Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway.

Adipose tissue is a dynamic endocrine organ, secreting a plethora of adipokines which play a key role in regulating metabolic homeostasis and other physiological processes. An altered adipokine secretion profile from adipose tissue depots has been associated with obesity and related cardio-metabolic diseases. Asprosin is a recently described adipokine that is released in response to fasting and can elicit orexigenic and glucogenic effects. Circulating asprosin levels are elevated in a number of cardio-metabolic diseases, including obesity and type 2 diabetes. In vitro studies have reported pro-inflammatory effects of asprosin in a variety of tissues. The present study aimed to further elucidate the role of asprosin in inflammation by exploring its potential effect(s) in THP-1 macrophages. THP-1 monocytes were differentiated to macrophages by 48 h treatment with dihydroxyvitamin D3. Macrophages were treated with 100 nM recombinant human asprosin, 100 ng/mL lipopolysaccharide (LPS), and 10 µM caffeic acid phenethyl ester (CAPE; an inhibitor of NFκB activation) or 1 µM TAK-242 (a Toll-like receptor 4, TLR4, inhibitor). The expression and secretion of pertinent pro-inflammatory mediators were measured by qPCR, Western blot, ELISA and Bioplex. Asprosin stimulation significantly upregulated the expression and secretion of the pro-inflammatory cytokines: tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß), IL-8 and IL-12 in vitro. This pro-inflammatory response in THP-1 macrophages was partly attenuated by the treatments with CAPE and was significantly inhibited by TAK-242 treatment. Asprosin-induced inflammation is significantly counteracted by TLR4 inhibition in THP-1 macrophages, suggesting that asprosin exerts its pro-inflammatory effects, at least in part, via the TLR4 signalling pathway.

Functional cardiac orexin receptors: role of orexin-B/orexin 2 receptor in myocardial protection.

Orexins/hypocretins exert cardiovascular effects which are centrally mediated. In the present study, we tested whether orexins and their receptors may also act in an autocrine/paracrine manner in the heart exerting direct effects. Quantitative reverse transcription-PCR (RT-PCR), immunohistochemical and Western blot analyses revealed that the rat heart expresses orexins and orexin receptors (OXR). In isolated rat cardiomyocytes, only orexin-B (OR-B) caused an increase in contractile shortening, independent of diastolic or systolic calcium levels. A specific orexin receptor-2 (OX2R) agonist ([Ala11, d-Leu15]-Orexin B) exerted similar effects as OR-B, whereas a specific orexin receptor-1 (OX1R) antagonist (SB-408124) did not alter the responsiveness of OR-B. Treatment of the same model with OR-B resulted in a dose-dependent increase in myosin light chain and troponin-I (TnI) phosphorylation. Following ischaemia/reperfusion in the isolated Langendorff perfused rat heart model, OR-B, but not OR-A, exerts a cardioprotective effect; mirrored in an in vivo model as well. Unlike OR-A, OR-B was also able to induce extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2) and Akt phosphorylation in rat myocardial tissue and ERK1/2 phosphorylation in human heart samples. These findings were further corroborated in an in vivo rat model. In human subjects with heart failure, there is a significant negative correlation between the expression of OX2R and the severity of the disease clinical symptoms, as assessed by the New York Heart Association (NYHA) functional classification. Collectively, we provide evidence of a distinct orexin system in the heart that exerts a cardioprotective role via an OR-B/OX2R pathway.

Circulatory changes of the novel adipokine adipolin/CTRP12 in response to metformin treatment and an oral glucose challenge in humans.

