Nitric oxide modulates fracture healing.

The role of the messenger molecule nitric oxide has not been evaluated in fracture healing. NO is synthesized by three kinds of nitric oxide synthase (NOS): inducible NOS (iNOS), endothelial (eNOS), and neuronal (bNOS). We evaluated the role of these enzymes in a rat femur fracture-healing model. There was no messenger RNA (mRNA) expression, immunoreactivity, or enzymatic activity for NOS in unfractured femoral cortex. After fracture, however, mRNA, protein, and enzymatic activity for iNOS were identified in the healing rat femoral fracture callus, with maximum activity on day 15. The mRNA expression for eNOS and bNOS was induced slightly later than for iNOS, consistent with a temporal increase in calcium-dependent NOS activity that gradually increased up to day 30. mRNA expression for the three NOS isoforms also was found in six of six human fracture callus samples. To study the effect of suppression of NO synthesis on fracture healing, an experimental group of rats was fed an NOS inhibitor, L-nitroso-arginine methyl ester (L-NAME), and the control group was fed its inactive enantiomer, D-nitroso-arginine methyl ester (D-NAME). An 18% (p < or = 0.01) decrease in cross-sectional area and a 45% (p < or = 0.05) decrease in failure load were observed in the NOS-inhibited group on day 24 after fracture. Furthermore, the effect of NO supplementation to fracture healing was studied by delivering NO to the fracture site using carboxybutyl chitosan NONOate locally. On day 17 after fracture, there was a 30% (p < or = 0.05) increase in cross-sectional area in the NO-donor group compared with the NOS inhibition group. These results show for the first time that NO is expressed during fracture healing in rats and in humans, that suppression of NOS impairs fracture healing, and that supplementation of NO can reverse the inhibition of healing produced by NOS inhibitors.     

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.     

Postreceptor mechanisms of small-bowel water and electrolyte transport

Neurohumoral agents modulate intestinal transport by interactions with cell membrane receptors. Intracellular second messenger systems implicated in mediation of membrane receptor regulation of cellular events include the phosphoinositide and adenylate cyclase systems. In this study we have investigated the effects of direct postreceptor activation of key components of these systems on intestinal water and electrolyte transport. Rabbit ileal segments (n = 35) were arterially perfused ex vivo with an oxygenated sanguineous solution. The lumen was perfused with an isotonic solution containing 14C-polyethylene glycol as a nonabsorbable marker. Net fluxes of H2O, Na+, and Cl- in six experimental groups were calculated for three 20-minute periods: basal, drug infusion, and recovery. The control group had no drug infusion. Two phorbol esters--phorbol 12, 13-diacetate (PDA; 10(-5) mol), and phorbol 12, 13-dibutyrate (PDB; 10(-5) mol)--were used to activate protein kinase C, an important component of the phosphoinositide system. The inactive 4 alpha-phorbol 12, 13-didecanoate (PDD; 10(-5) mol) served as a drug-infused control. Forskolin at two doses (FOR; 10(-5) mol and 10(-6) mol) was used to activate adenylate cyclase. The control and PDD groups had no changes in the flux of water and electrolytes. Both PDA and PDB had proabsorptive effects, with the more lipophilic and potent phorbol ester (PDB) having a more pronounced, significant effect (p less than 0.05). FOR caused significant secretion of H2O, Na+, and Cl- in a dose-dependent fashion (p less than 0.05). These results indicate that direct protein kinase C activation causes a proabsorptive effect and that direct activation of adenylate cyclase causes a secretory effect in the isolated small bowel. The activation status of these second messenger systems has a major influence on the transport state of the intestine.     

Nitric oxide in rheumatology.

Nitric oxide (NO) is attracting considerable interest because it mediates many functions. This gas is ubiquitously produced in the body by three enzymes, called NO synthases. Two NO synthases are constitutively expressed, one in the nervous system and the other in the blood vessels, where it regulates tissue perfusion. The third NO synthase can be induced by several stimuli (bacterial endotoxins, cytokines), most notably in inflammatory cells and chondrocytes. The effects of NO produced by the inducible NO synthase range from T-cell response modulation to formation of free radicals responsible fortissue damage and cartilage matrix degradation. Administration of NO synthase inhibitors in animal models of arthritis yields ambiguous effects, often with prevention of arthritis, but sometimes with worsening of established arthritis. The data available to date do not support the use of such inhibitors in the treatment of human arthritis.     

Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing, and inflammation and exerts its effect via tyrosine kinase receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1 or KDR). We have previously shown that VEGF is up-regulated in a model of chronic cyclosporine (CsA) nephrotoxicity and that l-arginine (l-Arg) improved while N-nitro-l-arginine-methyl ester (L-NAME) worsened fibrosis. We examined the role of nitric oxide modulation on VEGF in this model. METHODS: Pair-fed salt-depleted rats were administered CsA, CsA + L-NAME, CsA +l-Arg, vehicle (VH), VH + L-NAME or VH +l-Arg and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot and the protein expression of VEGF by Western blot and immunohistochemical staining. RESULTS: While L-NAME worsened renal function and histology, l-Arg had the opposite beneficial effect in CsA-treated rats. VEGF mRNA and protein expressions increased with CsA, further increased with L-NAME and became significantly reduced with L-Arg. Flt-1 expression was similar in all groups. On the other hand, KDR/Flk-1 mRNA expression was modulated in a fashion similar to VEGF. Also, nitric oxide modulation did not have an effect on VH-treated rats. CONCLUSIONS: VEGF expression in chronic CsA nephrotoxicity is increased by nitric oxide blockade and decreased by nitric oxide enhancement. Moreover, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but it is probable that VEGF plays a role, either independently or through nitric oxide, in CsA-induced fibrosis.     

Nitric oxide in osteoarthritis.

Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.     

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.     

Nitric oxide modulates renal vasoconstrictor effect of endothelin-1 in conscious lambs

To test the hypothesis that nitric oxide (NO) buffers the renal vasoconstrictor effects of endothelin-1 (ET-1) early in life, renal haemodynamic responses to ET-1 were measured in the presence and absence of endogenously produced NO in conscious lambs. Renal haemodynamic effects of ET-1 were measured for 5 min before (control) and 20 min after intraarterial injection of ET-1 before and after pretreatment with 20 mg/kg of the l-arginine analogue N^G-nitro-l-arginine methyl ester (l-NAME), (experiment 1) and its inactive isomer D-NAME (experiment 2) in conscious lambs aged ~1 week (N=7) and ~6 weeks (N=6). The two experiments were carried out in random order at intervals of 24–48 h. In lambs aged ~6 weeks, a marked increase in renal vascular resistance (RVR) was elicited by ET-1 administration; this response was enhanced twofold following pretreatment with l-NAME. In 1-week-old lambs, however, an increase in RVR in response to ET-1 occurred only after pretreatment with l-NAME. Therefore, we accept our hypothesis and conclude that NO buffers the renal vasoconstrictor effects of ET-1 early in life.The current address of Dr. Liesbeth van der Velde is UCLA Medical Center, Los Angeles, CA, USA. The current address of Dr. Alp Sener is University of Western Ontario, London, ON, Canada     

Nitric oxide/platelet activating factor cross-talk in mesangial cells modulates the interaction with leukocytes

