Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Glutamate and nitric oxide as regulatory transmitters in developing rabbit retina

Glutamate is the major excitatory neurotransmitter in the retina and serves as the synaptic messenger for the three classes of neurons which constitute the vertical pathway--the photoreceptors, bipolar cells and ganglion cells. In addition, the glutamate system has been localized morphologically, pharmacologically as well as molecularly during the first postnatal week of development before synaptogenesis occurs. The role which glutamate plays in the maturing visual system is complex but ranges from mediating developmental neurotoxicity to inducing neurite outgrowth.^ Nitric oxide/cGMP is a novel intercellular messenger which is thought to act in concert with the glutamate system in regulating a variety of cellular processes in the brain as well as retina, most notably neurotoxicity. Several developmental activities including programmed cell death, synapse elimination and synaptic reorganization are possible functions of cellular regulation modulated by nitric oxide as well as glutamate.^ The purpose of this thesis is to (1) biochemically characterize the endogenous pools of glutamate and determine what fraction exists extracellularly; (2) examine the morphological expression of NO producing cells in developing retina; (3) test the functional coupling of the NMDA subtype of glutamate receptor to the NO system by examining neurotoxicity which has roles in both the maturing and adult retina.^ Biochemical sampling of perfusates collected from the photoreceptor surface of ex vivo retina demonstrated that although the total pool of glutamate present at birth is relatively modest, a high percentage resides in extracellular pools. As a result, immature neurons without significant synaptic connections survive and develop in a highly glutamatergic environment which has been shown to be toxic in the adult retina.^ The interaction of the glutamate system with the NO system has been postulated to regulate neuronal survival. We therefore examined the developmental expression of the enzyme responsible for producing NO, nitric oxide synthase (NOS), using an antibody to the constitutive form of NOS found in the brain. The neurons thought to produce the majority of NO in the adult retina, a subpopulation of widefield amacrine cells, were not immunoreactive until the end of the second postnatal week. However, a unique developmental expression was observed in the ganglion cell layer and developing outer nuclear layer of the retina during the first postnatal week. We postulate NO producing neurons may not be present in a mature configuration therefore permitting neuronal survival in a highly glutamatergic microenvironment and allowing NO to play a development-specific role at this time.^ The next set of experiments constituted a functional test of the hypothesis that the absence of the prototypic NO producing cells in developing retina protects immature neurons against glutamate toxicity. An explant culture system developed in order to examine cellular responses of immature and adult neurons to glutamate toxicity showed that immature neurons were affected by NMDA but were less responsive to NMDA and NO mediated toxicity. In contrast, adult explants exhibited significant NMDA toxicity which was attenuated by NMDA antagonists, 2-amino-5-phosphonovaleric acid (APV), dextromethorphan (Dex) and N$\rm\sp{G}$-D-methyl arginine (metARG). These results indicated that pan-retinal neurotoxicity via the NMDA receptor and/or NO activation occurred in the adult retina but was not significant in the neonate. (Abstract shortened by UMI.) ^

The role of nitric oxide in Listeria encephalitis of ruminants and in rats intracisternally infected with Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular bacteria which infects a wide range of hosts. In ruminants, infection with L. monocytogenes frequently causes encephalitis, which is usually fatal in sheep and goat, while cattle often recover with antibiotic therapy. Since the role of NO in the control of Listeria is controversial, we have studied the expression of iNOS in the brains of cattle, sheep and goats which had succumbed to listeria encephalitis. iNOS was demonstrated in decreasing intensity in the M phi of microabscesses from cattle, sheep and goat. iNOS expression was accompanied by NT in the microabscesses of cattle, but was only present to a low degree in sheep and was absent in goats. This is indirect evidence for differences in the ability to produce NO in the three species. Presence of iNOS and NT were inversely correlated with the numbers of bacteria. While microabscesses of goats contained high amounts of L. monocytogenes they occurred only rarely in cattle. To corroborate our hypothesis that NO is involved in the control of listeria encephalitis a new animal model was developed. Eleven day old infant rats were infected intracisternally with a low dose of L. monocytogenes. This resulted in a transient meningoencephalitis with moderate clinical signs and low mortality. Listeria proliferated strongly in the inflammatory lesions during the first days of infection, reached a peak at day 4 and were eliminated until day 7. The presence of bacteria was closely accompanied by high numbers of iNOS-expressing M phi and the formation of NT. Administration of the iNOS inhibitor L-NIL or the radical scavenger PBN resulted in rapid death of the treated animals. However, the increase in bacterial numbers was one order of magnitude higher for animals treated with PBN compared with L-NIL administration. This shows that NO plays an important role in the control of a brain infection with Listeria, but suggests that reactive oxidants other than NO are also involved. In conclusion, our findings point to a possible involvement of the differences in the ability to express iNOS and subsequent NO production in the different clinical outcome of listeria encephalitis in cattle and small ruminants.

