Cardiomyocyte-derived small extracellular vesicles can signal eNOS activation in cardiac microvascular endothelial cells to protect against Ischemia/Reperfusion injury.

Rationale: The crosstalk between cardiac microvascular endothelial cells (CMECs) and cardiomyocytes (CMs) has emerged as a key component in the development of, and protection against, cardiac diseases. For example, activation of endothelial nitric oxide synthase (eNOS) in CMECs, by therapeutic strategies such as ischemic preconditioning, plays a critical role in the protection against myocardial ischemia/reperfusion (I/R) injury. However, much less is known about the signals produced by CMs that are able to regulate CMEC biology. Here we uncovered one such mechanism using Tongxinluo (TXL), a traditional Chinese medicine, that alleviates myocardial ischemia/reperfusion (I/R) injury by activating CMEC eNOS. The aim of our study is to identify the signals produced by CMs that can regulate CMEC biology during I/R. Methods:Ex vivo, in vivo, and in vitro settings of ischemia-reperfusion were used in our study, with the protective signaling pathways activated in CMECs identified using genetic inhibition (p70s6k1 siRNA, miR-145-5p mimics, etc.), chemical inhibitors (the eNOS inhibitor, L-NNA, and the small extracellular vesicles (sEVs) inhibitor, GW4869) and Western blot analyses. TritonX-100 at a dose of 0.125% was utilized to inactivate the eNOS activity in endothelium to investigate the role of CMEC-derived eNOS in TXL-induced cardioprotection. Results: We found that while CMEC-derived eNOS activity was required for the cardioprotection of TXL, activation of eNOS in CMECs by TXL did not occur directly. Instead, eNOS activation in CMECs required a crosstalk between CMs and CMECs through the uptake of CM-derived sEVs. We further demonstrate that TXL induced CM-sEVs contain increased levels of Long Intergenic Non-Protein Coding RNA, Regulator Of Reprogramming (Linc-ROR). Upon uptake into CMECs, linc-ROR downregulates its target miR-145-5p leading to activation of the eNOS pathway by facilitating the expression of p70s6k1 in these cells. The activation of CMEC-derived eNOS works to increase survival in both the CMECs and the CMs themselves. Conclusions: These data uncover a mechanism by which the crosstalk between CMs and CMECs leads to the increased survival of the heart after I/R injury and point to a new therapeutic target for the blunting of myocardial I/R injury.

Differential mechanisms of action of the trace amines octopamine, synephrine and tyramine on the porcine coronary and mesenteric artery.

Trace amines such as p-tyramine, p-octopamine and p-synephrine are found in low concentrations in animals and plants. Consumption of pre-workout supplements containing these plant-derived amines has been associated with cardiovascular side effects. The aim of this study was to determine the mechanisms of action of these trace amines on porcine isolated coronary and mesenteric arteries. Noradrenaline caused contraction of mesenteric arteries and relaxation of coronary arteries. In both tissues, all three trace amines induced contractions with similar potencies and responses were unaffected by the ß-adrenoceptor antagonist propranolol (1 µM), the nitric oxide synthase inhibitor L-NNA (100 µM), or the TAAR-1 antagonist, EPPTB (100 nM). However, the contractile responses of mesenteric arteries, but not coronary arteries, were significantly reduced by depletion of endogenous noradrenaline. Mesenteric responses to all three amines were abolished in the presence of prazosin (1 µM) whereas residual contractile responses remained in the coronary artery which were inhibited by a high concentration (100 µM) of EPPTB. The results suggest complex responses of the coronary artery to the trace amines, with activity at α1-adrenoceptors and potentially TAARs other than TAAR-1. In contrast the actions of the amines on the mesenteric artery appeared to involve indirect sympathomimetic actions and direct actions on α1-adrenoceptors.

Biodegradable amino acid-based poly(ester amine) with tunable immunomodulating properties and their in vitro and in vivo wound healing studies in diabetic rats' wounds.

