EFFECTS OF EXPERIMENTALLY INDUCED DIABETES MELLITUS ON PHARMACOLOGICALLY AND ELECTRICALLY ELICITED MYOMETRIAL CONTRACTILITY

• 1 Diabetes is one of the most frequent complications of gestation, affecting approximately 7% of pregnancies. However, little is known about its effects on electrically and pharmacologically stimulated myometrial contractility. The aim of the present study was to investigate the consequences of streptozotocin (STZ)‐induced diabetes on: (i) electrical field stimulation (EFS)‐evoked contraction of isolated uterine rings as a function of gestational age; and (ii) the uterotonic and tocolytic actions of α‐ and β‐adrenoceptor stimulation, respectively. The effects of oxytocin in late pregnancy were also investigated.
• 2 During pregnancy, EFS‐evoked contractions of isolated uterine rings from intact rats declined, whereas isolated uterine rings from diabetic rats exhibited continuously low sensitivity to EFS.
• 3 In non‐pregnant rats, diabetes resulted in increased noradrenaline‐mediated contractility and a decreased relaxation response to terbutaline. At the mRNA level, diabetes enhanced the expression of α_1B‐adrenoceptors in non‐pregnant rats from 14.65 to 18.39 µg/mL (P < 0.05), whereas the expression of α_1D‐adrenoceptors decreased (from 42.87 to 35.67 µg/mL; P < 0.05). During pregnancy, the responses to these sympathomimetics did not differ between diabetic and intact rats.
• 4 In late pregnancy (on Days 15 and 21), oxytocin caused greater maximum contractility of uterine rings from diabetic rats without affecting the EC₅₀. In addition, on Day 15 of pregnancy, the expression of oxytocin receptors in the myometrium of diabetic rats was higher than that in intact rats.
• 5 The results of the present study indicate that experimental diabetes facilitates gestation‐induced denervation and increases myometrial sensitivity to oxytocin in late pregnancy. If similar mechanisms operate in humans, this could contribute to a tendency to premature uterine contractions in diabetes‐complicated pregnancies.

     

Antihypertensive methyldopa,labetalol, hydralazine,and clonidine reversed tumour necrosis factor‐α inhibited endothelial nitric oxide synthase expression in endothelial‐trophoblast cellular networks

Medications used to control hypertension in pregnancy also improve trophoblast and endothelial cellular interaction in vitro. Tumour necrosis factor‐α (TNF‐α) inhibits trophoblast and endothelial cellular interactions and simultaneously decreases endothelial nitric oxide synthase (eNOS) expression. This study investigated whether antihypertensive medications improved these cellular interactions by modulating eNOS and inducible nitric oxide synthase (iNOS) expression. Human uterine myometrial microvascular endothelial cells (UtMVECs) were pre‐incubated with (or without) low dose TNF‐α (0.5 ng/mL) or TNF‐α plus soluble fms‐like tyrosine kinase‐1 (sFlt‐1) (100 ng/mL). The endothelial cells were cultured on Matrigel. After endothelial cellular networks appeared, trophoblast derived HTR‐8/SVneo cells were co‐cultured in the presence of clinically relevant doses of methyldopa, labetalol, hydralazine or clonidine for 24 hours. Cells were retrieved from the Matrigel to extract mRNA and eNOS and iNOS expression were examined by quantitative PCR. Methyldopa, labetalol, hydralazine and clonidine reversed the inhibitory effect of TNF‐α on eNOS mRNA expression. After pre‐incubating endothelial cells with TNF‐α and sFlt‐1, all the medications except methyldopa lost their effect on eNOS mRNA expression. In the absence of TNF‐α, antihypertensive medications did not change eNOS expression. The mRNA expression of iNOS was not affected by TNF‐α or any medications. This study shows that selected antihypertensive medications used in the treatment of hypertension in pregnancy increase eNOS expression in vitro when induced by the inflammatory TNF‐α. The anti‐angiogenic molecule sFlt‐1 may antagonise the potential benefit of these medications by interfering with the NOS pathway.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

