Vasorelaxant activity of twenty-one physiologically relevant (poly)phenolic metabolites on isolated mouse arteries.

Polyphenols are beneficial for health, but are metabolised after consumption. We compared the vasorelaxant capacity of twenty-one physiologically relevant polyphenol metabolites in isolated mouse arteries. Hesperetin, urolithins and ferulic acid-4-O-sulfate - not their glucuronidated forms or ferulic acid - caused vasorelaxation. Therefore, we advise the use of relevant conjugates in future mechanistic research.

Response to exercise and mechanical efficiency in non-ischaemic stunning, induced by short-term rapid pacing in dogs: a role for calcium?

AIM: Rapid pacing (RP) is a regularly used model to induce heart failure in dogs. The aim of the study was to evaluate Ca2+ handling, left ventricular (LV) contractile response during Ca2+ administration compared to exercise, as well as oxygen consumption and mechanical efficiency after 48 h of RP. METHODS: Fifty-three mongrel dogs were instrumented to measure LV pressure, LV fractional shortening, regional wall thickening and coronary blood flow. Contractile reserve was measured with isoproterenol and intravenous (IV) Ca2+ administration. To assess the function of the sarcoplasmic reticulum (SR), post-extrasystolic potentiation (PESP) and SR Ca2+ uptake were measured. A graded treadmill test was performed in baseline and after RP (n = 14). In a separate group of animals (n = 5), myocardial performance and oxygen consumption were measured using a wide range of loading conditions. RESULTS: Left ventricular contractility was significantly decreased upon cessation of pacing. The contractile response to isoproterenol was blunted compared to a preserved response to IV Ca2+ . Post-extrasystolic potentiation was slightly increased after RP. Maximal velocity (Vmax ) of SR Ca2+ uptake was unchanged. Contractile response during exercise is attenuated after RP. External work is reduced, whereas oxygen consumption is preserved, provoking a reduced mechanical efficiency. CONCLUSION: Forty-eight-hours RP provokes a reversible LV dysfunction, while the SR function and response to exogenous Ca2+ are preserved. This is compatible with an intracellular functional remodelling to counteract Ca2+ overload provoked by RP. Left ventricular dysfunction is accompanied by a reduced contractile reserve, but an unchanged oxygen consumption, illustrating an alteration in oxygen utilization.

Treatment of erectile dysfunction: new targets and strategies from recent research.

In recent years, research on penile erection has increasingly been centered on the molecular mechanisms involved. Major progress has been made in the field and at present a whole number of neurotransmitters, chemical effectors, growth factors, second-messenger molecules, ions, intercellular proteins, and hormones have been characterized as components of the complex process of erection. This knowledge has led to the discovery of several new therapeutic targets and multiple medical approaches for the treatment of erectile dysfunction (ED). This review focuses on the progress made in this field within the last few years.

Expression of lipolytic genes in adipose tissue is differentially regulated during multiple phases of dietary intervention in obese women.

The aim of this study was to investigate the time-course of the expression of key lipolysis-regulating genes in the subcutaneous adipose tissue (SCAT) during different phases of a 6-month dietary intervention. Fifteen obese women (BMI 34.7+/-1.0 kg.m(-2)) underwent a 6-month dietary intervention consisting of 1 month very low calorie diet (VLCD), followed by 2 months low calorie diet (LCD) and 3 months weight maintenance diet (WM). At each phase of the dietary intervention, a needle microbiopsy of the abdominal SCAT was obtained to evaluate mRNA expression of key lipolysis-regulating genes and a hyperinsulinemic euglycemic clamp (HEC) was performed. Dietary intervention induced a body weight reduction of 9.8 % and an improvement of insulin sensitivity as assessed by a HEC. Compared to pre-diet levels, mRNA levels of the adrenergic beta(2)-receptor in SCAT were higher at the end of VLCD and not different at the end of LCD and WM. In contrast, the expression of the adrenergic alpha(2)-receptor was lower at the end of VLCD and LCD compared to the pre-diet levels and did not differ at WM. Adipose triglyceride lipase and hormone-sensitive lipase levels were lower than the pre-diet levels at the end of LCD only, while phosphodiesterase-3B and the insulin receptor levels did not change throughout the dietary intervention. The results suggest that the regulation pattern of the genes that are involved in the control of lipolysis is different at the respective phases of the dietary intervention and depends on the duration of the diet and the status of energy balance.

Effect of hypoxia in mice mesenteric arteries surrounded by adipose tissue.

