Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide(NO) is synthesized in normal muscle fibers by the neuronal (nNOS) andthe endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NOcontributes to the regulation of several processes such asexcitation-contraction coupling and mitochondrial respiration. Weassessed in this study whether NO production is regulated in responseto an acute increase in muscle activation. Three groups ofanesthetized, tracheostomized, spontaneously breathing rats wereexamined after an experimental period of 3 h. Group 1 served as a control (no loading), whereasgroups 2 and3 were exposed to moderate and severeinspiratory resistive loads, respectively, which elicited trachealpressures of 30 and 70% of maximum, respectively. Ventilatory(diaphragm, intercostal, and transverse abdominis) and limb(gastrocnemius) muscles were excised at the end of the experimentalperiod and examined for NOS activity and NOS protein expression.Neither submaximal nor maximum tracheal pressures were altered after 3 h of resistive loading. Diaphragmatic and intercostal muscle NOSactivities declined significantly in response to moderate and severeloading, whereas those of transverse abdominis and gastrocnemiusmuscles remained unchanged. On the other hand, resistive loading had nosignificant effect on ventilatory and limb muscle NOS isoformexpression. We propose that a contraction-induced decline in muscle NOSactivity represents a compensatory mechanism through which musclecontractility and mitochondrial function are protected from theinhibitory influence of NO.

     

Mechanisms underlying the nitric oxide inhibitory effects in mouse ileal longitudinal muscle

We investigated the mechanisms involved in the nitric oxide (NO)-induced inhibitory effects on longitudinal smooth muscle of mouse ileum, using organ bath technique. Exogenously applied NO, delivered as sodium nitroprusside (SNP; 0.1-100 micromol/L) induced a concentration-dependent reduction of the ileal spontaneous contractions. 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ; 1 micromol/L), a guanilyl cyclase inhibitor, reduced the SNP-induced effects. Tetraethylammonium chloride (20 mmol/L), a non-selective K+ channel blocker, and charybdotoxin (0.1 micromol/L), blocker of large conductance Ca2+-dependent K+ channels, significantly reduced SNP-induced inhibitory effects. In contrast, apamin (0.1 micromol/L), blocker of small conductance Ca2+-dependent K+ channels, was not able to affect the response to SNP. Ciclopiazonic acid (10 micromol/L) or thapsigargin (0.1 micromol/L), sarcoplasmatic reticulum Ca2+-ATPase inhibitors, decreased the SNP-inhibitory effects. Ryanodine (10 micromol/L), inhibitor of Ca2+ release from ryanodine-sensitive intracellular stores, significantly reduced the SNP inhibitory effects. The membrane permeable analogue of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (100 micromol/L), also reduced spontaneous mechanical activity, and its effect was antagonized by ryanodine. The present study suggests that NO causes inhibitory effects on longitudinal smooth muscle of mouse ileum through cGMP which in turn would activate the large conductance Ca2+-dependent K+ channels, via localized ryanodine-sensitive Ca2+ release.     

Inhibition of nitric oxide synthesis attenuates thermally induced asthma.

To determine whether the inhibition of nitric oxide (NO) synthesis attenuates thermally induced obstruction, we had 10 asthmatic volunteers perform isocapnic hyperventilation with frigid air after inhaling 1 mg of N(G)-monomethyl-L-arginine (L-NMMA) or isotonic saline in a blinded fashion. The challenges were identical in all respects, and there were no differences in baseline lung function [1-s forced expiratory volume (FEV(1)); saline 2.8 +/- 0.3 liters, L-NMMA 2.9 +/- 0.3 liters; P = 0.41] or prechallenge fractional concentration of nitric oxide in the exhaled air (FENO) [saline 23 +/- 6 parts/billion (ppb), L-NMMA 18 +/- 4 ppb; P = 0.51]. Neither treatment had any impact on the FEV(1), pulse, or blood pressure. After L-NMMA, FENO fell significantly (P < 0.0001), the stimulus-response curves shifted to the right, and the minute ventilation required to reduce the FEV(1) 20% rose 53.5% over control (P = 0.02). The results of this study demonstrate that NO generated from the airways of asthmatic individuals may play an important role in the pathogenesis of thermally induced asthma.     