REDUCED LUNG WATER TRANSPORT RATE ASSOCIATED WITH DOWNREGULATION OF AQUAPORIN-1 AND AQUAPORIN-5 IN AGED MICE

• 1 The purpose of the present study was to examine lung water transport properties and the expression and regulation of the alveolar endothelial water channel aquaporin (AQP)‐1 and the epithelial water channel AQP‐5 in aged mouse lung using gene expression analysis and water permeability measurements.
• 2 In aged (20–24‐month‐old) mice, AQP‐1 and AQP‐5 mRNA expression decreased by 55.5 and 50.3%, respectively, compared with that in young (8–10‐week‐old) mice (P < 0.01). In addition, AQP‐1 and AQP‐5 protein expression decreased in aged mice by 36.9 and 44.6%, respectively, compared with that in young mice (P < 0.01).
• 3 The osmotically driven water transport rate between the airspace and capillary compartments was reduced by 31.7% in aged mice compared with young mice (2.8 ± 0.3 vs 4.1 ± 0.3 mg/s, respectively; P < 0.01). The hydrostatically driven lung water accumulation rate in response to a 10 cmH₂O increase in pulmonary artery pressure was also reduced in aged mice by 21.9% compared with young mice (0.32 ± 0.06 vs 0.41 ± 0.04 mg/s, respectively; P < 0.01).
• 4 There was a 62.7% decrease in serum glucocorticoids in aged mice compared with young mice (67.6 ± 26.8 vs 181.3 ± 44.4 nmol/L, respectively; P < 0.01). In vivo administration of dexamethasone (4 mg/kg) for 5 consecutive days to aged mice increased lung AQP‐1 mRNA and protein expression by 2.1 ± 0.1 fold (P < 0.01) and 1.8 ± 0.2 fold (P < 0.01), respectively. Accordingly, osmotically and hydrostatically driven water transport rates increased by 35.6% (P < 0.01) and 31.2% (P < 0.01), respectively.
• 5 The present study provides the first evidence of altered lung water transport associated with downregulation of AQPs in aged lung. Blood glucocorticoid hormone levels are important to maintain normal AQP‐1 expression in the lung microvascular endothelium. Corticosteroid‐induced AQP‐1 upregulation may contribute to the role of corticosteroids in accelerating oedema clearance in aged lung.

     

OBESITY INDUCED RENAL OXIDATIVE STRESS CONTRIBUTES TO RENAL INJURY IN SALT-SENSITIVE HYPERTENSION

• 1 In the present study, we determined the role of hypertension, oxidative stress and inflammation on kidney damage in a rodent model of obesity and diabetes. Hypertension was induced in male obese (db/db) mice and lean (db/m) mice by implantation of deoxycorticosterone acetate (DOCA) pellets and mice were allowed to drink water containing 1% salt. Mice were divided into six groups as follows: obese and lean control, obese and lean 1% salt (salt) and obese and lean DOCA plus 1% salt (DOCA‐salt).
• 2 Blood pressure was significantly increased in lean and obese DOCA‐salt groups relative to their respective controls; however, there was no difference in blood pressure between the lean and obese control and salt groups. Urinary 8‐isoprostane was increased in obese control compared with lean control mice (1464 ± 267 vs 493 ± 53 pg/µmol creatinine, respectively) and this elevation was further increased in the obese DOCA‐salt treated mice (2430 ± 312 pg/µmol creatinine). Urinary monocyte chemoattractant protein‐1 excretion and CD68‐positive cells were also increased in both obese and lean DOCA‐salt groups compared with their respective controls. Furthermore, DOCA‐salt treatment increased collagen IV excretion in both obese and lean mice compared with controls, but there was no difference between obese and lean DOCA‐salt groups. Urinary albumin excretion was significantly increased in the obese compared with the lean DOCA‐salt mice (507 ± 160 vs 202 ± 48 µg/day, respectively).
• 3 These data suggest that obese DOCA‐salt hypertensive mice exhibit greater renal injury than lean DOCA‐salt hypertensive mice in a manner independent of blood pressure and that this renal injury is associated with obesity related pre‐existing renal oxidative stress.

     