BACKGROUND: The secondary hyperparathyroidism of chronic kidney disease (CKD) produces a high turnover osteodystrophy that is associated with peritrabecular fibrosis. The nature of the cells involved in the development of peritrabecular fibrosis may represent osteoprogenitors expressing a fibroblastic phenotype that are retarded from progressing through osteoblast differentiation. METHODS: To test the hypothesis that osteoblast differentiation is retarded in secondary hyperparathyroidism due to CKD producing bone marrow fibrosis, we administered bone morphogenetic protein 7 (BMP-7), a physiologic regulator of osteoblast regulation, to C57BL6 mice that had CKD produced by electrocautery of one kidney followed by contralateral nephrectomy two weeks later. Following the second surgical procedure, a subgroup of mice received daily intraperitoneal injections of BMP-7 (10 microg/kg). Three to six weeks later, the animals were sacrificed, blood was obtained for measurements of blood urea nitrogen (BUN) and parathyroid hormone (PTH) levels, and the femora and tibiae were processed for histomorphometric analysis. RESULTS: The animals had significant renal insufficiency with BUN values of 77.79 +/- 22.68 mg/dL, and the level of renal impairment between the CKD untreated mice and the CKD mice treated with BMP-7 was the same in the two groups. PTH levels averaged 81.13 +/- 51.36 and 75.4 +/- 43.61 pg/mL in the CKD and BMP-7 treated groups, respectively. The animals with CKD developed significant peritrabecular fibrosis. In addition, there was an increase in osteoblast surface and osteoid accumulation as well as increased activation frequency and increased osteoclast surface consistent with high turnover renal osteodystrophy. Treatment with BMP-7 eliminated peritrabecular fibrosis, increased osteoblast number, osteoblast surface, mineralizing surface and single labeled surface. There was also a significant decrease in the eroded surface induced by treatment with BMP-7. CONCLUSIONS: These findings indicate that BMP-7 treatment in the setting of high turnover renal osteodystrophy prevents the development of peritrabecular fibrosis, affects the osteoblast phenotype and mineralizing surfaces, and decreases bone resorption. This is compatible with a role of osteoblast differentiation in the pathophysiology of osteitis fibrosa.     

Pathophysiological study of the ileum after total colectomy in combination with mucosal proctectomy and ileoanostomy--the alteration in water and electrolyte absorption by the ileum after ileoanostomy

We have developed a new procedure, total colectomy in combination with mucosal proctectomy and ileoanostomy, to preserve anorectal function for the patients with adenomatosis coli and ulcerative colitis. Using dialysis method, we evaluated the alteration of the ability to absorb water and electrolytes at the ileum after ileoanostomy in 12 patients with adenomatosis coli and 4 patients with ulcerative colitis, and compared the result with fecal volume and content to investigate compensatory function of the small intestine after total colectomy. Volume of water absorbed at the ileum was 0.389 g in an average soon after the first stage operation but gradually increased after the intestinal continuity was restored following the second stage operation with the volume of 0.666 g after 1 year and 0.702 g after 2 years (1.8 fold increase). Fecal volume excreted was 852 g/day in an average after loop ileostomy (first stage operation) but decreased to 316 g/day in a year after the second stage operation with simultaneous change of fecal character from watery to solid. The decrease of fecal volume was inversely correlated with the increase of the volume of water absorbed at the ileum (p less than 0.001). The ability to absorb electrolytes increased after the second stage operation compared to that of after the first stage operation but subsequently minimal change was noted. And the absorption pattern was different from that of the large intestine.     

Nitric oxide and glomerulonephritis

Glomerulonephritis is a common clinical condition that is caused by immune-mediated injury to the kidney and is characterized by dysfunction of the glomerular capillary filtration barrier. Nitric oxide (NO), a ubiquitous molecule with many biological functions throughout the body, has been evaluated as an inflammatory mediator in these circumstances. NO may induce glomerular injury directly or may act via stimulation of a host of other inflammatory mediators. A variety of experimental models of glomerulonephritis have been studied including those induced by infusion of antibodies to the Thy1.1 antigen or glomerular basement membrane, Heymann nephritis, and autoimmune nephritis. In virtually all of these cases there is evidence of increased NO production. Excessive production of NO by inducible nitric oxide synthase (iNOS), derived from infiltrating immune cells or resident glomerular cells, nearly always is associated with increased glomerular injury. Interventions that inhibit this enzyme result in less proteinuria and diminished glomerular damage. In contrast, NO derived from endothelial nitric oxide synthase (eNOS) may limit glomerular disease by preserving endothelial cell integrity. There are only a limited number of studies that have evaluated the impact of NO in patients with glomerulonephritis. Although the bulk of evidence supports a role of NO as a pro-inflammatory mediator in glomerulonephritis, additional work is needed to show an association between altered NO production and the severity and outcome of disease in patients with this disease. It is hoped that better understanding of the role of NO in glomerulonephritis will lead to the development of therapies to ameliorate the disease.     