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to β-adrenergic agonists is eliminated when the heart is perfused with N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the β-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 μM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with l-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed β-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the β-stimulated calcium current.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Effect of inhibition of nitric oxide synthesis on the diaphragmatic microvascular response to hypoxia

Ward, Michael E. Effect of inhibition of nitric oxidesynthesis on the diaphragmatic microvascular response to hypoxia. J. Appl. Physiol. 81(4):1633-1641, 1996.The purpose of this study was to determine theeffect of inhibition of nitric oxide (NO) release on the diaphragmaticmicrovascular responses to hypoxia. In -chloralose-anesthetizedmongrel dogs, the microcirculation of the vascularly isolated ex vivoleft hemidiaphragm was studied by intravital microscopy. The diaphragmwas pump perfused with blood diverted from the femoral artery through aseries of membrane oxygenators. The responses to supramaximalconcentrations of sodium nitroprusside, moderate hypoxia (phrenicvenous PO₂ 27 Torr), andsevere hypoxia (phrenic venous PO₂ 15 Torr) were recorded before and after an infusion ofN^G-nitro-L-arginine(L-NNA; 6 × 10⁴ M) into the phreniccirculation for 20 min. Under control conditions, diaphragmatic bloodflow was 12.4 ± 1.1 ml ^· min^{1 ·} 100 g¹. Diaphragmatic bloodflows recorded during moderate and severe hypoxia were 15.6 ± 1.2 and 24.3 ± 1.5 ml ^· min^{1 ·} 100 g¹, respectively(P < 0.05 for both compared withcontrol values). Treatment withL-NNA reduced diaphragmaticblood flow to 9.6 ± 0.8 ml ^· min^{1 ·} 100 g¹ under control conditions(P < 0.05) and caused arteriolarvasoconstriction to a degree that was dependent on vessel size (i.e.,larger vessels constricted more than smaller vessels).L-NNA eliminated the increase inblood flow during moderate hypoxia and inhibited arteriolar dilation byan amount that was related to vessel size (i.e., dilation of largervessels was inhibited more than that of smaller vessels). Inhibition ofNO synthesis had no effect on the increase in diaphragmatic blood flow(23.6 ± 1.9 ml ^· min^{1 ·} 100 g¹;P > 0.05 compared with that duringsevere hypoxia before treatment withL-NNA) or arteriolar diametersduring severe hypoxia. NO release plays a role in the diaphragmaticvascular response to hypoxia, but this role is limited to dilation oflarger arterioles during hypoxia of moderate severity.

     

Statin blocks Rho/Rho-kinase signalling and disrupts the actin cytoskeleton: relationship to enhancement of LPS-mediated nitric oxide synthesis in vascular smooth muscle cells

We previously demonstrated statins to enhance cytokine-mediated nitric oxide (NO) synthesis in vascular smooth muscle cells (VSMC). To clarify the mechanism by which this occurs, we evaluated the effects of fluvastatin in lipopolysaccharide (LPS)-stimulated VSMC. NO production induced by LPS was dose-dependently enhanced by fluvastatin, as were iNOS mRNA levels and iNOS protein expression. Exogenous mevalonate and geranylgeranylpyrophosphate (GGPP) dampened the stimulatory effect of fluvastatin. A pull-down assay demonstrated fluvastatin to decrease levels of GTP-bound Rho A. Moreover, a Rho-kinase inhibitor, Y-27632, was observed to enhance LPS-induced NO production. We recently demonstrated that disrupting F-actin formation dramatically potentiates the ability of LPS to induce iNOS mRNA and protein expression. In the present study, staining of F-actin with nitrobenzoxadiazole (NBD)-phallacidin demonstrated that fluvastatin significantly impairs F-actin stress fiber formation. In light of these results, the ability of statins to increase NO production is due, at least in part, to their ability to block the biosynthesis of mevalonate, thereby preventing isoprenoid biosynthesis. This inhibits Rho/Rho-kinase signalling and, in turn, disrupts the actin cytoskeleton. Further analysis of the signalling pathway by which the actin cytoskeleton affects iNOS expression might yield new insight into mechanisms of regulation of NO production.     