The objective of this study is to design a new family of biodegradable synthetic polymeric biomaterials for providing a tunable inhibition of macrophage's nitric oxide synthase (NOS) pathway. l-Arginine (Arg) is the common substrate for NOS and arginase. Both two metabolic pathways participate in the wound healing process. An impaired wound healing, such as diabetic or other chronic wounds is usually associated with an overproduction of NO by macrophages via the NOS pathway. In this study, a new family of l-nitroarginine (NOArg) based polyester amide (NOArg-PEA) and NOArg-Arg PEA copolymers (co-PEA) were designed and synthesized with different composition ratios. The NOArg-PEA and NOArg-Arg co-PEAs are biodegradable (more than 50% degradation in vitro in 4 days at 37 °C), biocompatible and did not activate the resting macrophage immune response per se. When classically activated or alternatively activated macrophages (CAM/AAM) were incubated with NOArg-PEA and NOArg-Arg co-PEAs, the treatments decreased the NO production of CAM, increased the arginase activity in both CAM and AAM, increased TGF-ß1 production of CAM to various degrees and had no significant effect on TNF-α production. Diabetic rat models were used to evaluate the efficacy of NOArg-PEA and NOArg-Arg co-PEAs on wound healing. Diabetic rats treated with 2-NOArg-4 PEA, 2-NOArg-4-Arg-4 20/80, and 2-NOArg-4-Arg-4 50/50 biomaterials achieved 40%-80% faster-wound healing when compared with the control on day 7. The data from the histological and immunohistochemical analysis showed that the 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments led to more AAM phenotypes (CD206) and arginase I production in wound tissue than the control during the first 7 days, i.e., suggesting pro-healing wound microenvironment with improved re-epithelialization of wound healing. A similar trend was retained until day 14. The 2-NOArg-4-Arg-4 20/80 and 2-NOArg-4-Arg-4 50/50 treatments also increased the collagen deposition and angiogenesis in the healing wound between day 7 and day 14. Both in vitro and in vivo data of this study showed that this new family of NOArg-Arg co-PEA biomaterials have the potential as viable alternatives for treating impaired wound healing, such as diabetic or other types of chronic wounds. STATEMENT OF SIGNIFICANCE: Diabetic or other chronic wounds is usually associated with an overproduction of NO and pro-inflammatory signals by macrophages. Arginine supplement or NOS inhibitors administration failed to achieve an expected improved wound healing because of the dynamic complexity of arginine catabolism, the difficulty in transition from pro-inflammatory to pro-healing, and the short-term efficacy. We designed and synthesized a new family of water-soluble and degradable nitroarginine-arginine polyester amides to rebalance NOS/arginase metabolism pathways of macrophages. They showed tunable immunomodulating properties in vitro. The in vivo studies were performed to evaluate their efficacy in accelerating the healing. These new biomaterials have the potential as viable alternatives for treating impaired wound healing. The general audience of Acta Biomaterialia should be interested in these findings.

Nitric oxide synthase and cyclooxygenase modulate ß-adrenergic cutaneous vasodilatation and sweating in young men.

KEY POINTS: ß-Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in ß-adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol. We show that while NOS contributes to ß-adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation. We also show that combined inhibition of NOS and COX augments ß-adrenergic sweating These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of ß-adrenergic receptors in the skin. ABSTRACT: ß-Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to ß-adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to ß-adrenergic sweating. In 10 young males (25 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10 mm Nω -nitro-l-arginine (l-NNA), a non-specific NOS inhibitor, (3) 10 mm ketorolac, a non-specific COX inhibitor, or (4) a combination of l-NNA and ketorolac. All sites were co-administered with a high dose of isoproterenol (100 µm) for 3 min to maximally induce ß-adrenergic sweating (ß-adrenergic sweating is significantly blunted by subsequent activations). Approximately 60 min after the washout period, three incremental doses of isoproterenol were co-administered (1, 10 and 100 µm each for 25 min). Increases in CVC induced by the first and second 100 µm isoproterenol were attenuated by l-NNA alone, and those in response to all doses of isoproterenol were reduced by l-NNA with co-infusion of ketorolac (all P ≤ 0.05). Ketorolac alone augmented increases in CVC induced by 10 µm and by the second 100 µm isoproterenol (both P ≤ 0.05). While isoproterenol-induced sweating was not affected by the separate administration of l-NNA or ketorolac (all P > 0.05), their combined administration augmented sweating elicited by the first 3 min of 100 µm isoproterenol (P = 0.05). We show that while NOS contributes to ß-adrenergic cutaneous vasodilatation, COX restrains the vasodilatation. Finally, combined inhibition of NOS and COX augments ß-adrenergic sweating.

Contribution of Cyclooxygenase End Products and Oxidative Stress to Intrahepatic Endothelial Dysfunction in Early Non-Alcoholic Fatty Liver Disease.