Nitric oxide in wound-healing

Modulation of the complex process of wound-healing remains a surgical challenge. Little improvement beyond controlling infection, gentle tissue handling, and debridement of necrotic tissue has been had in the modern era. However, increasing appreciation of the process from a biomolecular perspective offers the potential for making significant strides in wound modulation. The bioactive molecule nitric oxide was found to have wide-ranging impact on cellular activities, including the cellular responses engendered by wound healing. Current research suggests that nitric oxide and several nitric oxide donors can exert biologic effects, although the particular net responses of cells contributing to wound repair are context-dependent.     

Nitric oxide in shock

Refractory hypotension with end-organ hypoperfusion and failure is an ominous feature of shock. Distributive shock is caused by severe infections (septic shock) or severe systemic allergic reactions (anaphylactic shock). In 1986, it was concluded that nitric oxide (NO) is the endothelium-derived relaxing factor that had been discovered 6 years earlier. Since then, NO has been shown to be important for the physiological and pathological control of vascular tone. Nevertheless, although inhibition of NO synthesis restores blood pressure, NO synthase (NOS) inhibition cannot improve outcome, on the contrary. This implies that NO acts as a double-edged sword during septic shock. Consequently, the focus has shifted towards selective inducible NOS (iNOS) inhibitors. The contribution of NO to anaphylactic shock seems to be more straightforward, as NOS inhibition abrogates shock in conscious mice. Surprisingly, however, this shock-inducing NO is not produced by the inducible iNOS, but by the so-called constitutive enzyme endothelial NOS. This review summarizes the contribution of NO to septic and anaphylactic shock. Although NOS inhibition may be promising for the treatment of anaphylactic shock, the failure of a phase III trial indicates that other approaches are required for the successful treatment of septic shock. Amongst these, high hopes are set for selective iNOS inhibitors. But it might also be necessary to shift gears and focus on downstream cardiovascular targets of NO or on other vasodilating phenomena.     

Nitric oxide and glomerulonephritis

The normal glomerulus expresses constitutive nitric oxide (NO) synthesis. Increased NO from inducible nitric oxide synthase (iNOS) occurs in acute immune glomerulonephritis (GN), in which rapid induction probably depends on local cytokines and/or oxygen radical production. Although intrinsic glomerular cells possess iNOS, in acute GN, a leukocyte origin for iNOS activity is most evident. Although NO potentially could be toxic or protective, there is as yet no clear understanding of how it affects the pathogenesis of GN. This may depend on the amount present, which in turn depends on NOS gene regulation, simultaneous production of other radicals, and the activity of arginase. High-output NO has been implicated in injury in only one model of GN, and no effect of iNOS knockout found in another. The concept is emerging that constitutive NO is critical for offsetting increased vasoconstriction in the injured glomerulus.     

Nitric oxide and hemodialysis

Nitric oxide (NO), previously thought of as a noxious gas, is now recognized as an important mediator of vascular responsiveness. Soon after its discovery, it was realized that the actions of NO are similar to the previously described endothelium-derived relaxing factor (EDRF). It is synthesized in the vascular endothelium utilizing the enzyme nitric oxide synthase (NOS) and diffuses in the adjacent vascular media, where it has a vasodilatory action. Opposing actions of NO and vasoconstrictor agents (such as endothelin-1, angiotensin IotaIota, and others) maintain the vascular tone of the renal arteries. The same balance at the level of the macula densa maintains glomerular filtration rate (GFR) during varying levels of salt excretion. Lack of NO can result in disruption of this fine balance, with resultant vasoconstriction and disease progression, hypertension, and accelerated atherosclerosis. In addition, hypertension may result from positive salt balance that occurs when macula densa NOS is inhibited. While most investigators report low levels of NO in uremic subjects, the levels in hemodialysis (HD) patients have not been characterized adequately. This is primarily because HD patients are exposed to both stimulatory and inhibitory factors for NO synthesis. Retention of inhibitors of NOS tends to decrease NO levels, whereas production of NO will be increased by cytokines generated during blood-dialyzer interaction. There is less disagreement, however, over the finding of elevated levels in those with dialyzer reactions and dialysis-induced hypotension. Recent developments in the isolation of inducible and constitutive forms of NOS makes understanding of its pathophysiologic effects more complete. Newer treatment directed at inhibiting only the inducible forms of NOS (sparing the constitutive forms) may soon be found useful for the treatment and prevention of hypotension and dialyzer reactions in HD patients.