects cGMP efflux, was absent in the antisense ODN-treated rats, a finding consistent with loss of MRP5 function. In conclusion, if one minimizes the confounding effects of basal cGMP production, a clearer picture emerges, one that indicates an important role for MRP5-mediated cGMP efflux in the regulation of NO-induced cerebral arteriolar relaxation.

Effect of the neuronal nitric oxide synthase inhibitor 7-nitroindazole on the righting reflex ED50 and minimum alveolar concentration during sevoflurane anaesthesia in rats.

BACKGROUND AND OBJECTIVE: The aim was to determine the effect of acute and chronic administration of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor, on the righting reflex ED50 and the minimum alveolar concentration during sevoflurane anaesthesia in rats. METHODS: 7-Nitroindazole was acutely (0, 50 and 100 mg kg(-1)) and chronically (0 and 150 mg kg(-1) day(-1), 4 days) administered to rats. After the preparation, the minimum alveolar concentration and the righting reflex ED50 were measured. The concentration of cGMP in the brain, cerebellum and spinal cord was also measured. RESULTS: Acute administration reduced the minimum alveolar concentration (50 mg kg(-1), 58.8% (95% CI: 50.3-67.3%) of the baseline value, P < 0.01; 100 mg kg(-1), 55.8 (46.9-64.7), P < 0.01) and the righting reflex ED50 (50 mg kg(-1), 27.2 (17.2-37.2), P < 0.01; 100 mg kg(-1), 14.3 (6.6-22.0), P < 0.01). Chronic administration did not reduce the minimum alveolar concentration; however, it reduced the righting reflex ED50 (65.3 (52.9-77.7), P < 0.01). Overall, the reduction in minimum alveolar concentration in the acute and chronic protocol did not correlate with that of the righting reflex ED50. 7-Nitroindazole (100 mg kg(-1), acute) reduced the cGMP concentration within the cerebellum by 55.4%; however, it did not decrease concentrations in the brain or spinal cord. CONCLUSIONS: Different mechanisms are responsible for the observed alterations to the minimum alveolar concentration and the righting reflex ED50 following treatment with 7-nitroindazole. The nitric oxide-cGMP pathway might play a less important role in the determination of minimum alveolar concentration than the righting reflex ED50.

Compensatory role of NO in cerebral circulation of piglets chronically treated with indomethacin.

We hypothesize that inhibitory effects exist between prostanoids and nitric oxide (NO) in their contributions to cerebral circulation. Piglets (1-4 days old) were divided into three chronically treated (6-8 days) groups: control piglets, piglets treated with indomethacin (75 mg/day), and piglets treated with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 mg x kg(-1) x day(-1)). Pial arterioles dilated in response to hypercapnia similarly among the three groups (41 +/- 4, 40 +/- 6, and 45 +/- 11%). Cerebrospinal fluid cAMP increased in control piglets, while cGMP increased in indomethacin-treated piglets. L-NAME, but not 7-nitroindazole, inhibited the response to hypercapnia only in indomethacin-treated piglets (40 +/- 6 vs. 17 +/- 5%). Topical sodium nitroprusside or iloprost restored dilation in response to hypercapnia. Similar results were obtained when the dilator was bradykinin. Pial arterioles of control and L-NAME-treated piglets constricted in response to ACh (-24 +/- 3%). However, those of indomethacin-treated piglets dilated in response to ACh (15 +/- 2%). This dilation was inhibited by L-NAME. NO synthase activity, but not endothelial NO synthase expression, increased after chronic indomethacin treatment. These data suggest that chronic inhibition of cyclooxygenase can increase the contribution of NO to cerebrovascular circulatory control in piglets.

Increased cerebrospinal fluid cAMP levels in Alzheimer's disease.