AIM: To investigate the influence of hypoxia on the vasoactive effect of peri-vascular white adipose tissue. METHODS: Isometric tension recordings were performed on mesenteric arteries from Swiss male mice with or without adherent adipose tissue. RESULTS: Hypoxia (bubbling with 95% N(2), 5% CO(2)) induced a biphasic response, i.e. vasoconstriction followed by vasorelaxation, in pre-contracted (noradrenaline, 10 µm) mesenteric arteries with adipose tissue in the presence of indomethacin (10 µm) and N(ω) -nitro-l-arginine (0.1 mm). Only a small vasorelaxation was observed in arteries without adipose tissue. Pre-contraction with 60 or 120 mm K(+) , incubation with tetraethylammoniumchloride (1 and 3 mm), apamin (1 µm) combined with charybdotoxin (0.1 µm) or 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) (10 µm) significantly impaired the hypoxic vasorelaxation. Removal of the endothelium only reduced the hypoxic vasorelaxation. Apamin (1 µm) and charybdotoxin (0.1 µm) did not influence the vasorelaxation of sodium hydrosulfide hydrate. Zinc protoporphyrin IX (10 µm), miconazole (10 µm), 8-(p-sulfophenyl)theophylline (0.1 mm), 1 H-[1, 2, 4]oxadiazolo[4,3- A]quinoxalin-1-one (10 µm), apocynin (0.3 mm), diphenyliodonium (1 µm), catalase (2500 U mL(-1)) and tempol (0.1 mm) did not influence the hypoxic vasorelaxation. In contrast to losartan (0.1 mm), indomethacin (10 µm) and SQ-29548 (10 µm) significantly reduced the hypoxic vasoconstriction. CONCLUSIONS: Moderate hypoxia induces a biphasic vasomotor response in mice mesenteric arteries surrounded by adipose tissue. The hypoxic vasoconstriction is endothelium independent, whereas the vasodilation is endothelium dependent, soluble guanylyl cyclase independent and in part mediated by opening K(Ca) channels. Cyclooxygenase metabolites mediate the hypoxic vasoconstriction, while endothelium-derived hyperpolarizing factor plays a small role in the hypoxic vasorelaxation.

Role of the soluble guanylyl cyclase alpha1-subunit in mice corpus cavernosum smooth muscle relaxation.

Soluble guanylyl cyclase (sGC) is the major effector molecule for nitric oxide (NO) and as such an interesting therapeutic target for the treatment of erectile dysfunction. To assess the functional importance of the sGCalpha(1)beta(1) isoform in corpus cavernosum (CC) relaxation, CC from male sGCalpha(1)(-/-) and wild-type mice were mounted in organ baths for isometric tension recording. The relaxation to endogenous NO (from acetylcholine, bradykinin and electrical field stimulation) was nearly abolished in the sGCalpha(1)(-/-) CC. In the sGCalpha(1)(-/-) mice, the relaxing influence of exogenous NO (from sodium nitroprusside and NO gas), BAY 41-2272 (NO-independent sGC stimulator) and T-1032 (phosphodiesterase type 5 inhibitor) were also significantly decreased. The remaining exogenous NO-induced relaxation seen in the sGCalpha(1)(-/-) mice was significantly decreased by the sGC-inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. The specificity of the impairment of the sGC-related responses was demonstrated by the unaltered relaxations seen with forskolin (adenylyl cyclase activator) and 8-pCPT-cGMP (cGMP analog). In conclusion, the sGCalpha(1)beta(1) isoform is involved in corporal smooth muscle relaxation in response to NO and NO-independent sGC stimulators. The fact that there is still some effect of exogenous NO in the sGCalpha(1)(-/- mice suggests the contribution of (an) additional pathway(s).

Inhibition of vascular endothelial growth factor (VEGF) does not affect early renal changes in a rat model of lean type 2 diabetes.

Type 2 diabetes is the most frequent cause of end-stage renal failure in many Western countries. Approximately 10-15 % of all type 2 diabetics are lean. Various growth factors and cytokines have been implicated in the pathophysiology of diabetic kidney disease, including vascular endothelial growth factor (VEGF). To elucidate a role for VEGF in the renal changes associated with type 2 diabetes, we examined the effect of a VEGF-antibody (ab) on early renal changes in the Goto-Kakizaki (GK) rat, a lean type 2 diabetes model. GK-rats were treated for 6 weeks with the VEGF-ab or with an isotype-matched irrelevant IgG. Wistar rats were used as non-diabetic controls. Placebo-treated GK-rats showed a pronounced increase in glomerular volume and urinary albumin excretion (UAE) and no change in the renal expression of endothelial nitric oxide synthase (eNOS) compared to placebo-treated non-diabetic controls. Kidney weight and creatinine clearance were no different between the groups. VEGF-ab treatment had no effect on glomerular volume, UAE, eNOS expression, body weight, blood glucose levels or food intake, but lowered serum insulin levels in non-diabetic and diabetic animals. We conclude that VEGF inhibition has minimal effects on early renal changes in GK-rats.

Contractility studies on isolated bovine choroidal small arteries: determination of the active and passive wall tension-internal circumference relation.

