Ethics statement: All procedures were approved by the Animal Experimentation Ethics Committee of RMIT University (approval number 0822) and conformed to the National Health and Medical Research Council of Australia code of practice for the care and use of animals for scientific purposes.
Induction of diabetes
Male 6–8 week old Wistar rats weighing approximately 200 g (Animal Resource Centre, Perth, WA, Australia) were randomly divided into two groups: normal and diabetic. Type 1 diabetes was induced by a single injection of streptozotocin (STZ, 50 mg/kg) into the tail vein after the rats were fasted overnight. The control groups received an equivalent volume of the vehicle (0.1 mol/l citrate buffer, pH 4.5) alone. Once the rats were rendered diabetic (blood glucose >25 mmol/l) the following week after STZ injection, all diabetic rats were maintained on a low insulin dose (4–5 IU, s.c., 3 injections per week, Protaphane, Novo Nordisk, NSW, Australia) to promote weight gain and reduce mortality 
. At the end of the experimental period, blood samples were obtained from the left ventricle and the glucose concentration and glycated haemogloblin (HbA1c
) were measured using a one touch glucometer (Roche, Sydney, NSW, Australia) and Micromat HbA1c
analyser (Biorad, Sydney, NSW, Australia) respectively.
After seven weeks of STZ-induced diabetes, the 2 groups of rats were further divided into 2 groups (Normal, Normal+DiOHF, Diabetic, Diabetic+DiOHF) receiving either vehicle (10% DMSO+90% peanut oil) or (DiOHF, 1mg/kg s.c. per day) for a period of 7 days. The last dose of DiOHF was administered at least 24 hours prior to the start of experimentation.
Isolation of mesenteric arteries
Eight weeks after STZ treatment, the rats were killed with pentobarbitone sodium (325 mg/kg, i.p, Virbac, Australia). The mesenteric arcade was isolated and immediately placed in ice cold Krebs bicarbonate solution (118 mmol/l NaCl, 4.7 mmol/l KCl, 1.18 mmol/l MgSO4
, 1.2 mmol/l KH2
, 25 mmol/l NaHCO3
, 11.1 mmol/l D-glucose, and 1.6 mmol/l CaCl2
) containing indomethacin (10 µmol/l), a non-selective cyclo-oxygenase (COX) inhibitor, to inhibit the synthesis of prostanoids. Our preliminary data suggested that there is no significant contribution of prostanoids to endothelium-dependent in mesenteric arteries from all groups of rats (data not shown). Small mesenteric arteries (third-order branch of the superior mesenteric artery, internal diameter ~300 µm) were isolated, cleared of fat and connective tissue, cut into 2 mm long rings and mounted on a Mulvany-Halpern myograph (model 610M, Danish Myo Technology, Aarhus, Denmark). After the arteries were mounted, the vessels were allowed to stabilize at zero tension for 15 min before normalisation. The passive tension-internal circumference was determined by stretching to achieve an internal circumference equivalent to 90% of that of the blood vessel under a transmural pressure of 100 mmHg 
. All experiments were performed at 37°C and the baths were bubbled with carbogen (95% O2
and 5% CO2
Assessment of vascular reactivity
Thirty minutes after normalization, vessels were maximally contracted with KPSS (123 mmol/l). After several washouts using normal Krebs solution, basal tension was regained. To assess the integrity of the endothelium, mesenteric arteries were precontracted to ~50–60% of the KPSS response with phenylephrine (0.1–3 µmol/l) and a high concentration of acetylcholine (ACh, 10 µmol/l) was used to relax the artery rings. ACh-induced relaxation was greater than 80% of the precontracted tone in all cases, indicating that the endothelium was functionally intact. After further washouts, arteries were again precontracted to a similar level using phenylephrine (0.1–3 µmol/l) or in some cases 30 mmol/l K+, and cumulative concentration-response curves to ACh (0.1 nmol/l–10 µmol/l) and SNP (0.01 nmol/l–10 µmol/l) were determined. In addition, responses to ACh and SNP were examined after 20 minutes incubation with different combinations of L-NNA (100 µmol/l), a non-selective nitric oxide synthase (NOS) inhibitor, ODQ (10 µmol/l), a soluble guanylate cyclase (sGC) inhibitor, TRAM-34 (1 µmol/l), a selective blocker of the intermediate-conductance calcium-activated K+ channel (IKCa or KCa3.1), ibtx (100 nmol/l), a selective blocker of maxi KCa and apamin (1 µmol/l), a SKCa inhibitor.
To evaluate the constrictor reactivity, cumulative concentration-response curves to ET-1 (0.1 nmol/l–0.1 µmol/l) were constructed in the absence of indomethacin.