ACUTE EFFECTS OF NICOTINE ON ARTERIAL STIFFNESS AND WAVE REFLECTION IN HEALTHY YOUNG NON-SMOKERS

• 1 Recently, we have demonstrated that cigarette smoke exposure proportionally increases plasma nicotine levels and arterial wave reflection to the aorta. However, the exact contribution of nicotine to the smoke‐induced enhancement of wave reflection and the potential underlying mechanisms have not been fully investigated.
• 2 The present study was a prospective study in 15 healthy male non‐smokers. All received a placebo and a 2 mg nicotine tablet, according to a randomized double‐blind cross‐over study design. Each subject underwent repeated measurements at baseline and for 1 h after nicotine or placebo intake, using carotid–femoral pulse wave velocity (PWV) to assess arterial compliance. Concurrently, aortic pressures and the augmentation index were evaluated using applanation tonometry.
• 3 Plasma nicotine concentrations achieved 1 h after intake of the nicotine tablet reached comparable levels to those achieved after 1 h exposure to passive smoke (3.6 ± 0.4 vs 3.2 ± 0.4 ng/mL, respectively; P = 0.4).
• 4 Nicotine enhanced arterial wave reflection to the aorta, as assessed by the augmentation index corrected for heart rate (4.2 ± 1.3 vs–0.7 ± 0.8% with placebo; P = 0.001). In addition, a progressive increase in carotid–femoral PWV was noted after nicotine administration (0.3 ± 0.1 vs–0.02 ± 0.1 m/s with placebo; P = 0.04). This remained significant even after adjustment for changes in mean blood pressure and heart rate (P = 0.01).
• 5 Plasma nicotine concentrations comparable to those achieved after exposure to passive smoke enhance arterial wave reflection to the aorta. This is accompanied by an increase in carotid–femoral PWV, denoting a deterioration of arterial compliance by nicotine.

     

Is there a role for ambulatory blood pressure monitoring in pregnancy?

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EFFECTS OF 1400W AND/OR NITROGLYCERIN ON RENAL OXYGENATION AND KIDNEY FUNCTION DURING ENDOTOXAEMIA IN ANAESTHETIZED RATS

• 1 The pathogenesis of acute renal failure (ARF) in sepsis is multifactorial. The role of nitric oxide (NO) in septic ARF has been a source of controversy. We hypothesized that endotoxaemia‐induced exacerbation of inducible nitric oxide synthase (iNOS)‐related NO release impairs renal oxygenation and contributes to ARF in anaesthetized rats.
• 2 In the present study, rats received lipopolysaccharide (2.5 mg/kg) for 30 min. Two hours later, fluid resuscitation was started (HES130; 5 mL/kg per h after a 5 mL/kg bolus) supplemented either by the NO donor nitroglycerin (NTG; 0.5 µg/kg per min after a 2 µg/kg bolus), the selective iNOS inhibitor 1400W (3 mg/kg per h after a 3 mg/kg bolus) or both. Systemic haemodynamics and renal microvascular Po ₂ (µPo ₂) were recorded continuously. Furthermore, creatinine clearance, plasma NO_x (nitrate + nitrite + S‐nitrosothiols) levels and the expression of iNOS mRNA were measured.
• 3 Endotoxaemia reduced renal blood flow, decreased mean arterial pressure, resulted in anuria and was associated with an increase in plasma NO_x levels and renal iNOS expression. Renal µPo ₂ deteriorated gradually during endotoxaemia and there was a significant decrease in renal O₂ delivery and consumption. Manipulation of NO levels had no beneficial effect on systemic haemodynamics, renal µPo ₂ or creatinine clearance over standard fluid resuscitation. The application of 1400W+NTG significantly reduced plasma NO_x levels compared with fluid resuscitation and NTG alone.
• 4 Neither iNOS inhibition, NO donation nor a combination of both showed beneficial effects on systemic haemodynamics, renal oxygenation and renal function compared with fluid resuscitation alone. Our results question the proposed key role of NO in the pathogenesis of septic ARF in rats.

     

Effects of Matrix Metalloproteinase (MMP)-2 Polymorphisms on Responsiveness to Antihypertensive Therapy of Women with Hypertensive Disorders of Pregnancy

Imbalanced matrix metalloproteinase (MMP) expression, including MMP‐2, has been demonstrated in pre‐eclampsia. However, little is known about the effect of polymorphisms in MMP‐2 gene on hypertensive disorders of pregnancy. We examined whether two functional MMP‐2 polymorphisms (g.‐1306C>T and g.‐735C>T) are associated with pre‐eclampsia and/or gestational hypertension and whether these polymorphisms affect therapeutic responses in women with these conditions. We studied 216 healthy pregnant women (HP), 185 patients with gestational hypertension (GH) and 216 patients with pre‐eclampsia (PE). They were stratified as responsive or non‐responsive to antihypertensive therapy according to clinical and laboratorial parameters of therapeutic responsiveness. Genomic DNA was extracted from whole blood and genotypes for g‐1306C>T and g.‐735C>T polymorphisms were determined by real‐time PCR using Taqman allele discrimination assays. Haplotype frequencies were inferred using the PHASE 2.1 program. The distributions of MMP‐2 genotypes and haplotypes were similar in HP, GH and PE patients (p > 0.05). In addition, we found no significant differences in MMP‐2 genotype or haplotype frequencies when GH or PE patients were classified as responsive or non‐responsive to antihypertensive therapy (p > 0.05). Our results suggest that MMP‐2 polymorphisms do not affect the susceptibility to hypertensive disorders of pregnancy. In parallel, MMP‐2 polymorphisms apparently do not affect the responsiveness to antihypertensive therapy of women with these hypertensive disorders of pregnancy.     

