The novel anti-inflammatory agent VA694, endowed with both NO-releasing and COX2-selective inhibiting properties, exhibits NO-mediated positive effects on blood pressure, coronary flow and endothelium in an experimental model of hypertension and endothelial dysfunction.

Selective cyclooxygenase 2 (COX2) inhibitors (COXIBs) are effective anti-inflammatory and analgesic drugs with improved gastrointestinal (GI) safety compared to nonselective nonsteroidal anti-inflammatory drugs known as traditional (tNSAIDs). However, their use is associated with a cardiovascular (CV) hazard (i.e. increased incidence of thrombotic events and hypertension) due to the inhibition of COX2-dependent vascular prostacyclin. Aiming to design COX2-selective inhibitors with improved CV safety, new NO-releasing COXIBs (NO-COXIBs) have been developed. In these hybrid drugs, the NO-mediated CV effects are expected to compensate for the COXIB-mediated inhibition of prostacyclin. This study evaluates the potential CV beneficial effects of VA694, a promising NO-COXIB, the anti-inflammatory effects of which have been previously characterized in several in vitro and in vivo experimental models. When incubated in hepatic homogenate, VA694 acted as a slow NO-donor. Moreover, it caused NO-mediated relaxant effects in the vascular smooth muscle. The chronic oral administration of VA694 to young spontaneously hypertensive rats (SHRs) significantly slowed down the age-related development of hypertension and was associated with increased plasma levels of nitrates, stable end-metabolites of NO. Furthermore, a significant improvement of coronary flow and a significant reduction of endothelial dysfunction were observed in SHRs submitted to chronic administration of VA694. In conclusion, VA694 is a promising COX2-inhibiting hybrid drug, showing NO releasing properties which may mitigate the CV deleterious effects associated with the COX2-inhibition.

Effects of zileuton and montelukast in mouse experimental spinal cord injury.

BACKGROUND AND PURPOSE: 5-lipoxygenase (5-LO) is the key enzyme in leukotriene (LT) biosynthesis from arachidonic acid (AA). Here, we examined the role of the 5-LO-product, cysteinyl-LT (Cys-LT), with a 5-LO inhibitor (zileuton) and a Cys-LT, receptor antagonist (montelukast), in the inflammatory response and tissue injury associated with spinal cord injury (SCI). EXPERIMENTAL APPROACH: SCI was induced in mice by the application of vascular clips to the dura via a two-level T6 to T7 laminectomy for 1 min. Cord inflammation was assessed histologically and by measuring inflammatory mediators (ELISA) and apoptosis by annexin V, TUNEL, Fas ligand staining and Bax and Bcl-2 expression (immunohistochemistry and western blots). Motor function in hindlimbs was assessed by a locomotor rating scale, for 10 days after cord injury. KEY RESULTS: SCI in mice resulted in tissue damage, oedema, neutrophil infiltration, apoptosis, tumour necrosis-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) expression, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) production, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in injured tissue. Treatment of the mice with zileuton or montelukast reduced the spinal cord inflammation and tissue injury, neutrophil infiltration, TNF-alpha, COX-2 and pERK1/2 expression, PGE(2) and LTB(4) production, and apoptosis. In separate experiments, zileuton or montelukast significantly improved the recovery of limb function over 10 days. CONCLUSIONS AND IMPLICATIONS: Zileuton and montelukast produced a substantial reduction of inflammatory events associated with experimental SCI. Our data underline the important role of 5-LO and Cys-LT in neurotrauma.

Leptin increases serotonin turnover by inhibition of brain nitric oxide synthesis.

Leptin administration inhibits diencephalic nitric oxide synthase (NOS) activity and increases brain serotonin (5-HT) metabolism in mice. We evaluated food intake, body-weight gain, diencephalic NOS activity, and diencephalic content of tryptophan (TRP), 5-HT, hydroxyindoleacetic acid (5-HIAA), and 5-HIAA/5-HT ratio after intracerebroventricular (ICV) or intraperitoneal (IP) leptin injection in mice. Five consecutive days of ICV or IP leptin injections induced a significant reduction in neuronal NOS (nNOS) activity, and caused a dose-dependent increase of 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio. Diencephalic 5-HT metabolism showed a significant increase in 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio 3 hours after a single leptin injection. This effect was maintained for 3 hours and had disappeared by 12 hours after injection. After a single IP leptin injection, the peak for 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio was achieved at 6 hours. Single injections of ICV or IP leptin significantly increased diencephalic 5-HT content. Leptin-induced 5-HT increase was antagonized by the coadministration of L-arginine only when the latter was ICV injected, whereas D-arginine did not influence leptin effects on brain 5-HT content. Finally, in nNOS-knockout mice, the appetite-suppressant activity of leptin was strongly reduced, and the leptin-induced increase in brain 5-HT metabolism was completely abolished. Our results indicate that the L-arginine/NO pathway is involved in mediating leptin effects on feeding behavior, and demonstrate that nNOS activity is required for the effects of leptin on brain 5-HT turnover.