Assessment of basal release of NO in mesenteric arteries
In another separate set of experiments, the effect of diabetes on basal levels of NO release was also examined in the absence of indomethacin through the addition of L-NNA (100 µmol/l) in endothelium-intact rings precontracted with phenylephrine (10–100 nmol/l) to approximately ~20% KPSS. Under those conditions a contractile response to L-NNA was considered to reflect the level of the basal release of NO 
Western blots were performed as described previously 
with the following modifications. Endothelium-intact mesenteric arteries from 2 animals from the same treatment group were pooled and considered as n
1. Equal amounts of protein homogenate were subjected to SDS-PAGE and western blot analysis with mouse/rabbit primary antibodies (all 1
1000, overnight, 4°C) against endothelial NO synthase (eNOS), inducible-NOS, Nox2 (all BD Transduction Laboratories, Lexington, KY, USA). To normalize for the amount of protein, membranes were reprobed with a loading control antibody (actin). All proteins were detected by enhanced chemiluminescence (Amersham, GE Healthcare, Sydney, NSW, Australia) after incubation with anti-mouse/rabbit secondary antibody (Millipore, Billerica, MA, USA) for 1 hour at room temperature (1
2000). All protein bands were quantified by densitometry (Biorad Chemidoc, Sydney, NSW, Australia) and expressed as a ratio of the loading control. To investigate eNOS homodimer formation in the tissue, a non-boiled sample was resolved by 6% SDS-PAGE at 4°C 
, and the membranes were probed and visualized as described above.
Measurement of ROS in mesenteric artery
Two different methods of ROS measurement were employed. Superoxide production in the mesenteric artery was measured using L-012 as previously described 
. Mesenteric arteries were incubated at 37°C for 30 minutes in Krebs-HEPES buffer either alone, in the presence of apocynin, a ROS scavenger 
(300 µmol/l) or L-NNA (100 µmol/l), which determines NOS-derived superoxide production. 300 µL of Krebs-HEPES buffer, containing L-012 (100 µmol/l, Wako Pure Chemicals, Osaka, Japan) and the appropriate treatments were placed into a 96-well Optiplate, which was loaded into a Polarstar Optima photon counter (BMG Labtech, Melbourne, VIC, Australia) to measure background photon emission at 37°C. After background counting was completed, a single ring segment of mesenteric artery was added to each well and photon emission was re-counted. Reactive oxygen species (H2
) were measured with DCFDA 
with the following modification. Rings of mesenteric artery were loaded with DCFDA solution (10 µmol/l) for 60 min, followed by 3 washes in Krebs-HEPES buffer. Background fluorescence was measured after excitation at 485 nm and emission at 520 nm. After being rinsed three times with Krebs buffer to remove excess probe, a single segment of the mesenteric artery was added to each well, and fluorescence intensity was recounted. The luminescence and fluorescence counts were normalized with dry tissue weight.
NADPH oxidase activity
NADPH oxidase-driven superoxide production in the mesenteric artery was measured using lucigenin-enhanced chemiluminescence. Mesenteric arteries were preincubated for 45 min at 37°C in Krebs–HEPES buffer containing diethylthiocarbamic acid (1 mmol/l), to inactivate superoxide dismutase, and NADPH (100 µmol/l) as a substrate for NADPH oxidase, and either alone or in the presence of diphenylene iodonium (5 µmol/l), as a flavoprotein inhibitor that inhibits NADPH oxidase. 300 µL of Krebs-HEPES buffer containing lucigenin (5 µmol/l) and the appropriate treatments were placed into a 96-well Optiplate, and superoxide production was measured and quantified as previously described 
All drugs were purchased from Sigma-Aldrich (St Louis, MO, USA), except for acetylcholine perchlorate (BDH Chemicals, Poole, Dorset, UK), DiOHF (Indofine Chemicals, Hillsborough, NJ, USA) and ODQ (Cayman Chemical, Ann Arbor, MI, USA). All drugs were all dissolved in distilled water, with the exception of indomethacin, which was dissolved in 0.1 mol/l sodium carbonate, L-NNA, which was dissolved in 0.1 mol/l sodium bicarbonate, ODQ and TRAM-34, which were dissolved in dimethyl sulfoxide (DMSO).
All results are expressed as the mean±s.e.m., n represents the number of animals per group or the number of assays when tissue from animals was pooled. Concentration-response curves from rat isolated mesenteric arteries were computer fitted to a sigmoidal curve using nonlinear regression (Prism version 5.0, GraphPad Software, San Diego, CA, USA) to calculate the sensitivity of each agonist (pEC50). Maximum relaxation (Rmax) to ACh or SNP was measured as a percentage of precontraction to phenylephrine. Group pEC50 and Rmax values were compared by one-way ANOVA with post-hoc analysis using Dunnett's test or Bonferroni's selected comparison test (normal vs. normal+DiOHF, normal vs. diabetic, normal+DiOHF vs. diabetic+DiOHF and diabetic vs. diabetic+DiOHF) as appropriate. P<0.05 was considered statistically significant.