MEASUREMENT OF PULMONARY FLOW RESERVE IN HIGHER PRIMATES

• 1 There are currently limited diagnostic methods for assessing the integrity of the pulmonary microvasculature. We hypothesized that a novel, invasively determined physiological index of ‘pulmonary flow reserve’ (PFR = maximal hyperaemic pulmonary blood flow divided by basal pulmonary flow) may facilitate microvascular assessment in the lung. Therefore, we developed a baboon model in which to: (i) validate the use of Doppler flow velocity for PFR assessment; (ii) define the optimal drug and dose regimen for attainment of maximal pulmonary hyperaemia; and (iii) demonstrate the feasibility of measuring PFR in healthy higher primates.
• 2 Doppler sensor guidewires were placed in segmental pulmonary arteries of 11 ketamine‐anaesthetized baboons. Vessel diameter, flow velocity and haemodynamics were recorded before and after direct intrapulmonary artery administration of saline, adenosine (50–500 µg/kg per min) and papaverine (3–60 mg), enabling calculation of PFR.
• 3 Saline (either bolus injection or infusion) did not alter vessel diameter or flow velocity (P > 0.1), validating local drug administration. Both adenosine and papaverine induced dose‐dependent increases in flow velocity from baseline (from 22.5 ± 2.3 to 32.7 ± 4.8 cm/s for 400–500 µg/kg per min adenosine; and from 23.9 ± 1.1 to 34.6 ± 4.0 cm/s for 24 mg papaverine; both P < 0.0001), without affecting pulmonary artery pressure or vessel diameter (P > 0.3). Healthy primate PFR values were 1.35 ± 0.10 and 1.39 ± 0.10 using 200 µg/kg per min adenosine and 24 mg papaverine, respectively (P > 0.8).
• 4 In conclusion, pulmonary flow reserve in higher primates can be assessed using Doppler sensor guidewire and either adenosine or papaverine as microvascular hyperaemic agents. Measurements of PFR may facilitate pulmonary microvascular assessments.

     

CARDIOVASCULAR EFFECTS OF CENTRALLY ADMINISTERED ARACHIDONIC ACID IN HAEMORRHAGE-INDUCED HYPOTENSIVE RATS: INVESTIGATION OF A PERIPHERAL MECHANISM

• 1 The aims of the present study were to determine the cardiovascular effects of arachidonic acid (AA) and to investigate the peripheral mechanisms mediating these effects in haemorrhage‐induced hypotensive rats.
• 2 Acute haemorrhage was induced by withdrawing a total volume of 2.2 mL blood/100 g bodyweight over a period of 10 min. Rats were then injected with 75–300 µg, i.c.v., AA and cardiovascular changes were monitored over the next 60 min. Plasma catecholamine and vasopressin levels, as well as plasma renin activity (PRA), were measured 10 min after injection of 150 µg AA in haemorrhage‐induced hypotensive awake rats. In addition, rats were pretreated with saline (1 mL/kg, i.v.), the vasopressin V₁ receptor antagonist [β‐mercapto‐β,β‐cyclopentamethylenepropionyl¹,O‐Me‐Tyr²,Arg⁸]‐vasopressin (10 µg/kg, i.v.), the α₁‐adrenoceptor antagonist prazosin (500 µg/kg, i.v.), the non‐specific angiotensin II receptor antagonist saralasin (250 µg/kg, i.v.) or a combination of these three antagonists 5 min before injection of AA (150 µg, i.c.v.). The effects of these antagonists on responses to AA were determined.
• 3 Arachidonic acid caused dose‐ and time‐dependent increases in mean arterial pressure and heart rate and reversed hypotension in haemorrhaged rats. Haemorrhage itself produced an increase in plasma catecholamine and vasopressin levels, as well as PRA; injection of AA produced further increases in these parameters, ranging from 39–123%, under hypotensive conditions. Under hypotensive conditions, pretreatment of rats with all three receptor antagonists produced similar partial blockade of the pressor response to AA, but not the increase in heart rate. Moreover, combined administration of all three receptor antagonists prior to the i.c.v. injection of 150 µg AA completely abolished the pressor response to AA in haemorrhage‐induced hypotensive rats.
• 4 These results indicate that centrally administered AA reverses hypotension by increasing blood pressure and heart rate in the hypotensive setting. The observed increases in plasma catecholamine and vasopressin levels, as well as PRA, mediate the pressor response to AA in haemorrhage‐induced hypotensive rats.