OBJECTIVE: Adipolin/CTRP12 is a novel adipokine with anti-inflammatory and glucose-lowering properties in rodents. We sought to investigate the effects of metformin treatment (850 mg twice daily for 6 months) and a 2 h 75 g oral glucose tolerance test (OGTT) on serum adipolin concentrations in humans. DESIGN: Cross-sectional study [PCOS (n = 83) and control (n = 39) subjects]. Serum adipolin was measured by ELISA. Metformin treatment (850 mg twice daily for 6 months) was offered to all women with PCOS, 34 women participated but 21 women completed 6 months of metformin therapy. Reasons for subjects not completing the study were nausea and gastrointestinal side effects (n = 4), pregnancies (n = 5), noncompliance (n = 2) and loss of contact (n = 2). RESULTS: Metformin treatment (850 mg twice daily for 6 months) substantially increased serum adipolin concentrations (P < 0·05) in women with polycystic ovary syndrome (PCOS), a pro-inflammatory state associated with obesity, diabetes, dyslipidaemia and atherosclerosis. Furthermore, changes in waist-hip ratio, glucose, triglycerides, CRP and carotid intima media thickness showed significant negative associations with changes in adipolin levels (P < 0·05, P < 0·01); in multiple regression analyses, only changes in glucose were predictive of changes in adipolin levels (ß = -0·570, P = 0·009). Serum adipolin decreased significantly in response to the OGTT in PCOS and control subjects at 90 min (P < 0·05) and 120 min (P < 0·01). CONCLUSIONS: Adipolin and/or novel pharmacologic agents that increase adipolin's circulating concentrations might constitute a novel approach in the treatment of insulin resistant states.

NUCB2/Nesfatin-1 Reduces Obesogenic Diet Induced Inflammation in Mice Subcutaneous White Adipose Tissue.

BACKGROUND: Excess adipose tissue accumulation and obesity are characterised by chronic, low-grade, systemic inflammation. Nestfatin-1 is a neuropeptide derived from the precursor protein nucleobindin-2 (NUCB2), which was initially reported to exert anorexigenic effects. The present study aimed to investigate the effects of an obesogenic diet (OD; high-fat, high-sugar) in NUCB2 knockout (KO) mice and of nesfatin-1 treatment in LPS-stimulated 3T3-L1 preadipocytes. METHODS: Subcutaneous white adipose tissue (Sc-WAT) samples from wild type (WT) and NUCB2 KO mice that were fed a normal diet (ND), or the OD for 12 weeks were used for RNA and protein extraction, as well as immunohistochemistry. 3T3-L1 cells were treated with 100 nM nesfatin-1 during differentiation and stimulated with 1 µg/mL LPS for measuring the expression and secretion of pro-inflammatory mediators by qPCR, western blotting, immunofluorescence, Bioplex, and ELISA. RESULTS: Following the OD, the mRNA, protein and cellular expression of pro-inflammatory mediators (Tnfα, Il-6, Il-1ß, Adgre1, Mcp1, TLR4, Hmbgb1 and NF-kB) significantly increased in the ScWAT of NUCB2 KO mice compared to ND controls. Adiponectin and Nrf2 expression significantly decreased in the ScWAT of OD-fed NUCB2 KO, without changes in the OD-fed WT mice. Furthermore, nesfatin-1 treatment in LPS-stimulated 3T3-L1 cells significantly reduced the expression and secretion of pro-inflammatory cytokines (Tnfα, Il-6, Il-1ß, Mcp1) and hmgb1. CONCLUSION: An obesogenic diet can induce significant inflammation in the ScWAT of NUCB2 KO mice, involving the HMGB1, NRF2 and NF-kB pathways, while nesfatin-1 reduces the pro-inflammatory response in LPS-stimulated 3T3-L1 cells. These findings provide a novel insight into the metabolic regulation of inflammation in WAT.

Direct evidence of nitric oxide release from neuronal nitric oxide synthase activation in the left ventricle as a result of cervical vagus nerve stimulation.