Inhibition of nitric oxide synthesis in primary cultured mouse hepatocytes by alpha-lipoic acid

Recent work shows that septic or endotoxic shock is associated with lipopolysaccharide and cytokine mixture-induced nitric oxide (NO) synthesis in liver. Here we found that DL-alpha-lipoic acid inhibited but other thiol-containing antioxidants such as glutathione and N-acetylcysteine enhanced lipopolysaccharide and cytokine mixture (referred as LPS/CM)-induced NO synthesis in hepatocytes. The inhibitory action of alpha-lipoic acid on hepatocyte NO synthesis was as potent as that of NG-monomethyl-L-arginine without obvious cytotoxicity. Deletion by diethylmaleate or inhibition by buthionine sulfoximine of intracellular glutathione caused a significant decrease in hepatocyte NO synthesis, implying that increased intracellular reduced glutathione levels could not be the reason for alpha-lipoic acid inhibited NO synthesis. alpha-Lipoic acid inhibition of NO synthesis seems to be from alpha-lipoic acid improved carbohydrate metabolism in hepatocytes. Since alpha-lipoic acid is an essential compound existing naturally in physiological systems, it may serve as both a research and therapeutic agent for sepsis.     

Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia.

Systemic hypoxia results in oxidative stress due to a change in the reactive oxygen species (ROS)-nitric oxide (NO) balance. These experiments explored two mechanisms for the altered ROS-NO balance: 1) decreased NO synthesis by NO synthase due to limited O(2) substrate availability and 2) increased superoxide generation. ROS levels and leukocyte adherence in mesenteric venules of rats during hypoxia were studied in the absence and presence of an NO donor [spermine NONOate (SNO)] and of the NO precursor L-arginine. We hypothesized that if the lower NO levels during hypoxia were due to O(2) substrate limitation, L-arginine would not prevent hypoxia-induced microvascular responses. Graded hypoxia (produced by breathing 15, 10, and 7.5% O(2)) increased both ROS (123 +/- 6, 148 +/- 11, and 167 +/- 3% of control) and leukocyte adherence. ROS levels during breathing of 10 and 7.5% O(2) were significantly attenuated by SNO (105 +/- 6 and 108 +/- 3%, respectively) and L-arginine (117 +/- 5 and 115 +/- 2%, respectively). Both interventions reduced leukocyte adherence by similar degrees. The fact that the effects of L-arginine were similar to those of SNO does not support the idea that NO generation is impaired in hypoxia and suggests that tissue NO levels are depleted by the increased ROS during hypoxia.     

Inhibition of inducible nitric oxide synthesis by azathioprine in a macrophage cell line

Azathioprine is used as an anti-inflammatory agent. Although there are numerous data demonstrating cytotoxic and immunosuppressive properties of azathioprine and its metabolite 6-mercaptopurine, the mechanism of the anti-inflammatory action of azathioprine has not yet been fully clarified. During our study, we investigated the effects of azathioprine on the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated murine macrophages (RAW 264.7) by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), and NO production (nitrite levels). Azathioprine (0-210 muM) induces a concentration dependent inhibition of inducible nitric oxide synthesis (IC50: 33.5 muM). iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of azathioprine. Azathioprine decreases iNOS mRNA levels as shown by semiquantitative competitive RT-PCR. In contrast, 6-mercaptopurine showed no inhibition of inducible nitric oxide synthesis. Azathioprine did not reduce iNOS mRNA stability after the addition of actinomycin D. Enzymatic activity assays with increasing concentrations of azathioprine (0-210 muM) showed no statistically significant inhibition of iNOS enzyme activity compared to cell lysates without azathioprine. Nuclear translocation of NF-kappaB p65 subunit and binding of NF-kappaB p50 subunit from nuclear extracts to a biotinylated-consensus sequence was unaffected by azathioprine treatment. iNOS inhibition by azathioprine was associated with a decreased expression of IRF-1 (interferon regulatory factor 1) and IFN-beta (beta-interferon) mRNA. Azathioprine induced iNOS inhibition seems to be associated with an action of the methylnitroimidazolyl substituent. This suggests a route to the rational design of nontoxic anti-inflammatory agents by replacing the 6-mercaptopurine component of azathioprine with other substituents. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of azathioprine.     