     

EFFECTS OF SODIUM FERULATE ON HUMAN OSTEOARTHRITIC CHONDROCYTES AND OSTEOARTHRITIS IN RATS

• 1 Oxidation, inflammation and apoptosis are involved in the aetiology and pathology of osteoarthritis (OA). Sodium ferulate (SF) has been demonstrated to have anti‐oxidant, anti‐inflammatory and anti‐apoptotic actions in cardiovascular, hepatic and diabetic disorders. These findings suggest that SF may have beneficial effects on OA. Therefore, present study investigated the effects of SF in an in vivo rat OA model, as well as in vitro in human OA chondrocytes.
• 2 Rats were divided into the following groups: (i) an untreated control group; (ii) papain‐induced OA; (iii) OA rats treated with 0.1 or 0.5% SF; and (iv) normal rats injected with 0.5% SF intra‐articularly. Human chondrocytes from OA patients were cultured before being stimulated with 2 ng/mL interleukin‐1β and subsequently treated with SF (125, 250 and 500 µmol/L). The effects of SF were evaluated both in vivo and in vitro.
• 3 In OA rats, SF treatment dose‐dependently reversed pathological changes in OA cartilage, decreased BAX‐immunopositive chondrocytes and increased Bcl‐2‐immunopositive chondrocytes. Both in vivo and in vitro analyses demonstrated a significant decrease in matrix metalloproteinase‐1 and an increase in tissue‐specific inhibitor of metalloproteinase‐1. In vitro, SF enhanced chondrocyte proliferation and decreased nitric oxide production and apoptosis.
• 4 The results demonstrate that SF dose‐dependently suppresses pathological processes in both in vitro and in vivo OA models. Thus, SF could be a therapeutic strategy for the treatment of OA.

     

NOX2-CONTAINING NADPH OXIDASE AND XANTHINE OXIDASE ARE SOURCES OF SUPEROXIDE IN MOUSE TRACHEA

• 1 Superoxide anion plays an important role in host defence against invading pathogens and in the inflammation that arises in lungs. The aim of the present study was to elucidate whether the two key candidate superoxide‐producing enzymes in mammalian cells, namely Nox2‐containing NADPH oxidase and xanthine oxidase, are responsible for superoxide production in mouse trachea.
• 2 Superoxide production by isolated trachea, as measured by L‐012‐dependent chemiluminescence, was markedly reduced by superoxide dismutase (300 U/mL) and the xanthine oxidase inhibitor allopurinol (100 µmol/L). Tracheas from Nox2^?/– mice had significantly lower levels (~60%) of superoxide than control mice.
• 3 These novel findings suggest that superoxide production by mouse trachea is attributed to both Nox2‐containing NADPH oxidase and xanthine oxidase.

     

EPIGALLOCATECHIN-3-GALLATE ATTENUATES CARDIAC HYPERTROPHY IN HYPERTENSIVE RATS IN PART BY MODULATION OF MITOGEN-ACTIVATED PROTEIN KINASE SIGNALS

• 1 It has been demonstrated that epigallocatechin‐3‐gallate (EGCG) inhibits cardiac hypertrophy through its antihypertensive and anti‐oxidant effects. However, the underlying molecular mechanism is not clear.
• 2 In the present study, we tested the hypothesis that EGCG attenuates transaortic abdominal aortic constriction (TAC)‐induced ventricular hypertrophy by regulating mitogen‐activated protein kinase (MAPK) signal pathways in hypertensive rats. Four groups of rats were used: (i) a sham‐operated control group; (ii) an EGCG‐treated (50 mg/kg per day, i.p., for 21 days) sham‐operated group; (iii) a TAC group; and (iv) an EGCG‐treated TAC group. Histological analysis of whole hearts and biochemical analyses of left ventricular (LV) tissue were used to investigate the effects of EGCG.
• 3 The results showed that the LV myocyte diameter and the expression of atrial natriuretic peptide, brain natriuretic peptide and β‐myocardial heavy chain were significantly decreased in the EGCG‐treated (50 mg/kg per day, i.p.) TAC group. Levels of reactive oxygen species and malondialdehyde in the lV were significantly reduced by EGCG in the TAC group. Total superoxide dismutase, catalase and glutathione peroxidase activities were decreased in the TAC group, and this decrease was significantly restored by EGCG treatment. Phosphorylation of extracellular signal‐regulated kinase 2, p38 and c‐Jun N‐terminal kinase 1 was significantly reversed in the LV of EGCG‐treated TAC rats (40%, 53% and 52%vs TAC, respectively), accompanied by significant inhibition of nuclear factor‐κB and activator protein‐1. Transaortic abdominal aortic constriction significantly upregulated LV expression of matrix metalloproteinase‐9 from 32 ± 6 to 100 ± 12% and this increase was inhibited by EGCG treatment (from 100 ± 12 to 50 ± 15%). In addition, TAC decreased mitochondrial DNA copy number and the activity of respiratory chain complexes I (from 100 ± 7 to 68 ± 5%), III (from 100 ± 4 to 2 ± 5%) and IV (from 766 ± 2 to 100 ± 5%); this decrease was reversed by EGCG treatment to levels seen in sham‐operated rats.
• 4 In conclusion, EGCG attenuates TAC‐induced ventricular hypertrophy in hypertensive rats in part by suppression of anti‐oxidant enzymes and regulation of MAPK signals.