Information regarding vagal innervation in the cardiac ventricle is limited and the direct effect of vagal stimulation on ventricular myocardial function is controversial. We have recently provided indirect evidence that the anti-fibrillatory effect of vagus nerve stimulation on the ventricle is mediated by nitric oxide (NO). The aim of this study was to provide direct evidence for the release of nitric oxide in the cardiac ventricle during stimulation of the efferent parasympathetic fibres of the cervical vagus nerve. The isolated innervated rabbit heart was employed with the use of the NO fluorescent indicator 4,5-diaminofluorescein diacetate (DAF-2 DA) during stimulation of the cervical vagus nerves and acetylcholine perfusion in the absence and presence of the non-specific NO synthase inhibitor NG-nito-L-arginine (L-NNA) and the neuronal NO synthase selective inhibitor 1-(2-trifluormethylphenyl)imidazole (TRIM). Using the novel fluorescence method in the beating heart, we have shown that NO-dependent fluorescence is increased by 0.92 +/- 0.26, 1.20 +/- 0.30 and 1.91 +/- 0.27% (during low, medium and high frequency, respectively) in the ventricle in a stimulation frequency-dependent manner during vagus nerve stimulation, with comparable increases seen during separate stimulation of the left and right cervical vagus nerves. Background fluorescence is reduced during perfusion with L-NNA and the increase in fluorescence during high frequency vagal stimulation is inhibited during perfusion with both L-NNA (1.97 +/- 0.35% increase before L-NNA, 0.00 +/- 0.02% during L-NNA) and TRIM (1.78 +/- 0.18% increase before TRIM, -0.11 +/- 0.08% during TRIM). Perfusion with 0.1 microM acetylcholine increased NO fluorescence by 0.76 +/- 0.09% which was blocked by L-NNA (change of 0.00 +/- 0.03%) but not TRIM (increase of 0.82 +/- 0.21%). Activation of cardiac parasympathetic efferent nerve fibres by stimulation of the cervical vagus is associated with NO production and release in the ventricle of the rabbit, via the neuronal isoform of nitric oxide synthase.

Empagliflozin improves primary haemodynamic parameters and attenuates the development of atherosclerosis in high fat diet fed APOE knockout mice.

The effects of long-term treatment with empagliflozin on biochemical and immunohistochemical markers related to atherosclerosis and atherosclerosis development in the aorta of apolipoprotein E knockout [Apo-E (-/-)] mice were evaluated in this study. Empagliflozin-treated mice had lower total cholesterol (P < 0.05), fasting glucose (P < 0.01), heart rate (P < 0.01) and diastolic blood pressure (DBP) (P < 0.05) compared to controls. Histomorphometry revealed reduced atherosclerotic lesion progress approaching statistical significance (P = 0.06) and approximately 50% wider lumen area for the Empagliflozin treated mice group. Although empagliflozin significantly reduced Vcam-1 and Mcp-1 (P < 0.05, P < 0.01, respectively) and marginally induced Timp-1 and Timp-2 mRNA expression (P < 0.08, P = 0.1 respectively), immunohistochemistry revealed a marginal reduction in VCAM-1 and MMP-9 (P = 0.1) without affecting the expression of TIMP-2 and MCP-1 in atherosclerotic lesions. Empagliflozin improves primary haemodynamic parameters and attenuates the progression of atherosclerosis by reducing hyperlipidemia and hyperglycemia, while direct actions in aorta vessel mediated via SGLT-1 are strongly hypothesized.

Autonomic modulation of electrical restitution, alternans and ventricular fibrillation initiation in the isolated heart.

OBJECTIVE: Abnormal autonomic nerve activity is a strong prognostic marker for ventricular arrhythmias but the mechanisms underlying the autonomic modulation of ventricular fibrillation (VF) initiation are poorly understood. We examined the effects of direct sympathetic (SS) and vagus (VS) nerve stimulation on electrical restitution, alternans and VF threshold (VFT) in a novel isolated rabbit heart preparation with intact dual autonomic innervation. METHODS: Monophasic Action Potentials (MAPs) were recorded from a left ventricular epicardial site on innervated, isolated rabbit hearts (n=16). Standard restitution, effective refractory period (ERP), electrical alternans and VFT were measured at baseline and during SS and VS separately. RESULTS: The restitution curve was shifted downwards and made steeper with SS whilst VS caused an upward shift and a flattening of the curve. The maximum slope of restitution was increased from 1.30+/-0.10 at baseline to 1.86+/-0.17 (by 45+/-12%, P<0.01) with SS and decreased to 0.69+/-0.10 (by 51+/-6%, P<0.001) with VS. ERP was decreased from 127.3+/-2.5 ms to 111.8+/-1.8 ms with SS (by 12+/-2%, P<0.001) and increased to 144.0+/-2.2 ms with VS (by 13+/-2%, P<0.001). VFT was decreased from 4.7+/-0.6 mA to 1.9+/-0.5 mA with SS (by 64+/-5%, P<0.001) and increased to 8.7+/-1.1 mA with VS (by 89+/-14%, P<0.0005). There was a significant inverse relationship between the maximum slope of restitution and VFT (r=-0.63, P<0.0001). When compared with baseline, SS caused electrical alternans at longer pacing cycle lengths (139.0+/-8.4 vs. 123.0+/-7.8 ms, P<0.01) with greater degree of alternans (32.5+/-9.9 vs. 15.4+/-3.2%, P<0.05). It also caused a wider range of cycle lengths where alternans occurred (53.0+/-6.2 vs. 41.0+/-7.0 ms, P<0.05) whilst vagus nerve stimulation shortened this range (33.0+/-7.3 ms, P<0.001). CONCLUSIONS: Sympathetic stimulation increased maximum slope of restitution and electrical alternans but decreased ERP and VF threshold whilst vagus nerve stimulation had opposite effects. The interaction between action potential duration and beat-to-beat interval may play an important role in the autonomic modulation of VF initiation.