INTRODUCTION: Metabolic syndrome induces endothelial dysfunction, a surrogate marker of cardiovascular disease. In parallel, metabolic syndrome is frequently associated with non-alcoholic fatty liver disease (NAFLD), which may progress to cirrhosis. The aim of the present study was to evaluate intrahepatic endothelial dysfunction related to cyclooxygenase end products and oxidative stress as possible mechanisms involved in the pathophysiology of NAFLD. MATERIALS AND METHODS: Sprague-Dawley rats were fed standard diet (control-diet, CD) or high-fat-diet (HFD) for 6 weeks. Metabolic syndrome was assessed by recording arterial pressure, lipids, glycemia and rat body weight. Splanchnic hemodynamics were measured, and endothelial dysfunction was evaluated using concentration-effect curves to acetylcholine. Response was assessed with either vehicle, L-NG-Nitroarginine (L-NNA), indomethacin, tempol, or a thromboxane receptor antagonist, SQ 29548. We quantified inflammation, fibrosis, oxidative stress, nitric oxide (NO) bioavailability and thromboxane B2 levels. RESULTS: HFD rats exhibited metabolic syndrome together with the presence of NAFLD. Compared to control-diet livers, HFD livers showed increased hepatic vascular resistance unrelated to inflammation or fibrosis, but with decreased NO activity and increased oxidative stress. Endothelial dysfunction was observed in HFD livers compared with CD rats and improved after cyclooxygenase inhibition or tempol pre-incubation. However, pre-incubation with SQ 29548 did not modify acetylcholine response. CONCLUSIONS: Our study provides evidence that endothelial dysfunction at an early stage of NAFLD is associated with reduced NO bioavailability together with increased cyclooxygenase end products and oxidative stress, which suggests that both pathways are involved in the pathophysiology and may be worth exploring as therapeutic targets to prevent progression of the disease.

Antidepressant Effect of Cnestis ferruginea Vahl ex DC (Connaraceae): Involvement of Cholinergic, Monoaminergic and L-arginine-nitric Oxide Pathways.

BACKGROUND: We have previously reported antidepressant effect of Cnestis ferruginea (CF) in behavioral models of depression. Due to the promise shown by this extract, this study was carried out to investigate the contribution of monoaminergic, cholinergic and nitrergic systems to the antidepressant-like effect elicited by CF. METHODS: Male albino mice were pretreated with monoaminergic or cholinergic receptor antagonists, L-arginine or N(G)-nitro-L-arginine (nitric oxide synthase inhibitor) (at doses reported to block the in vivo effect of the agonists), 15 min before oral administration of CF (100 mg/kg), 1 h later, the forced swim test (FST) in mice was carried out. RESULTS: CF treatment produced significant changes in the duration of swimming (F(5,42)=9.86, P<0.001), climbing behaviour (F(5,42)=4.51, P=0.004) and mean time spent immobile (F(5,42)=11.55, P<0.001) vs. vehicle-treated control. Co-administration of CF with fluoxetine or imipramine potentiated their effect. However, pretreatment of mice with reserpine (F(1,16)=119.20, P<0.001), prazosin (F(1,16)=68.98, P<0.001), sulpiride (F(1,16)=15.46, P<0.01), RS 127445 ((F(1,20)=8.22, P<0.01), SB 399885 ((F(1,20)=38.44, P<0.001), atropine (F(1,16)=53.77, P<0.001), or L-arginine (nitric oxide precursor) (F(1,16)=10.35, P<0.01) prevented CF-induced antidepressant-like effect in mice. In addition, pretreatment of mice with L-NNA (10 mg/kg) augmented the effect of CF. CONCLUSION: C. ferruginea exerts its antidepressant-like action through interaction with α-adrenoceptor, dopamine D2, 5-HT2B, 5-HT6 and muscarinic cholinergi1c receptors as well as L-arginine-nitric oxide systems. C. ferruginea could be used as adjuvant with conventional antidepressants in the treatment of major depressive disorder.

Effect of Sodium Nitrite and L-NNA on the Outcome of Experimental Ischemic Stroke.