     

Effects of sodium valproate on synaptic transmission and neuronal excitability in rat hippocampus

• 1 Valproate (VPA) has long been used in the treatment of both generalized and partial seizures. However, its cellular mechanisms of action remain unclear.
• 2 In the present study, the effects of VPA on synaptic transmission and neuronal excitability were examined in the hippocampal CA1 region using whole‐cell patch clamp recordings.
• 3 Perfusion with VPA, at therapeutically attainable concentrations (i.e. 0.3 and 0.6 mmol/L), significantly increased the frequency (112 ± 2 and 133 ± 2% of control, respectively; n = 5; both P < 0.05), but not the average amplitude, of miniature inhibitory post‐synaptic currents (mIPSCs). Perfusion with VPA had no effect on either the amplitude or the frequency of miniature excitatory post‐synaptic currents (mEPSCs).
• 4 In acutely dissociated CA1 pyramidal neurons, VPA had no effect on 10 µmol/L GABA‐induced currents. Furthermore, following the administration of 0.3 and 0.6 mmol/L VPA, the frequency of action potential firing was significantly reduced from 18.0 ± 1.1 to 15.3 ± 0.9 and from 18.6 ± 0.9 to 12.6 ± 0.6, respectively (n = 8; both P < 0.05). In contrast, 0.3 and 0.6 mmol/L VPA significantly increased spike frequency adaptation from 4.02 ± 0.47 to 4.72 ± 0.55 and from 3.47 ± 0.41 to 4.48 ± 0.58, respectively (n = 8; P < 0.05).
• 5 The results of the present study suggest that VPA presynaptically increases inhibitory synaptic activity without modifying excitatory synaptic transmission and reduces neuronal excitability. Any or all of these effects may contribute to its anticonvulsant action.

     