Short-Chain Fatty Acid Acetate Stimulates Adipogenesis and Mitochondrial Biogenesis via GPR43 in Brown Adipocytes.

Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gßγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)ßγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43.

Nesfatin-1 inhibits proliferation and enhances apoptosis of human adrenocortical H295R cells.

NUCB2/nesfatin and its proteolytically cleaved product nesfatin-1 are recently discovered anorexigenic hypothalamic neuroproteins involved in energy homeostasis. It is expressed both centrally and in peripheral tissues, and appears to have potent metabolic actions. NUCB2/nesfatin neurons are activated in response to stress. Central nesfatin-1 administration elevates circulating ACTH and corticosterone levels. Bilateral adrenalectomy increased NUCB2/nesfatin mRNA levels in rat paraventricular nuclei. To date, studies have not assessed the effects of nesfatin-1 stimulation on human adrenocortical cells. Therefore, we investigated the expression and effects of nesfatin-1 in a human adrenocortical cell model (H295R). Our findings demonstrate that NUCB2 and nesfatin-1 are expressed in human adrenal gland and human adrenocortical cells (H295R). Stimulation with nesfatin-1 inhibits the growth of H295R cells and promotes apoptosis, potentially via the involvement of Bax, BCL-XL and BCL-2 genes as well as ERK1/2, p38 and JNK1/2 signalling cascades. This has implications for understanding the role of NUCB2/nesfatin in adrenal zonal development. NUCB2/nesfatin may also be a therapeutic target for adrenal cancer. However, further studies using in vivo models are needed to clarify these concepts.

Novel insights into the cardio-protective effects of FGF21 in lean and obese rat hearts.

AIMS: Fibroblast growth factor 21 (FGF21) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF21 on cardio-protection in obese and lean hearts in response to ischemia. METHODS AND RESULTS: FGF21, FGF21-receptor 1 (FGFR1) and beta-Klotho (ßKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF21 was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-ßKlotho components. Cardiac-FGF21 expression and secretion were increased in response to global ischemia. In contrast ßKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF21 activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF21 administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF21 induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF21 was reduced in obesity, although the cardiac expression profiles and circulating FGF21 levels were increased. CONCLUSION: In an ex vivo Langendorff system, we show that FGF21 induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF21-induced cardiac effects in obesity and ischemia.

Metformin increases the novel adipokine adipolin/CTRP12: role of the AMPK pathway.