We studied the effect of sodium nitrite in doses of 5 and 50 mg/kg and NO synthase inhibitor L-NNA in a dose of 20 mg/kg on the course of experimental ischemic stroke caused by occlusion of both carotid arteries. Sodium nitrite and NO synthase inhibitor were administered 1 h prior to occlusion of еру carotid arteries and 5 sec after brain ischemia. Sodium nitrite in a dose of 5 mg/kg had a protective effect on the time course of neurological disorders and reduced animal mortality. NO synthase inhibitor L-NNA aggravated the neurological symptoms.

Puerarin enhances proliferation and osteoblastic differentiation of human bone marrow stromal cells via a nitric oxide/cyclic guanosine monophosphate signaling pathway.

Puerarin, a major active isoflavone extracted from the Traditional Chinese Medicine Radix Puerariae, has been studied for its comprehensive biological effects. However, to date, its effect on bone formation and the underlying mechanism of action have not been well investigated. The present study investigated the effect of puerarin on cell proliferation and osteoblastic maturation in cultured human bone marrow stromal cells (hBMSC) in vitro. Puerarin (2.5-100 µM) increased hBMSC growth in a dose-dependent manner, as indicated by an MTT assay, and stimulated osteoblastic maturation as indicated by assessment of alkaline phosphatase (ALP) activity, as well as calcium deposition into the extracellular matrix detected by alizarin red S staining. Furthermore, polymerase chain reaction analysis showed that the expression of osteoblastic markers, including Runt-related transcription factor 2/core-binding factor alpha 1, osterix and osteocalcin, were increased in hBMSCs following incubation with puerarin. Further experiments indicated that puerarin increased the nitric oxide (NO) production and cyclic guanosine monophosphate (cGMP) content in hBMSCs. The effects of puerarin were mimicked by 17ß-estrodiol (10(-8) M) and were abolished in the presence of estrogen receptor antagonist ICI182780 (10(-7) M). A NO synthase inhibitor, Nx-nitro-L-arginine methylester (6 x 10(-3) M), significantly attenuated puerarin-induced increases in NO production and cGMP content, in parallel with a reduction of cell proliferation and osteoblastic differentiation as well as the expression of osteoblastic markers. These results suggested that puerarin may prevent osteoporosis by exerting stimulatory effects on bone formation and the NO/cGMP pathway, which has an important role in puerarin-induced hBMSC proliferation and osteoblastic differentiation.

5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the Lewis rat.

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using 'gold standard' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.

Colonic electrical stimulation: potential use for treatment of delayed colonic transit.

AIM: Recently there has been an increased interest in using electrical stimulation to regulate gut motility generally and particularly for the treatment of slow-transit constipation. In this preliminary canine study, we aimed to study the effects of colonic electrical stimulation (CES) on colonic motility and transit. METHOD: Nine dogs, each equipped with a pair of serosal colon electrodes and a proximal colon cannula were randomized to receive: (i) sham-CES, (ii) long pulse CES (20 cpm, 300 ms, 6 mA) or (iii) pulse train CES (40 Hz, 6 ms, 6 mA). Animals underwent assessment of colonic contractions via manometry, and of colonic transit by inserting 24 radiopaque markers via the colonic cannula and radiographically monitoring the markers at 2, 4 and 6 h following their insertion. The colonic transit was assessed by the geometric centre. RESULTS: We found that, compared with sham-CES, pulse train CES, but not long pulse CES, significantly increased the overall colonic motility index twofold and accelerated the colonic transit by 104% at 2 h, by 60% at 4 h and by 31% at 6 h (P = 0.01, P = 0.02 and P = 0.03 vs sham-CES at 2, 4 and 6 h, respectively). The accelerating effect of pulse train CES was found to be mediated via both cholinergic and nitrergic pathways. CONCLUSION: CES with pulse trains has prokinetic effects on colonic contractions and transit in healthy dogs, mediated via the cholinergic and nitrergic pathways. Further clinical studies are warranted to explore the therapeutic potential of CES for slow colonic transit constipation.

Effects of Kaempferia parviflora Wall. Ex Baker on endothelial dysfunction in streptozotocin-induced diabetic rats.