17beta-estradiol antiinflammatory activity in carrageenan-induced pleurisy.

We have recently demonstrated that 17beta-estradiol (E2) opposes cytokine-dependent increase of inducible nitric oxide synthase (iNOS) activity in rat smooth muscle cells and proposed that this effect might be associated to an antiinflammatory activity of this hormone. In the present study, we examine the E2 effects on a well-known in vivo model of inflammation. We show that, in carrageenan treatment of ovariectomized rats, prior exposure to E2 significantly attenuated inflammatory response as measured by histological examination and exudate production. The effect was visible with a single injection of a physiological dose of E2 1 h before the carrageenan treatment and was blocked by coadministration of the estrogen receptor antagonists ICI 182,780 or tamoxifen. This latter observation suggests that the effect is receptor mediated. The mechanisms by which estradiol has beneficial effects in this model of inflammation are unclear: we show that in hormonally treated rats there is a decrease in polymorphonuclear cells migration as shown by cell counting and myeloperoxidase measurement. In addition, E2 pretreatment opposes carrageneen-induced high lipid peroxidation maintaining malondialdehyde activity at control levels. E2 treatment decreases NO production and the activity of iNOS with consequent diminished nitrite synthesis and nitrosine accumulation. Finally, immunohistochemical analysis for poly (ADP-ribose) synthase revealed a positive staining in lungs from carrageenan-treated rats that was blocked by estradiol treatment. We conclude that E2 attenuates the degree of inflammation and tissue damage associated with carrageenan-induced pleurisy in the rat.

Preferential utilization of endogenous arachidonate by cyclo-oxygenase in incubations of human platelets.

Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.

Involvement of NF-kappaB in the regulation of cyclooxygenase-2 protein expression in LPS-stimulated J774 macrophages.

We investigated the involvement of NF-kappaB in the regulation of COX-2 protein expression and prostaglandin production in LPS-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microg/ml) for 24 h caused an increase of COX-2 protein expression and accumulation of both PGE2 and 6-keto-PGF1alpha in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 microM) and N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK, 1, 10, 100 microM), two inhibitors of NF-kappaB activation, suppressed in a concentration-dependent manner both LPS-induced COX-2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS-induced increase of NF-kappaB DNA binding activity and prevented IkappaB-alpha degradation. Our results show for the first time that NF-kappaB is involved in COX-2 protein expression in LPS-stimulated J774 macrophages and suggest that inhibitors of NF-kappaB activation may represent a useful tool for the pharmacological control of inflammation.

Prostaglandins prevent inducible nitric oxide synthase protein expression by inhibiting nuclear factor-kappaB activation in J774 macrophages.

We investigated the effect of PGE2 and iloprost (a prostacyclin analogue) on inducible nitric oxide synthase (iNOS) protein expression and nuclear factor-kappaB (NF-kappaB) activation in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (10 microg/ml) caused an increase of iNOS protein expression which was prevented in a concentration-dependent fashion by PGE2 (0.1, 1, 10 microM) and iloprost (0.01, 0.1, 1 microM). Electrophoretic mobility shift assay indicated that both prostanoids blocked the activation of NF-kappaB, a transcription factor necessary for NO synthase induction. PGE2 and iloprost also blocked disappearance of I kappaB-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show for the first time that PGE2 and iloprost down-regulate iNOS protein expression by inhibiting NF-kappaB activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged NO production in pathological events.

Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats.