Nitric oxide metabolite production in the cranial cruciate ligament, synovial membrane, and articular cartilage of dogs with cranial cruciate ligament rupture

OBJECTIVE To measure concentrations of nitric oxide metabolites (nitrite-nitrate [NOt]) in cartilage, synovial membrane, and cranial cruciate ligament (CCL) in dogs and evaluate associations with osteoarthritis in dogs with CCL rupture. ANIMALS 46 dogs with CCL rupture and 54 control dogs without joint disease. PROCEDURE Tissue specimens for histologic examination and explant culture were harvested during surgery in the CCL group or immediately after euthanasia in the control group; NOt concentrations were measured in supernatant of explant cultures and compared among dogs with various degrees of osteoarthritis and between dogs with and without CCL rupture. RESULTS Osteoarthritic cartilage had significantly higher NOt concentration (1,171.6 nmol/g) than did healthy cartilage (491.0 nmol/g); NOt concentration was associated with severity of macroscopic and microscopic lesions. Synovial membrane NOt concentration did not differ between dogs with and without CCL rupture. Ruptured CCL produced less NOt than did intact ligaments. In control dogs, NOt concentrations were similar for intact ligaments (568.1 nmol/g) and articular cartilage (491.0 nmol/g). Synthesis of NOt was inhibited substantially by coincubation with inhibitors. CONCLUSIONS AND CLINICAL RELEVANCE Results suggest that NOt in canine joint tissues originates from the inducible nitric oxide synthase pathway. Nitric oxide metabolite production in cartilage was greater in dogs with osteoarthritis than in healthy dogs and was associated with lesion severity, suggesting that nitric oxide inhibitors may be considered as a treatment for osteoarthritis. The CCL produces substantial concentrations of NOt; the importance of this finding is unknown.

The effects of doxycycline on nitric oxide and stromelysin production in dogs with cranial cruciate ligament rupture

OBJECTIVE To investigate the potential of doxycycline to reduce stromelysin and inducible nitric oxide synthase (iNOS) activity in dogs with osteoarthritis (OA) secondary to spontaneous cranial cruciate ligament (CCL) rupture. STUDY DESIGN Prospective, clinical study. ANIMALS Eighty-one dogs with OA secondary to CCL rupture and 54 normal dogs. METHODS Dogs with OA secondary to CCL rupture were divided into 2 groups before surgery. The Doxy-CCl group received 3 to 4 mg/kg doxycycline orally every 24 hours for 7 to 10 days (n = 35). The CCL group received no treatment (n = 46). Synovial fluid, articular cartilage, synovial membrane, and CCL samples were collected during surgery (Doxy-CCL group and CCL group) or immediately after euthanasia from healthy dogs (control group). Synovial fluid samples were examined cytologically. Total nitric oxide (NOt) concentrations were measured in the supernatant of explant cultures of all tissue samples, and stromelysin activity was measured in the supernatant of explant cultures of cartilage. RESULTS NOt concentrations measured in cartilage were significantly lower in the Doxy-CCL group than in the CCL group, but were not different from those measured in the control group. Doxycycline treatment did not have a significant effect on cartilage stromelysin levels. CONCLUSION The findings in this study indicate that doxycycline inhibits NO production in cartilage in dogs with CCL rupture. CLINICAL RELEVANCE Doxycycline may have a role in the treatment of canine OA by inhibiting NO production.

Endogenous nitric oxide production in canine osteoarthritis: Detection in urine, serum, and synovial fluid specimens

OBJECTIVE To measure nitric oxide (NO) concentrations in serum, urine, and synovial fluid (SF) of dogs with naturally occurring cranial cruciate ligament (CCL) rupture and normal dogs, and to compare these with clinical and histologic changes of osteoarthritis (OA). STUDY DESIGN Prospective clinical study including 2 groups of animals selected from the hospital population. ANIMALS Forty-three dogs (CCL group) with OA secondary to CCL rupture; 30 healthy dogs (control group) without CCL rupture. METHODS Serum, urine, and SF were collected before and during surgery in the CCL group or immediately after euthanasia in the control group. Articular cartilage and synovial membrane tissue specimens were prepared for routine histologic examination. The stable end products of NO, total nitrite and nitrate (NOt) activity, were measured in body fluids and compared with macroscopic and histologic degrees of OA. Urinary NOt concentration was compared with urinary creatinine concentration and stated as urinary NOt:creatinine ratio (UNCR). RESULTS-SF NOt concentrations were not significantly different between the 2 groups. Serum NOt concentrations (45.6 vs 28.9 micromol/L; P =.042) and the UNCR (0.007 vs 0.004; P =.035) were significantly higher in dogs of the CCL group compared with the control population. An association between UNCR and histologic and macroscopical OA grades could be demonstrated. CONCLUSION UNCR might be a useful indicator of nitrite and nitrate production and, therefore, osteoarthritic changes in joints. CLINICAL RELEVANCE UNCR could be used as a tool to evaluate the NOt production by joint tissues over time and might therefore provide a method of evaluating the effects of drugs in the control of osteoarthritis.