Effects of dipeptidyl peptidase iv inhibition on arterial blood pressure

• 1 The aim of the present study was to determine whether inhibition of dipeptidyl peptidase IV (DPP IV) elevates arterial blood pressure and whether any such effect is dependent on genetic background, the sympathetic nervous system and Y₁ receptors. The rationale behind this study was that: (i) neuropeptide (NP) Y_1–36 and peptide YY_1–36 (PYY_1–36) are endogenous Y₁ receptor agonists and are metabolised by DPP IV to NPY_3–36 and PYY_3–36, which are not Y₁ but rather selective Y₂ receptor agonists; (ii) Y₁ receptors mediate vasoconstriction, whereas Y₂ receptors have little effect on vascular tone; (iii) vaso‐constrictor effect of the Y₁ receptor is enhanced in spontaneously hypertensive rats (SHR) compared with normotenisve Wistar‐Kyoto (WKY) rats; and (iv) NPY_1–36 is released from sympathetic nerve terminals.
• 2 We examined the effects of acute administration of 3‐N‐[(2S,3S)‐2‐amino‐3‐methylpentanoyl]‐1,3‐thiazolidine (P32/98; a DPP IV inhibitor) on arterial blood pressure in anaesthetized adult SHR and WKY rats in the absence and presence of either captopril, hydralazine or chlorisondamine to lower basal mean arterial blood pressure (MABP) by different mechanisms (inhibition of angiotensin‐converting enzyme, direct vasodilation and ganglionic blockade, respectively).
• 3 In naïve SHR with severely elevated basal blood pressures (MABP = 176 ± 3 mmHg; n = 4), i.v. boluses (1, 3 and 10 mg/kg) of P32/98 did not affect blood pressure.
• 4 When basal blood pressure was reduced by pretreatment of SHR with either captopril (30 mg/kg, i.v.; MABP = 116 ± 3 mmHg; n = 9) or hydralazine (5 mg/kg, i.p.; MABP = 84 ± 3 mmHg; n = 7), P32/98 (1, 3 and 10 mg/kg) caused significant dose‐related increases in arterial blood pressure (4 ± 2, 10 ± 2 and 12 ± 3 mmHg in the captopril‐pretreated group, respectively (P < 0.01); 5 ± 2, 8 ± 3 and 11 ± 4 mmHg in the hydralazine‐pretreated group, respectively (P < 0.01)).
• 5 The increases in arterial blood pressure induced by P32/98 in captopril‐ or hydralazine‐pretreated SHR were entirely blocked by pretreatment with the selective Y₁ receptor antagonist N2‐(diphenylacetyl)‐N‐[(4‐hydroxyphenyl)methyl]‐d ‐arginine amide (BIBP 3226; 6 mg/kg per h).
• 6 When basal blood pressure was reduced in SHR by pretreatment with chlorisondamine (10 mg/kg, s.c.; MABP = 108 ± 4 mmHg; n = 7), inhibition of DPP IV with P32/98 did not affect arterial blood pressure. Basal heart rate in chlorisondamine‐treated SHR was significantly reduced compared with naïve SHR, captopril‐pretreated SHR and hydralazine‐pretreated SHR, indicating effectiveness of ganglionic blockade.
• 7 Unlike the results in genetically hypertensive animals, in normotensive WKY rats pretreated with captopril (30 mg/kg, i.v.; MABP = 81 ± 4 mmHg; n = 6), or hydralazine (5 mg/kg, i.p.; MABP = 63 ± 4 mmHg; n = 4) or chlorisondamine (10 mg/kg, s.c.; MABP = 63 ± 4 mmHg; n = 5), P32/98 did not affect arterial blood pressure.
• 8 We conclude that, in genetically susceptible animals, inhibition of DPP IV increases arterial blood pressure via Y₁ receptors when elevated blood pressure is reduced with antihypertensive drugs provided that the sympathetic nervous system is functional. The results suggest vigilance because DPP IV inhibitors are used more widely in hypertensive patients treated with antihypertensive drugs.

     

Frusemide and Its Acyl Glucuronide Show a Short and Long Phase in Elimination Kinetics and Pharmacodynamic Effect in Man

The pharmacokinetics of 80 mg frusemide given orally were investigated in normal subjects using a direct HPLC method for parent drug and its acyl glucuronide conjugate. Two half-lives could be distinguished in the plasma elimination of both frusemide and its conjugate, with values of 1.25 ± 0.75 and 30.4 ± 11.5 h for frusemide and 1.31 ± 0.60 and 33.2 ± 28.0 h for the conjugate. The renal excretion rate-time profile showed two phases; the rapid elimination phase lasted from 0–15 h and the second and slow phase, from 15–96 h. During the first 15 h, 33.3 ± 4.8% of the dosed frusemide was excreted; in the remaining period 15–96 h, 4.6 ± 1.5% was excreted. In the same two periods the excretion of the glucuronide was 13.4 ± 4.7 and 1.9 ± 1.1%, respectively. The mean renal clearance of frusemide was 90.2 ± 16.9 mL min^?1 during the first period and 91.5 ± 29.3 mL min^?1 in the remaining period, during which the stimulation of urine production was absent. The renal clearance of the acyl glucuronide was 702 ± 221 mL min^?1 in the first period, but only 109 ± 51.0 mL min^?1 in the second period. The stimulated urine production in the first 6 h after administration amounted to 2260 ± 755 mL (measured urine production minus baseline value of 1 mL min^?1 (360 mL). During the second or rebound period (6–96 h after drug administration), the quantity of urine was 990 ± 294 mL lower than what would have been expected from the baseline production of 5400 mL. This reduced production (0.82 mL min^?1) is equivalent to an 18% reduction in the average urine flow rate of 1 mL min^?1.     