AIM OF THE STUDY: The aim of the present study was to investigate an ethanolic extract of Kaempferia parviflora (KPE) reduces oxidative stress and preserves endothelial function in aortae from diabetic rats. MATERIALS AND METHODS: Diabetes was induced in Sprague-Dawley rats by streptozotocin (STZ) treatment (55 mg/kg i.v.). Vascular reactivity and superoxide generation were assessed in aortic rings using standard organ bath techniques and lucigenin-enhanced chemiluminescence, respectively. RESULTS: Eight weeks after STZ treatment blood glucose was elevated compared to citrate treated control rats and there was an increased aortic generation of superoxide anion. In aortic rings acetylcholine-induced relaxation was impaired whereas endothelium-independent relaxation to sodium nitroprusside was unaffected. When aortic rings were acutely exposed to KPE (1, 10 and 100 µg/ml) there was a significant reduction in the detection of superoxide anion and enhanced relaxation to acetylcholine. Two separate groups of rats (control and diabetic) were orally administered daily with KPE (100 mg/kg body weight) for 4 weeks. KPE treatment reduced superoxide generation and increased the nitrite levels in diabetic aortae, and enhanced acetylcholine-induced relaxation. In the presence of N(G)-nitro-L-arginine (L-NNA), the relaxation to acetylcholine in aortic rings of diabetic rats was only partially inhibited, but was totally abolished in aortic rings from the KPE-treated diabetic rats. Indomethacin did not affect relaxation to acetylcholine in aortic rings of any group. CONCLUSIONS: These results suggest that KPE, acutely in vitro or after 4 weeks administration in vivo, reduces oxidant stress, increases NO bioavailability and preserves endothelium-dependent relaxation in aortae from diabetic rats.

Nitric oxide synthase inhibitors improve prepulse inhibition responses of Wistar rats.

INTRODUCTION: Cognitive and attentional deficits in schizophrenia include impairment of the sensorimotor filter as measured by prepulse inhibition (PPI). In this way, the study of animals that naturally present low PPI responses could be a useful approach for screening new antipsychotic drugs. Several pieces of evidence suggest that dopamine and nitric oxide (NO) can modulate PPI but their role in those animals is unknown. OBJECTIVES: The aim of this study was to investigate the role of dopamine and NO in Wistar rats with naturally low PPI response. METHODS: Male Wistar rats with low PPI responses received an i.p. injection of the antipsychotics haloperidol (0.1, 0.3 or 1mg/kg) or clozapine (0.5, 1.5 or 5mg/kg), the anxiolytic diazepam (1 or 3mg/kg) or the NO synthase (NOS) inhibitors, N(G)- nitro-l-arginine (l-NOARG; 40mg/kg, acutely or sub-chronically) or 7-Nitroindazole (7-NI; 3, 10 or 30mg/kg). All animals were submitted to the PPI test 1h after injection. Striatal and cortical dopamine, DOPAC, and noradrenaline levels of rats with low PPI responses were compared to rats with normal PPI responses. RESULTS: We found increased levels of catecholamines on the striatum and prefrontal cortex of Wistar rats with low PPI. In these animals, both antipsychotics, typical and atypical, and NOS inhibitors significantly increased PPI. CONCLUSION: Taken together, our findings suggest that the low PPI phenotype may be driven by an overactive catecholamine system. Additionally, our results corroborate the hypothesis of dopamine and NO interaction on PPI modulation and suggest that Wistar rats with low PPI may represent an interesting non-pharmacological model to evaluate new potential antipsychotics.

Antiamnesic effect of B. monniera on L-NNA induced amnesia involves calmodulin.

Amnesia may result from ageing, chronic drug abuse or head injury and there are limited therapeutic strategies to such conditions. We have shown that Bacopa monniera, a memory enhancing drug can reverse both diazepam and scopolamine induced amnesia in mice. In order to understand the downstream effects of B. monniera, this study was designed to investigate how B. monniera antagonizes MK801, an NMDA receptor antagonist and N(omega)-Nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor. We compared the degree of reversal B. monniera imparts on MK801 and L-NNA induced anterograde amnesia in experimental mice. Our data revealed that L-NNA induced anterograde amnesia was significantly reversed by B. monniera, however, it did not attenuate the MK 801 induced anterograde amnesia. B. monniera significantly increased calmodulin (CaM) and pCREB/CREB levels when the whole brain lysates of B. monniera pretreated amnesic mice were compared with those of L-NNA treated mice. We conclude that antiamnesic effect B. monniera on L-NNA induced amnesia may be mediated by NO pathyway involving CaM, which is required for LTP sustenance. These studies evoke interest in their future development as potential antiamnesic drugs.

Purinergic and nitrergic neuromuscular transmission mediates spontaneous neuronal activity in the rat colon.