Correlation of urinary nitric oxide concentrations with the development of hip dysplasia in Labrador retrievers

Urinary nitric oxide was determined in terms of the ratio of the concentrations of total urinary nitrite and nitrate:creatinine in 40 juvenile labrador retrievers, and radiographic signs of hip dysplasia and distraction indices were investigated in the same dogs in later life. The ratio was correlated significantly with the Norberg angle and with subchondral sclerosis in both hips, and with the grade of dysplasia in each hip and the dogs' overall grade of hip dysplasia. No correlations were observed between the ratio and the distraction indices or other radiographic criteria for hip dysplasia.

Estrogen induces nitric oxide production via nitric oxide synthase activation in endothelial cells.

INTRODUCTION 17β-estradiol (E2) has been found to induce vasodilation in the cardiovascular system and at physiological levels, resulting in prevention of cerebral vasospasm following subarachnoid hemorrhage (SAH) in animal models. The goal of this study was to analyze the cellular mechanism of nitric oxide (NO) production and its relation to E2, in vitro in brain and peripheral endothelial cells. METHODS Human umbilical endothelial cells (HUVEC) and brain endothelial cells (bEnd.3) were treated with estradiol (E2, 0.1, 10, 100, and 1,000 nM), and supernatant was collected at 0, 5, 15, 30, 60, and 120 min for nitric oxide metabolome (nitrite, NO₂) measurements. Cells were also treated with E2 in the presence of 1400W, a potent eNOS inhibitor, and ICI, an antagonist of estradiol receptors (ERs). Effects of E2 on eNOS protein expression were assessed with Western blot analysis. RESULTS E2 significantly increased NO2 levels irrespective of its concentration in both cell lines by 35 % and 42 % (p < 0.05). The addition of an E2 antagonist, ICI (10 μM), prevented the E2-induced increases in NO2 levels (11 % p > 0.05). The combination of E2 (10 nM) and a NOS inhibitor (1400W, 5 μM) inhibited NO2 increases in addition (4 %, p > 0.05). E2 induced increases in eNOS protein levels and phosphorylated eNOS (eNOS(p)). CONCLUSIONS This study indicates that E2 induces NO level increases in cerebral and peripheral endothelial cells in vitro via eNOS activation and through E2 receptor-mediated mechanisms. Further in vivo studies are warranted to evaluate the therapeutic value of estrogen for the treatment of SAH-induced vasospasm.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Nitric oxide inhalation reduces brain damage, prevents mortality, and improves neurological outcome after subarachnoid hemorrhage by resolving early pial microvasospasms.

Subarachnoid hemorrhage is a stroke subtype with particularly bad outcome. Recent findings suggest that constrictions of pial arterioles occurring early after hemorrhage may be responsible for cerebral ischemia and - subsequently - unfavorable outcome after subarachnoid hemorrhage. Since we recently hypothesized that the lack of nitric oxide may cause post-hemorrhagic microvasospasms, our aim was to investigate whether inhaled nitric oxide, a treatment paradigm selectively delivering nitric oxide to ischemic microvessels, is able to dilate post-hemorrhagic microvasospasms; thereby improving outcome after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to experimental SAH. Three hours after subarachnoid hemorrhage pial artery spasms were quantified by intravital microscopy, then mice received inhaled nitric oxide or vehicle. For induction of large artery spasms mice received an intracisternal injection of autologous blood. Inhaled nitric oxide significantly reduced number and severity of subarachnoid hemorrhage-induced post-hemorrhage microvasospasms while only having limited effect on large artery spasms. This resulted in less brain-edema-formation, less hippocampal neuronal loss, lack of mortality, and significantly improved neurological outcome after subarachnoid hemorrhage. This suggests that spasms of pial arterioles play a major role for the outcome after subarachnoid hemorrhage and that lack of nitric oxide is an important mechanism of post-hemorrhagic microvascular dysfunction. Reversing microvascular dysfunction by inhaled nitric oxide might be a promising treatment strategy for subarachnoid hemorrhage.