We studied nitric oxide (NO) synthase activity and serotonin content in the diencephalon of 24 hr food deprived rats. NO synthase activity was significantly increased whereas serotonin levels together with those of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) were reduced in food deprived rats when compared to control rats. NG-Nitro-L-arginine (L-NO Arg), an inhibitor of NO synthase, was used as a tool to study the role of NO in food deprivation. Twenty-four hr food deprived male Sprague-Dawley rats were intraperitoneally (i.p.) administered L-NO Arg (12.5, 25 and 50 mg/kg) before food presentation. Control rats received a NaCl (0.9%) solution. Food consumption was monitored 1 and 2 hr after food presentation. L-NO Arg administration produced a dose-dependent reduction in food intake. Pretreatment with metergoline (2 mg/kg) but not with ritanserin (1 mg/kg) antagonized the anorectic effect of L-NO Arg. Moreover, in the diencephalon L-NO Arg significantly reduced NO synthase activity whereas it increased serotonin levels. Our data indicate that NO might have a physiological role in the regulation of food intake and suggest that brain NO may modulate the central serotoninergic system.

Modulation of the induction of nitric oxide synthase by eicosanoids in the murine macrophage cell line J774.

The effect of eicosanoids on the induction of nitric oxide synthase in the murine macrophage cell line J774 has been studied. We found that prostaglandin E2 (PGE2) and iloprost (a stable analogue of prostacyclin) both at nanomolar concentrations inhibited the lipopolysaccharide stimulated induction of NO synthase. In contrast PGF2 alpha, U46619, a stable analogue of thromboxane A2, leukotrienes B4 and C4 had no effect. These data demonstrate that the L-arginine: NO pathway in macrophages may be modulated by prostanoids.

Modulation by nitric oxide of prostaglandin biosynthesis in the rat.

1. Modulation of prostaglandin biosynthesis in vivo by either exogenous or endogenous nitric oxide (NO) has been studied in the rat using arachidonic acid (AA)-induced paw oedema and measuring both the foot volume and the amount of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), the stable metabolite of prostacyclin (PGI2), in the oedematous fluid recovered from inflamed paws. 2. Paw injections of 150 or 300 nmol of AA were virtually inactive whereas 600 nmol produced a moderate oedema which was greatly reduced by the NO synthase inhibitor L-NG-nitro arginine methyl ester (L-NAME, 100 nmol/paw) and the NO scavenger haemoglobin (Hb, 30 mumol/paw), but unaffected by the inhibitor of the soluble guanylate cyclase, methylene blue (Mb, 3 mumol/paw) and L-arginine (15 mumol/paw). 3. The NO-donors (10 mumol/paw) 3-morpholino-sydnonimine-hydrochloride (SIN-1), S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and sodium nitroprusside (SNP) significantly potentiated the paw oedema induced by AA (300 nmol/paw). 4. SIN-1 (2.5, 5 and 10 mumol/paw) produced a significant dose-dependent increase of the oedema induced by AA which was correlated with increased amounts of 6-keto-PGF1 alpha in the fluid recovered from inflamed paws. 5. Both oedema and prostaglandin biosynthesis induced by the combination AA+SIN-1 were greatly suppressed by either Hb (30 mumol/paw) or indomethacin (3 mumol/paw or 5 mg kg-1 s.c.) but unaffected by Mb (3 mumol/paw). 6. In LPS-treated rats (6 mg kg-1, i.p.) doses of AA inactive in normal animals produced a remarkable oedema which was reduced by L-NAME or Hb, unaffected by Mb and increased by L-arginine.7. These results demonstrate that NO increases prostaglandin biosynthesis in vivo through a guanosine 3': 5'-cyclic monophosphate (cyclic GMP)-independent mechanism and suggest that the interaction between NO synthase and cyclo-oxygenase (COX) pathways may represent an important mechanism for the modulation of the inflammatory response.

Vasocortin: a novel glucocorticoid-induced anti-inflammatory protein.

The preliminary characterization of ;vasocortin' a novel glucocorticoid-induced anti-inflammatory protein, is described. Vasocortin is released into the rat peritoneal cavity following systemic dexamethasone administration, has an apparent mol. wt. of 100 kD and inhibits rat dextran oedema. Vasocortin is distinct from lipocortin and is likely to be associated with the anti-inflammatory effect of glucocorticoids.

Selective inhibition by vasocortin of histamine release induced by dextran and concanavalin-A from rat peritoneal cells.