一氧化氮对心肌的抑制性保护作用

内皮型与神经型一氧化氮合酶(eNOS,nNOS)在心肌细胞内持续表达,而细胞应激可引起诱导型NOS(iNOS)表达.心肌细胞结构型eNOS与nNOS源性NO,在生理条件下对心肌主要发挥4方面的抑制作用:减缓心肌细胞搏动频率,轻度抑制心肌细胞收缩功能,加速心肌细胞舒张并增加顺应性,以及轻度抑制线粒体电子传递而增强氧利用效...     

Ovarian hormone secretory response to gonadotropins and nitric oxide following chronic nitric oxide deficiency in the rat

Ovarian hormone secretion is regulated by gonadotropins, and it has been demonstrated that this response is modulated by nitric oxide (NO). The focus of this study was to determine the effect of chronic NO deficiency on the secretion of ovarian steroids. Female rats were given N-nitro-L-arginine (L-NNA; 0.6 g/L) in their drinking water, and vaginal smears were obtained daily. By 4 wk of treatment, all the rats were in constant estrus or proestrus. At 6-8 wk the animals were killed; the ovaries were removed and incubated in the presence of eCG (1 IU/ml) and hCG (1 IU/ml) and/or S-nitroso-L-acetyl penicillamine (an NO donor, S-NAP; 0.1 mM) for 4 h. Medium was collected at 30-min intervals, and estradiol, progesterone, and androstenedione were measured. Ovaries from proestrous rats served as controls. Ovaries from L-NNA-treated animals had a greater basal and gonadotropin-stimulated release of estradiol but not of androstenedione or progesterone in comparison to ovaries from untreated controls. S-NAP decreased the gonadotropin-stimulated estradiol, progesterone, and androstenedione in ovaries from NO-deficient rats. Steroid secretion in controls was not responsive to S-NAP. We conclude that chronic NO inhibition produces constant estrus due to increased estradiol production and that NO acts to inhibit estradiol and androstenedione production.     

The dynamics of the nitric oxide release-transient from stretched muscle cells

Potent nitric oxide (NO) signals are described for many forms of cell-cell communication. Although NO plays a significant role in skeletal muscle metabolism and contractility and in precursor activation during muscle formation and stretching, there is no direct evidence of stretch-induced NO release from muscle. Differentiated muscle cell cultures from normal and dystrophic mdx mice were preloaded with the NO-specific dye DAF-2 (diaminofluorescein-2) before stretching. NO release was detected by video-microscopy. NO was released rapidly from wild-type (WT) cells after stretch and intensity declined rapidly to a plateau. Mdx cells showed much less NO release. Direct observations of the time-course of stretch-induced NO release in WT cells is congruent with the hypothesis of NO-mediated stretch activation of satellite cells in normal skeletal muscle. Distinct differences in the time-course between normal and dystrophic cells indicate visualization methods for NO release will be a sensitive measure of NOS-1 restoration following diverse treatment approaches to muscular dystrophy.     