Adipolin is a novel adipokine with anti-inflammatory and glucose-lowering properties. Lower levels of adipolin are found in obese and diabetic mice. Polycystic ovary syndrome (PCOS) is a pro-inflammatory state associated with obesity and diabetes. To date, there are no human studies on adipolin. Therefore, we measured serum (ELISA) and adipose tissue adipolin mRNA expression (RT-PCR) and protein concentrations (western blotting) in PCOS and control subjects. We also investigated the ex vivo effect of glucose and metformin on adipolin protein production in human subcutaneous adipose tissue explants. We report novel data that serum and subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were significantly lower in women with PCOS compared with control subjects. Furthermore, Spearman's rank analysis showed that serum adipolin concentrations were significantly negatively correlated with BMI, waist-to-hip ratio, and glucose (P<0.05). However, when subjected to multiple regression analysis, none of these variables were predictive of serum adipolin concentrations (P>0.05). Also, subcutaneous adipose tissue adipolin mRNA expression and protein concentrations were only significantly negatively correlated with glucose (P<0.05). No significant correlations were found with omental adipose tissue adipolin mRNA expression and protein concentrations (P>0.05). Moreover, glucose profoundly reduced and metformin significantly increased adipolin protein production in human adipose tissue explants respectively. Importantly, metformin's effects appear to be via the AMP-activated protein kinase signaling pathway.

Metformin increases the novel adipokine cartonectin/CTRP3 in women with polycystic ovary syndrome.

CONTEXT: Recently cartonectin was reported as a novel adipokine, with lower levels in diet-induced obese mice, glucose-lowering effects, and antiinflammatory and cardioprotective properties. Polycystic ovary syndrome (PCOS) is a proinflammatory state associated with obesity, diabetes, dyslipidemia, and cardiovascular complications. OBJECTIVES: The objective of the study was to investigate cartonectin levels and regulation in sera and adipose tissue (AT) as well as the effects of metformin of women with PCOS and control subjects. DESIGN: This was a cross-sectional study [PCOS (n = 83) and control (n = 39) subjects]. Real-time PCR and Western blotting were used to assess mRNA and protein expression of cartonectin. Serum cartonectin was measured by an ELISA. RESULTS: Serum and omental adipose tissue cartonectin were significantly lower in women with PCOS compared with control subjects (P < .05 and P < .01, respectively). Furthermore, cartonectin showed a significant negative association with body mass index, waist to hip ratio, glucose, insulin, total cholesterol, low-density lipoprotein-cholesterol, triglycerides, High sensitivity C-reactive protein (hs-CRP) and intima-media thickness (P < .05 and P < .01, respectively); in multiple regression analyses, triglycerides (P =.040) and hs-CRP (P = .031) were predictive of cartonectin levels (P < .05). After 6 months of metformin treatment, there was an associated increase in serum cartonectin (P < .05). Importantly, changes in hs-CRP were significantly negatively correlated with changes in serum cartonectin (P = .033). Finally, cartonectin protein production and secretion into conditioned media were significantly increased by metformin in control human omental AT explants (P < .05). CONCLUSIONS: Serum and omental AT cartonectin are lower in women with PCOS. Metformin treatment increases serum cartonectin levels in these women and in omental AT explants.

G-protein coupled estrogen receptor 1 expression in rat and human heart: Protective role during ischaemic stress.

G-protein coupled estrogen receptor 1, GPER, formerly known as GPR30, is a seven transmembrane domain receptor that mediates rapid estrogen responses in a wide variety of cell types. To date, little is known about the role of GPER during ischaemia/reperfusion injury. In this study, we report both mRNA and protein expression of GPER in the rat and human heart. The role of GPER in estrogen protection against ischaemic stress in the rat heart was also assessed using the isolated Langendorff system. Pre-treatment with 17beta-estradiol (E2) significantly decreased infarct size, (61.48+/-2.2% to 27.92+/-2.9% (P<0.001). Similarly, treatment with the GPER agonist G1 prior to 30-min global ischaemia followed by 120-min reperfusion significantly reduced infarct size from 61.48+/-2.2% to 23.85+/-3.2% (P<0.001), whilst addition of GPR30 antibody, abolished the protective effect of G1 (infarct size: 55.42+/-1.3%). The results suggest that GPER under cardiac stress exerts direct protection in the heart and may serve as a potential therapeutic target for cardiac drug therapy.

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart.