Vasocortin, a glucocorticoid-induced anti-inflammatory protein, has been purified from the peritoneal lavage fluid of dexamethasone-treated rats. Vasocortin inhibited the release of histamine from rat peritoneal cells stimulated by dextran or concanavalin A but did not alter the release induced by calcium ionophore A23187 or compound 48/80. This selective effect exhibited by vasocortin mimics the glucocorticoid inhibition of histamine release from rat mast cells.

Effects of intracerebroventricular leptin administration on food intake, body weight gain and diencephalic nitric oxide synthase activity in the mouse.

1. Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects. 2. To investigate the role of the brain L-arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5-2 microg) alone or in association with L-arginine (10 microg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25-2 microg) injection and after consecutive doses of leptin (0.25-2 microg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated. 3. I.c.v. injected leptin (1 and 2 microg) significantly and dose-dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97+/-0.16 g 24 h(-1) and 4.27+/-0.18 g 24 h(-1), respectively, vs 8.05+/-0.34 g 24 h(-1), P<0.001, n=6 for each group; body weight gain: -10.7+/-0.46% and -15.7+/-0.65%, respectively, vs 5.14+/-0.38%, P<0.001, n=6 for each group). This effect was antagonized by L-arginine (food intake: 7.90+/-0.37 g 24 h; body weight gain: 5.11+/-0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90+/-0.04 nmol citrulline min(-1) g(-1) tissue; leptin 1 microg: 0.62+/-0.03 nmol citrulline min(-1) g(-1) tissue, P<0.001; leptin 2 microg: 0.44+/-0.03 nmol citrulline min(-1) g(-1) tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h). 4. Our results suggest that the brain L-arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.

Relationship between nitric oxide and prostaglandins in carrageenin pleurisy.

The correlation between endogenous nitric oxide (NO) generation and prostaglandin biosynthesis was studied in rat carrageenin pleurisy induced by the injection of 0.2 mL of 1% lambda-carrageenin into the pleural cavity. The pleural exudate was collected at 4 hr and the amounts of NO2- + NO3- (NOx) and prostaglandin E2 (PGE2) measured. The NOx present in the inflammatory exudate was determined by measuring the NO2- with the Griess reaction, after the reduction of NO3- to NO2- using acid-washed cadmium powder. PGE2 was measured by radioimmunoassay. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 1-3-10 mg/kg subcutaneously) reduced NOx by 20 +/- 7%, 41 +/- 6% and 55 +/- 9% (P < 0.01) and PGE2 by 9 +/- 6%, 41 +/- 11% and 74 +/- 9% (P < 0.001). Conversely, L-arginine (300 mg/kg SC) increasedNOx by 39 +/- 7% (P < 0.01) and PGE2 by 78 +/- 6% (P < 0.001). The NO scavenger haemoglobin (Hb), coinjected into the pleural cavity (3 mg/site) with carrageenin, produced a parallel inhibition of NOx (65 +/- 16%, P < 0.001) and PGE2 (71 +/- 18%, P < 0.001). The soluble guanylate cyclase inhibitor methylene blue (Mb; 2 mg/site) had no effect. Moreover haemoglobin, but not methylene blue, was able to significantly suppress the L-arginine-induced increase of both NOx and PGE2. In each pleural exudate, independently from the animal treatment, the amount of NOx was highly correlated to the amount of PGE2 (r = 0.93, P < 0.001). These results suggest that in rat carrageenin pleurisy the modulation of the L-arginine:NO pathway results in a parallel modulation of prostaglandin biosynthesis. The interaction between cyclooxygenase and the NO pathway may represent an important mechanism for the modulation of the inflammatory response.

Identification of specific binding sites for leukotriene C4 in membranes from human lung.

Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4 degrees, 3H-LTC4 binding is specific, saturable (Bmax = 32-41 pmoles/mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6-7 X 10(-8) M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10(-2)M CaCl2. Unlabelled LTC4 is able to complete with 3H-LTC4 for its binding sites with an IC50 of 7.8 X 10(-8) M. The addition of 10(-2) M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 (IC50 S = 2.2 X 10(-6), 2.4 X 10(-5) M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.

Multiple organ failure following zymosan-induced peritonitis is mediated by nitric oxide.