Since increasing evidence suggests that upregulation of the cAMP-second messenger system may be implicated in Alzheimer's disease neurodegeneration, we have compared the cAMP and cGMP levels in cerebrospinal fluid (CSF) from patients with dementia of the Alzheimer type (DAT, n=10) with those from nondemented age-matched controls (n=10). Our results show that cAMP levels, but not cGMP, are significantly (p<0.01) elevated in CSF from patients with DAT compared to those from nondemented controls. Moreover, a linear regression analysis demonstrated a significant correlation (r=0.62; p<0.01) between cAMP and tau protein levels in CSF when controls and patients with DAT were studied together. These results suggest that upregulation of cAMP-signaling pathway is implicated in Alzheimer's disease physiopathology.

Cerebrospinal fluid cyclic guanosine 3'5' monophosphate levels in Parkinson's disease.

We measured CSF and plasma levels of cGMP in 22 patients with Parkinson's disease (PD) and in 28 age and sex-matched controls. PD patients had similar plasma cGMP levels than those of controls, although they showed a non-significant trend towards higher CSF cGMP levels (P=0.07). PD patients treated with levodopa showed significantly higher CSF cGMP levels than those not treated with this drug (P<0.01), and controls (P<0.01). However, treatment with dopamine agonists did not influence CSF cGMP levels. Plasma and CSF levels of cGMP did not correlate with age at onset, duration, and severity of PD. These results suggest that changes in the concentration of cGMP in CSF of patients with PD are not related with the disease, but rather with levodopa therapy.

Role of neuronal NO synthase in relationship between NO and opioids in hypoxia-induced pial artery dilation.

Nitric oxide (NO) contributes to hypoxia-induced pial artery dilation, at least in part, via the formation of guanosine 3',5'-cyclic monophosphate (cGMP) and subsequent release of Met-enkephalin and Leu-enkephalin in the newborn pig. In separate studies, these opioids were also observed to elicit NO-dependent pial dilation. The present study was designed to investigate the role of the neuronal isoform of NO synthase (NOS) in hypoxic pial dilation, associated opioid release, and opioid dilation in piglets equipped with a closed cranial window. Tetrodotoxin (10(-6) M) attenuated the dilation resulting from hypoxia (PO2 approximately 35 mmHg; 25 +/- 1 vs. 14 +/- 1%). Similarly, 7-nitroindazole, sodium salt (7-NINA, 10(-6) M), a purported neuronal NOS inhibitor, attenuated hypoxic pial dilation (26 +/- 1 vs. 14 +/- 2%). Hypoxic dilation was accompanied by elevated cerebrospinal (CSF) cGMP, which was blocked by 7-NINA (433 +/- 19 and 983 +/- 36 vs. 432 +/- 19 and 441 +/- 19 fmol/ml for control and hypoxia in absence and presence of 7-NINA, respectively). Additionally, hypoxic dilation was also accompanied by elevated CSF Met-enkephalin, which was attenuated by 7-NINA (1,027 +/- 47 and 2,871 +/- 134 vs. 779 +/- 78 and 1,551 +/- 42 pg/ml for control and hypoxia in absence and presence of 7-NINA, respectively). In contrast, Met-enkephalin (10(-10), 10(-8), and 10(-6) M) induced dilation that was unchanged by 7-NINA (7 +/- 1, 12 +/- 1, and 18 +/- 1 vs. 6 +/- 1, 10 +/- 1, and 17 +/- 1%, respectively). N-methyl-D-aspartate (NMDA, 10(-8) and 10(-6) M), an activator of neuronal NOS, induced pial dilation that was blocked by 7-NINA (10 +/- 1 and 20 +/- 2 vs. 1 +/- 1 and 2 +/- 1%, respectively). However, sodium nitroprusside-induced dilation was unchanged by 7-NINA. These data indicate that neuronal NOS contributes to hypoxic pial artery dilation but not to opioid-induced dilation. Furthermore, these data suggest that neuronally derived NO contributes to hypoxic dilation, at least in part, via formation of cGMP and the subsequent release of opioids.