HYPERTONICITY INCREASES RABBIT ATRIUM AND PULMONARY VEIN ARRHYTHMOGENESIS: A POTENTIAL CONTRIBUTOR TO THE GENESIS OF ATRIAL FIBRILLATION

• 1 Pulmonary veins are the most important focus for the initiation of atrial fibrillation. Diabetes mellitus may be associated with hypertonicity and increased occurrence of atrial fibrillation.
• 2 The purpose of the present study was to investigate whether hypertonicity alters the electrophysiological characteristics of pulmonary veins and atria to enhance the genesis of atrial fibrillation.
• 3 A whole‐cell patch‐clamp technique was used to investigate action potentials and ionic currents in rabbit isolated single pulmonary vein and atrial cardiomyocytes during immersion in isotonic and hypertonic (1.2× normal osmolality) solutions.
• 4 Hypertonicity increased the spontaneous beating rates of pulmonary vein cardiomyocytes from 2.3 ± 0.3 to 3.4 ± 0.3 Hz (n = 11; P < 0.001). Hypertonicity prolonged action potential duration to a greater extent in atrial cardiomyocytes than in pulmonary vein cardiomyocytes. Compared with atrial cardiomyocytes, hypertonicity increased the transient inward currents and Na⁺/Ca²⁺ exchange currents to a greater extent in pulmonary vein cardiomyocytes, but decreased the delayed rectified potassium currents to a lesser extent.
• 5 Hypertonicity plays an important role in the electrical activity of pulmonary vein and atrial cardiomyocytes, which may have a potential role in the pathophysiology of atrial fibrillation.

     

Decreased mobilization of endothelial progenitor cells contributes to impaired neovascularization in diabetes

• 1 Circulating bone marrow (BM)‐derived endothelial progenitor cells (EPCs) play an important role in neovascularization. In the present study, we investigated the mechanisms underlying the reduction in circulating EPCs in a mouse model of diabetes induced by streptozotocin.
• 2 Compared with non‐diabetic controls, diabetic mice had reduced circulating EPCs (0.59 ± 0.11 vs 0.94 ± 0.21%, respectively; P < 0.01) and increased plasma endothelial microparticles (18 642 ± 6809 vs 5692 ± 1862/mL, respectively; P < 0.01). In a mouse bone marrow (BM) transplantation model, increased adhesion of transplanted BM cells to aortas of diabetic mice was observed compared with control (900 ± 194 vs 431 ± 109 cells/mm², respectively; P < 0.01).
• 3 Following hindlimb ischaemia, diabetic mice exhibited suppressed EPC mobilization, a reduction in the expected increase in capillary density and suppressed restoration of transcutaneous oxygen pressure in the ischaemic tissue. Diabetic mice also showed impaired ischaemia‐induced upregulation of vascular endothelial growth factor (VEGF), hypoxia‐inducible factor (HIF)‐1α and interleukin‐1β, an exaggerated increase in matrix metalloproteinase (MMP)‐2 and ‐9 and a suppressed increase in tissue inhibitor of matrix metalloproteinase (TIMP)‐1. On multivariate analysis, VEGF expression was the only independent factor related to circulating EPC count.
• 4 In conclusion, the data indicate that the decrease in basal circulating EPCs in diabetes may be attributable, in part, to consumptive loss of EPCs due to increased endothelial damage. Impairment of ischaemia‐induced EPC mobilization in the diabetic mouse model is associated with altered HIF‐1α/VEGF and MMP/TIMP regulation and represents a novel mechanism underlying defective postischaemic neovascularization in diabetes.

     

PREVENTION OF AORTIC ELASTIC LAMINA DEFECTS BY LOSARTAN IN APOLIPOPROTEIN E-DEFICIENT MOUSE

• 1 In a previous study, we identified prevalent internal elastic lamina (IEL) defects in the aorta of hyperlipidaemic apolipoprotein E (ApoE)‐deficient mice that are thought to provide a structural basis for the development of atherosclerosis and intimal thickening. In the present study, we examined the effects of losartan, an angiotensin AT₁ receptor antagonist, on the development of IEL defects.
• 2 Male 18‐week‐old ApoE‐deficient mice (maintained on a normal diet) were treated with losartan (3 or 30 mg/kg per day) for 10 weeks via the drinking water. The IEL defects were quantified histologically by measuring the continuity of the IEL within the inner curvature of the aortic arch.
• 3 In untreated animals, there was an age‐dependent increase in IEL defects from 7.2 ± 2.1% at 18 weeks to 13.8 ± 4.0% at 28 weeks. Treatment with the high dose of losartan significantly prevented the development of IEL defects (4.7 ± 1.3% at 28 weeks; P < 0.05 vs untreated). This effect was independent of changes in blood pressure or plasma lipid levels. Using quantitative real‐time polymerase chain reaction, we found that the effects of losartan were not associated with changes in levels of matrix metalloproteinase (MMP)‐2 and MMP‐9, tissue inhibitor of matrix metalloproteinase‐1 or inflammatory markers in the aorta.
• 4 The results suggest that the renin–angiotensin system may contribute to the development of aortic IEL defects in a blood pressure‐independent manner.