In the present study we tested the hypothesis that nitric oxide may play a role in the pathogenesis of multiple organ failure induced by peritoneal injection of zymosan in the rat. A severe inflammatory response characterized by peritoneal exudation, high plasma and peritoneal levels of nitrate/ nitrite (breakdown products of nitric oxide), prostaglandin E2 and leukocyte infiltration into peritoneal exudate was induced by zymosan administration. This inflammatory process started within 3 h of administration and onset occurred at 18 h, coinciding with damage of lung, small intestine and liver, as assessed by histological examination and by increase of myeloperoxidase activity, indicative of neutrophil infiltration. Furthermore, at 18 h after zymosan-induced peritonitis, expression of inducible nitric oxide synthase enzyme was found mainly in the macrophages of inflamed lungs. Subcutaneously administration of a nonisoform selective nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester, reduced formation of peritoneal exudate fluid, blocked plasma and peritoneal nitrate/nitrite accumulation, and attenuated the elevated release of peritoneal prostaglandin E2. In addition, nitric oxide synthase inhibition was effective in preventing the development of organ failure since tissue injury and neutrophil infiltration, by myeloperoxidase evaluation, was reduced in lung, small intestine, and liver. In conclusion, major findings of our study are that nitric oxide exerts a proinflammatory role in the development of multiple organ failure and nitric oxide synthase inhibition is an effective antiinflammatory therapeutic tool, since inhibits not only nitric oxide but also prostaglandin production and cellular infiltration in inflamed organs.

Inhibition of nitric oxide formation reduces voluntary ethanol consumption in the rat.

Brain nitric oxide is involved in the mechanisms that regulate ingestive behavior. To test whether this compound plays a role in alcohol preference, we studied the effects of different doses of NG-nitro-L-arginine (L-NO arg), an inhibitor of nitric oxide synthase (NOS), on voluntary consumption of ethanol and on blood alcohol levels produced by a single intraperitoneal dose of alcohol in the rat. L-NO arg produced a significant and dose-dependent reduction of ethanol intake (P < 0.001) without influencing total fluid consumption or feeding behavior. L-NO arg did not influence the kinetics of alcohol. Our data show that inhibition of nitric oxide formation accompanies reduction of ethanol intake and suggest a possible role for nitric oxide in ethanol self-administration.

Nitric oxide modulates prostacyclin biosynthesis in the lung of endotoxin-treated rats.

In this study we have investigated the correlation between prostaglandin generation and the L-arginine:nitric oxide (NO) pathway in the lung of rats challenged with lipopolysaccharide. We found that in rats treated with L-arginine, the amount of prostacyclin, measured as 6-keto-PGF1 alpha, was significantly increased. Conversely in rats treated with NG-nitro-L-arginine methyl ester the production of 6-keto-PGF1 alpha by lung was significantly decreased. Chopped lungs that had been removed from rats challenged with lipopolysaccharide and were incubated overnight with L-arginine or nitric oxide inhibitors, NG-nitro-L-arginine methyl ester or NG-monomethyl-L-arginine, released respectively increased or decreased amount of 6-keto-PGF1 alpha. These results suggest that, in the rat, lung endogenous nitric oxide may modulate prostanoid production.

Vasocortin-like proteins induced by glucocorticoids in vascular tissue.

Rat and bovine aorta rings incubated with 10(-5) M dexamethasone release proteins which inhibit rat dextran oedema. These proteins seem to be related to vasocortin, derived from the peritoneal fluid of dexamethasone-treated rats, and may contribute to the control that glucocorticoids exert on vascular tonus and permeability.

The role of constitutive and inducible nitric oxide synthase in senna- and cascara-induced diarrhoea in the rat.

The role of constitutive and inducible nitric oxide (NO) synthase in rats treated with senna and cascara was studied. Senna (60 mg/kg p.o.) and cascara (800 mg/kg p.o.) ex vivo significantly increased Ca(2+)-dependent constitutive NO synthase activity in the rat colon. Induction of NO synthase (12% of the total NO synthase) was associated with cascara, but not senna, administration. Dexamethasone (0.03-0.3 mg/kg i.p.), which inhibits the expression of inducible NO synthase, significantly and dose-dependently reduced cascara-(but not senna-) induced diarrhoea and colonic fluid secretion. These findings suggest that senna probably exerts its laxative effect through stimulation of the constitutive isoform of NO synthase, while the inducible isoform of NO synthase also seems to be involved in the laxative effect of cascara.