Nitric oxide (NO) and ATP mediate smooth muscle relaxation in the gastrointestinal tract. However, the involvement of these neurotransmitters in spontaneous neuronal activity is unknown. The aim of the present work was to study spontaneous neuromuscular transmission in the rat midcolon. Microelectrode experiments were performed under constant stretch both in circular and longitudinal directions. Spontaneous inhibitory junction potentials (sIJP) were recorded. Tetrodotoxin (1 microM) and apamin (1 microM) depolarized smooth muscle cells and inhibited sIJP. N(omega)-nitro-l-arginine (l-NNA, 1 mM) depolarized smooth muscle cells but did not modify sIJP. In contrast, the P2Y(1) antagonist MRS-2500 (1 microM) did not modify the resting membrane potential (RMP) but reduced sIJP (IC(50) = 3.1 nM). Hexamethonium (200 microM), NF-023 (10 microM), and ondansetron (1 microM) did not modify RMP and sIJP. These results correlate with in vitro (muscle bath) and in vivo (strain gauges) data where l-NNA but not MRS-2500 induced a sustained increase of spontaneous motility. We concluded that, in the rat colon, inhibitory neurons regulate smooth muscle RMP and cause sIJP. In vitro, the release of inhibitory neurotransmitters is independent of nicotinic, P2X, and 5-hydroxytryptamine type 3 receptors. Neuronal NO causes a sustained smooth muscle hyperpolarization that is responsible for a constant inhibition of spontaneous motility. In contrast, ATP acting on P2Y(1) receptors is responsible for sIJP but does not mediate inhibitory neural tone. ATP and NO have complementary physiological functions in the regulation of gastrointestinal motility.

Effects of sepiapterin supplementation and NOS inhibition on glucocorticoid-induced hypertension.

BACKGROUND: Glucocorticoid-induced hypertension is associated with imbalance between nitric oxide (NO) and superoxide. One of the pathways that causes this imbalance is endothelial NO synthase (eNOS) uncoupling. In the present study, adrenocorticotrophic hormone (ACTH)- and dexamethasone-treated rats were further treated with sepiapterin, a precursor of tetrahydrobiopterin, or N-nitro-L-arginine (NOLA), an inhibitor of NOS, to investigate the role of eNOS uncoupling in glucocorticoid-induced hypertension. METHODS: Male Sprague-Dawley (SD) rats (n = 7-13/group) were treated with either sepiapterin (5 mg/kg/day, IP) or saline (sham) 4 days before and during ACTH (0.2 mg/kg/day, SC), dexamethasone (0.03 mg/kg/day, SC), or saline treatment. NOLA (0.4 mg/ml in drinking water) was given to rats 4 days before and during dexamethasone treatment. Systolic blood pressure (SBP) was measured by the tail-cuff method. RESULTS: Both ACTH (116 +/- 2 to 135 +/- 3 mm Hg (mean +/- s.e.m.), P < 0.001) and dexamethasone (114 +/- 4 to 133 +/- 3 mm Hg, P < 0.0005) increased SBP. Sepiapterin alone did not alter SBP. Sepiapterin did not prevent ACTH- (129 +/- 4 mm Hg, NS) or dexamethasone-induced hypertension (135 +/- 3 mm Hg, NS), although plasma total biopterin concentrations were increased. NOLA increased SBP in rats prior to dexamethasone or saline treatment. NOLA further increased SBP in both saline- (133 +/- 4 to 157 +/- 3 mm Hg, P < 0.05) and dexamethasone-treated rats (135 +/- 5 to 170 +/- 6 mm Hg, P < 0.05). ACTH and dexamethasone increased plasma F(2)-isoprostane concentrations. Neither sepiapterin nor NOLA significantly affected this marker of oxidative stress. CONCLUSION: Sepiapterin did not prevent ACTH- or dexamethasone-induced hypertension. NOLA exacerbated dexamethasone-induced hypertension. These data suggest that eNOS uncoupling does not play a major role in the genesis of glucocorticoid-induced hypertension in the rat.

L-arginine enhances nitrative stress and exacerbates tumor necrosis factor-alpha toxicity to human endothelial cells in culture: prevention by propofol.