Characterization of inhibitory innervation in porcine colonic circular muscle

Effects of stimulation of intramural nerves in the circular smooth muscle layer of the porcine colon (Sus scrofa domestica) were studied using the sucrose-gap technique. Electrical field stimulation of the preparation, superfused with Krebs solution at 21 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane. This hyperpolarization was an inhibitory junction potential (IJP). The responses obtained from circular muscle originating from either the centripetal or centrifugal gyri of the ascending colon did not differ significantly. The IJP was characterized as being mediated by intramural, nonadrenergic, noncholinergic (NANC) nerves. The amplitude and latency of the IJP changed linearly with temperature (15-25 degrees C: +1 mV and -0.1 s per degree Celsius, respectively) reflecting a temperature-dependent synchronization of transmitter release. The membrane resistance decreased during the IJP. The IJP amplitude decreased or increased during conditioning hyperpolarizations or depolarizations, respectively, and reversed at membrane potentials about 30 mV more negative than the resting membrane potential. Potassium conductance blocking agents, barium (1 mM), tetraethylammonium chloride (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM), apamin (1 microM), and aminacrine (10(-4) M) added to the superfusion medium increased the membrane resistance. Only barium, TEA, and apamin depolarized the smooth muscle cell membrane. The IJP amplitude decreased in the presence of aminacrine and apamin to 75 and 35%, respectively, suggesting that apamin-sensitive Ca2+-activated K+ channels are involved in this response. ATP, adenosine, and related adenine nucleotides in concentrations up to 10(-3) M did not mimic the IJP. Superfusion with ATP for 15 min revealed a gradually increasing attenuation by up to 20% of the IJP. This might suggest that the release of neurotransmitter from intramural NANC nerves is modulated presynaptically via purinoceptors. Exogenously applied vasoactive intestinal polypeptide (VIP) in concentrations of 10(-9) to 10(-4) M did not affect the preparation. Also at elevated temperatures (up to 35 degrees C), VIP (10(-7) to 10(-4) M) did not cause measurable effects. It is concluded that the inhibitory mediator of the intramural NANC nerves present in the circular muscle layers of the porcine colon is neither a purine nor VIP.     

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.     

Possible role of nitric oxide in transmitting information from vasodilator nerve to cerebroarterial muscle

Treatment with L-NG-monomethyl arginine (L-NMMA), an inhibitor of nitric oxide (NO) synthesis from L-arginine, suppressed the relaxant response of dog cerebral artery strips to transmural electrical stimulation and nicotine, as did oxyhemoglobin. The inhibition by L-NMMA was reversed or prevented by L-, but not D-, arginine. It is concluded that NO or an NO-related compound may play a crucial role in transmitting information from excited vasodilator nerves to cerebroarterial smooth muscle.     

Calcitonin gene-related peptide and capsaicin inhibit the circular muscle of the guinea-pig ileum

Calcitonin gene-related peptide (CGRP) is known to excite, through the release of acetylcholine, the circular muscle (CM) of the guinea-pig ileum in vitro. In the present experiments, the effect of rat CGRP was investigated on the CM of tetrodotoxin-treated, spontaneously active ileum preparations. CGRP (1-10 nM) caused concentration-dependent inhibition of both the amplitude and frequency of spontaneous CM contractions. Capsaicin (a sensory stimulant known to release CGRP from primary afferents) also inhibited CM activity. The effect of 1 microM capsaicin underwent rapid desensitization, indicating specific action on afferent structures, whereas a high concentration of the drug (33 microM) inhibited CM activity most probably on the smooth muscle itself.     

Effects of capsaicin on nitric oxide synthase isoforms in prepubertal rat ovary

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.     

Inhibition of inducible nitric oxide synthesis by the herbal preparation Padma 28 in macrophage cell line

Padma 28 is a mixture of herbs used in traditional Tibetan medicine with anti-inflammatory activities. We investigated the effects of Padma 28 on nitric oxide (NO) production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated mouse macrophages (RAW 264.7). Padma 28 (0-900 microg/mL) induced a concentration dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of Padma 28. Padma 28 decreased iNOS mRNA levels as shown by RT-PCR. Aqueous extracts from costi amari radix (costus root, the dried root of Saussurea lappa) and the outer cover of myrobalani fructus (the dried fruit of Terminalia chebula), constituents of the complex herb preparation Padma 28, were found to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of Padma 28.