4,5-Diaminofluorescein (DAF-2) has been used to measure nitric oxide (NO) activity from a variety of preparations. The aim of this study was to develop a method to assess changes in NO fluorescence using DAF-2 in isolated rabbit hearts (2.0-2.5 kg, n = 8). Hearts were perfused in constant flow Langendorff mode and instrumented to record aortic perfusion pressure, left ventricular pressure and left ventricular epicardial fluorescence using a bifurcated light guide at excitation wavelengths of 470 +/- 10, 480 +/- 10, 490 +/- 10 and 500 +/- 10 nm collected at 535 nm. DAF-2 DA was loaded using a single bolus 150-microl (1 micromol) injection. Changes in NO-dependent fluorescence were determined using the NO donor sodium nitroprusside (SNP: 100 microM), NO-dependent vasodilator bradykinin (BK: 100 microM) and non-specific NO synthase inhibitor NG-nitro-L-arginine (LNA: 200 microM) before and after loading hearts with DAF-2 DA. Before loading, these agents did not alter epicardial fluorescence. After loading, SNP, BK and LNA produced a consistent change in each excitation wavelength. Together, this suggests that change in fluorescence represents changes in the level of NO. SNP and BK increased whilst LNA significantly decreased left ventricular epicardial NO-dependent fluorescence. At the excitation wavelength of 490 nm, SNP and BK increased fluorescence by 104.7 +/- 18.7 mV (1.1 +/- 0.2%) and 150.7 +/- 26.1 mV (1.5 +/- 0.3%) respectively, whilst LNA significantly decreased fluorescence by 90.3 +/- 17.0 mV (-0.9 +/- 0.2%). Changing the rate of aortic perfusion did not alter fluorescence suggesting that changes in aortic perfusion pressure per se do not contribute to the changes in DAF-2 fluorescence seen with SNP, BK or LNA. Our data suggest that DAF-2 DA is a useful fluorescence indicator for measuring NO activity in isolated hearts.

Nitric oxide mediates the vagal protective effect on ventricular fibrillation via effects on action potential duration restitution in the rabbit heart.

We have previously shown that direct vagus nerve stimulation (VNS) reduces the slope of action potential duration (APD) restitution while simultaneously protecting the heart against induction of ventricular fibrillation (VF) in the absence of any sympathetic activity or tone. In the current study we have examined the role of nitric oxide (NO) in the effect of VNS. Monophasic action potentials were recorded from a left ventricular epicardial site on innervated, isolated rabbit hearts (n = 7). Standard restitution, effective refractory period (ERP) and VF threshold (VFT) were measured at baseline and during VNS in the presence of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NA, 200 microm) and during reversing NO blockade with L-arginine (L-Arg, 1 mm). Data represent the mean +/- S.E.M. The restitution curve was shifted upwards and became less steep with VNS when compared to baseline. L-NA blocked the effect of VNS whereas L-Arg restored the effect of VNS. The maximum slope of restitution was reduced from 1.17 +/- 0.14 to 0.60 +/- 0.09 (50 +/- 5%, P < 0.0001) during control, from 0.98 +/- 0.14 to 0.93 +/- 0.12 (2 +/- 10%, P = NS) in the presence of L-NA and from 1.16 +/- 0.17 to 0.50 +/- 0.10 (41 +/- 9%, P = 0.003) with L-Arg plus L-NA. ERP was increased by VNS in control from 119 +/- 6 ms to 130 +/- 6 ms (10 +/- 5%, P = 0.045) and this increase was not affected by L-NA (120 +/- 4 to 133 +/- 4 ms, 11 +/- 3%, P = 0.0019) or L-Arg with L-NA (114 +/- 4 to 123 +/- 4 ms, 8 +/- 2%, P = 0.006). VFT was increased from 3.0 +/- 0.3 to 5.8 +/- 0.5 mA (98 +/- 12%, P = 0.0017) in control, 3.4 +/- 0.4 to 3.8 +/- 0.5 mA (13 +/- 12%, P = 0.6) during perfusion with L-NA and 2.5 +/- 0.4 to 6.0 +/- 0.7 mA (175 +/- 50%, P = 0.0017) during perfusion with L-Arg plus L-NA. Direct VNS increased VFT and flattened the slope of APD restitution curve in this isolated rabbit heart preparation with intact autonomic nerves. These effects were blocked using L-NA and reversed by replenishing the substrate for NO production with L-Arg. This is the first study to demonstrate that NO plays an important role in the anti-fibrillatory effect of VNS on the rabbit ventricle, possibly via effects on APD restitution.