Supplementation of L-arginine, a nitric oxide precursor, during the late phase of myocardial ischemia/reperfusion increases myocyte apoptosis and exacerbates myocardial injury, but the underlying mechanism is unclear. During myocardial ischemia/reperfusion, apoptosis of endothelial cells precedes that of cardiomyocyte. Tumor necrosis factor-alpha (TNF) production is increased during myocardial ischemia/reperfusion, which may exacerbate myocardial injury by inducing endothelial cell apoptosis. We postulated that L-arginine may exacerbate TNF-induced endothelial cell apoptosis by enhancing peroxynitrite-mediated nitrative stress. Cultured human umbilical vein endothelial cells were either not treated (control) or treated with TNF alone or with TNF in the presence of L-arginine, the nonselective nitric oxide synthase inhibitor N (omega)-nitro-L-arginine (L-NNA), propofol (an anesthetic that scavenges peroxynitrite), or L-arginine plus propofol, respectively, for 24 hours. TNF increased intracellular superoxide and hydrogen peroxide production accompanied by increases of inducible nitric oxide synthase (iNOS) protein expression and nitric oxide production. This was accompanied by increased protein expression of nitrotyrosine, a fingerprint of peroxynitrite and an index of nitrative stress, and increased endothelial cell apoptosis. L-arginine did not enhance TNF-induced increases of superoxide and peroxynitrite production but further increased TNF-induced increase of nitrotyrosine production and exacerbated TNF-mediated cell apoptosis. L-NNA and propofol, respectively, reduced TNF-induced nitrative stress and attenuated TNF cellular toxicity. The L-arginine-mediated enhancement of nitrative stress and TNF toxicity was attenuated by propofol. Thus, under pathological conditions associated with increased TNF production, L-arginine supplementation may further exacerbate TNF cellular toxicity by enhancing nitrative stress.

Transient nitric oxide reduction induces permanent cardiac systolic dysfunction and worsens kidney damage in rats with chronic kidney disease.

Left ventricular systolic dysfunction (LVSD) in patients with chronic kidney disease (CKD) is associated with poorer prognosis. Because patients with CKD often exhibit progressively decreased nitric oxide (NO) availability and inhibition of NO production can reduce cardiac output, we hypothesized that loss of NO availability in CKD contributes to pathogenesis of LVSD. Subtotally nephrectomized (SNX) rats were treated with a low dose of the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA; 20 mg/l water; SNX+L-NNA) and compared with relevant control groups. To study permanent changes separate from hemodynamic effects, L-NNA was stopped after week 8 and rats were followed up to week 15, until blood pressure was similar in SNX+L-NNA and SNX groups. To study effects of NO depletion alone, a control group with high-dose L-NNA (L-NNA-High: 100 mg/l) was included. Mild systolic dysfunction developed at week 13 after SNX. In SNX+L-NNA, systolic function decreased by almost 50% already from week 4 onward, together with markedly reduced whole body NO production and high mortality. In L-NNA-High, LVSD was not as severe as in SNX+L-NNA, and renal function was not affected. Both LVSD and NO depletion were reversible in L-NNA-High after L-NNA was stopped, but both were persistently low in SNX+L-NNA. Proteinuria increased compared with rats with SNX, and glomerulosclerosis and cardiac fibrosis were worsened. We conclude that SNX+L-NNA induced accelerated and permanent LVSD that was functionally and structurally different from CKD or NO depletion alone. Availability of NO appears to play a pivotal role in maintaining cardiac function in CKD.

Tolerance to the cataleptic effect that follows repeated nitric oxide synthase inhibition may be related to functional enzymatic recovery.

Systemic or intra-striatal acute administration of nitric oxide synthase (NOS) inhibitors causes catalepsy in rodents. This effect disappears after sub-chronic treatment. The aim of the present study was to investigate if this tolerance is related to changes in the expression of NOS or dopamine-2 (D2) receptor or to a recovery of NOS activity. Male albino Swiss mice (25-30 g) received single or sub-chronic (once a day for 4 days) i.p. injections of saline or L-nitro-arginine (L-NOARG, 40 mg/kg), a non-selective inhibitor of neuronal nitric oxide synthase (nNOS). Twenty-four hours after the last injection, the animals were killed and their brains were removed for immunohistochemistry assay to detect the presence of nNOS or for 'in-situ' hybridisation study using (35)S-labeled oligonucleotide probe complementary to D2 receptor mRNA. The results were analysed by computerised densitometry. Independent groups of animals received the same treatment, but were submitted to the catalepsy test and had their brain removed to measure nitrite and nitrate (NOx) concentrations in the striatum. Acute administration of L-NOARG caused catalepsy that disappeared after sub-chronic treatment. The levels of NOx were significantly reduced after acute L-NOARG treatment. The decrease in NOx after drug injection suffered a partial tolerance after sub-chronic treatment. The catalepsy time after acute or sub-chronic treatment with L-NOARG was negatively (r = -0.717) correlated with NOx levels. Animals that received repeated L-NOARG injections also showed an increase in the number of nNOS-positive neurons in the striatum. No change in D2 receptor mRNA expression was found in the dorsal striatum, nucleus accumbens and substantia nigra. Together, these results suggest that tolerance to L-NOARG cataleptic effects do not depend on changes in D2 receptors. They may depend, however, on plastic changes in nNOS neurons resulting in partial recovery of NO formation in the striatum.

Acupuncture modulates mechanical responses of smooth muscle produced by transmural nerve stimulation in gastric antrum of genetically hyperglycemic rats.

Effects of acupuncture treatment on mechanical responses produced by transmural nerve stimulation (TNS) and acetylcholine (ACh) were investigated in circular smooth muscle preparations isolated from the antrum of the stomach of genetically hyperglycemic rats. While control rats had blood glucose levels of about 140 mg/dl, this was approximately tripled in the genetically hyperglycemic rats, but only doubled in the acupuncture treated genetically hyperglycemic rats. Antrum smooth muscle produced phasic contractions spontaneously, with a similar frequency and amplitude in the three groups of rats. Effects of atropine and Nomega-nitro-L-arginine (L-NA) on TNS-induced responses revealed that in the antrum smooth muscle of the control rats, cholinergic excitatory, non-adrenergic non-cholinergic excitatory (NANCE), nitrergic inhibitory and off-responses produced projections: the last projection was considered to be non-adrenergic non-cholinergic non-nitrergic (NANCNN) in nature. In genetically hyperglycemic rats, nitrergic and NANCNN projections were enhanced and NANCE projections were absent. Acupuncture treated genetically hyperglycemic rats showed a reduction of NANCNN projection and enhancement of cholinergic projection, with no alteration to nitrergic projection, but a recovery of NANCE projection. ACh elicited inhibitory responses at low concentrations (1-30 nM) and excitatory responses at high concentrations (100-300 nM), in the three groups of rats. L-NA converted the ACh-induced inhibitory responses to excitatory responses. Immunohistochemical examination indicated no significant difference in the distribution of c-Kit expressing cells in the antrum smooth muscle from the three groups of rats. The results indicated that in antral smooth muscle, hyperglycemia was associated with enhanced activity in nitrergic and NANCNN projections and attenuation of NANCE projections, and that acupuncture treatment caused both a reduced blood glucose level and attenuated NANCNN projections. In genetically hyperglycemic rats, cholinergic responses were enhanced by acupuncture, possibly due to the enhanced cholinergic projections, with no change in the sensitivity of postjunctional muscarinic receptors to ACh.

Nitric oxide modulates the influx of extracellular Ca2+ and actin filament organization during cell wall construction in Pinus bungeana pollen tubes.

Nitric oxide (NO) plays a key role in many physiological processes in plants, including pollen tube growth. Here, effects of NO on extracellular Ca(2+) flux and microfilaments during cell wall construction in Pinus bungeana pollen tubes were investigated. Extracellular Ca(2+) influx, the intracellular Ca(2+) gradient, patterns of actin organization, vesicle trafficking and cell wall deposition upon treatment with the NO donor S-nitroso-N-acetylpenicillamine (SNAP), the NO synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NNA) or the NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were analyzed. SNAP enhanced pollen tube growth in a dose-dependent manner, while L-NNA and cPTIO inhibited NO production and arrested pollen tube growth. Noninvasive detection and microinjection of a Ca(2+) indicator revealed that SNAP promoted extracellular Ca(2+) influx and increased the steepness of the tip-focused Ca(2+) gradient, while cPTIO and L-NNA had the opposite effect. Fluorescence labeling indicated that SNAP, cPTIO and L-NNA altered actin organization, which subsequently affected vesicle trafficking. Finally, the configuration and/or distribution of cell wall components such as pectins and callose were significantly altered in response to L-NNA. Fourier transform infrared (FTIR) microspectroscopy confirmed the changes in the chemical composition of walls. Our results indicate that NO affects the configuration and distribution of cell wall components in pollen tubes by altering extracellular Ca(2+) influx and F-actin organization.