Studies on isolated choroidal arteries could help to understand the regulatory mechanisms in the choroidal circulation. The aim of the present study was therefore to assess whether contractility studies on isolated choroidal arteries were feasible and to determine the active and passive wall tension-internal circumference relation of these arteries. This relation is essential for reliable further pharmacodynamic studies on these vessels. Isolated choroidal arteries were mounted on a wire myograph for isometric tension recording. After the vessel was mounted, the L(100) (the circumference of the vessel at a transmural pressure of 100 mmHg) was determined. Then the passive and active wall tension-internal circumference relation of the choroidal vessels was obtained by stepwise increasing the internal circumference. The changes in the internal circumference were expressed as a percentage of L(100). After each increase in circumference, the passive tone (in a calcium free medium), the spontaneous tone (in a Krebs--Ringer bicarbonate solution) and the active tone (in a solution containing K(+) 120 mM and prostaglandin F(2 alpha) 30 microM) was measured. The passive tone of the vessel increased exponentially with the circumference of the vessel. Both the spontaneous tone and the active tone also increased when the vessel was stretched. They peaked when the internal circumference approached 90% of the L(100) and diminished again when the circumference was further increased. The peak value of the active tension curve averaged 2.24+/-0.47 Nm(-1) (n=10). The passive tension was 0.57+/-0.08 Nm(-1) (n=10) at this circumference. The peak value of the spontaneous tension curve averaged 0.37+/-0.08 Nm(-1) (n=10). It can be concluded that in vitro contractility studies on isolated choroidal arteries are feasible. The optimal length or preload of the choroidal arteries is attained when the internal circumference of the artery is set to 90% of the L(100).

Regional differences in anandamide- and methanandamide-induced membrane potential changes in rat mesenteric arteries.

The possibility that anandamide is an endothelium-derived hyperpolarizing factor was explored in the rat mesenteric vasculature by use of conventional microelectrode techniques. In the main mesenteric artery, anandamide and its more stable analog methanandamide hardly caused a measurable change in membrane potential of the smooth muscle cells, which promptly hyperpolarized to EDHF liberated by acetylcholine. Inhibition of endogenous anandamide breakdown by phenylmethylsulfonyl fluoride did not increase membrane responses to acetylcholine. The CB(1) receptor antagonist SR141716 did not significantly influence EDHF-mediated hyperpolarization except at extremely high concentrations. Smooth muscle cells of third to fourth order branches of the mesenteric artery, which have a more negative resting membrane potential and show smaller responses to acetylcholine, hyperpolarized by about 6 mV to both anandamide and methanandamide, whereas another CB(1) receptor agonist, WIN 55,212-2, had no effect. Mechanical endothelium removal or pre-exposure to SR141716A did not affect anandamide- and methanandamide-induced hyperpolarizations. However, in the presence of capsazepine, a selective vanilloid receptor antagonist, these membrane potential changes were reversed to a small depolarization, whereas EDHF-induced hyperpolarizations were not affected. Pretreating small vessels with capsaicin, causing desensitization of vanilloid receptors and/or depletion of sensory neurotransmitter, completely blocked methanandamide-induced hyperpolarizations. These findings show that anandamide cannot be EDHF. In smooth muscle cells of small arteries, anandamide-induced changes in membrane potential are mediated by vanilloid receptors on capsaicin-sensitive sensory nerves. The different membrane response to the cannabinoids between the main mesenteric artery and its daughter branches might be explained by the different density of perivascular innervation.

The impaired renal vasodilator response attributed to endothelium-derived hyperpolarizing factor in streptozotocin--induced diabetic rats is restored by 5-methyltetrahydrofolate.

AIMS/HYPOTHESIS: Endothelial dysfunction contributes to the development of diabetic vascular complications. A better understanding of the pathophysiology of endothelial dysfunction in diabetes could lead to new approaches to prevent microvascular disease. METHODS: Endothelium-dependent and endothelium-independent vasodilator responses were investigated in the renal microcirculation of streptozotocin-induced diabetic rats. We measured renal blood flow changes with an electromagnetic flow probe. In addition, the responses of the different segments of the renal microcirculation were evaluated with videomicroscopy using the hydronephrotic kidney technique. Because endothelial cells release different relaxing factors (nitric oxide, prostacyclin and an unidentified endothelium-derived hyperpolarizing factor), responses to acetylcholine were measured before and after treatment with the nitric oxide synthase inhibitor L-NG-nitroarginine methylester HCI (L-NAME) and the cyclooxygenase inhibitor indomethacin. We evaluated with the effect of 5-methyltetrahydrofolate, the active form of folate, on the responses. RESULTS: The L-NAME- and indomethacin-resistant vasodilation to intra-renal acetylcholine was significantly reduced in the diabetic compared with control rats, suggesting impaired endothelium-derived hyperpolarizing factor-mediated vasodilation. The responses to the nitric oxide donor (Z)-1-[-2-(aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-i um-1,2-diolate (DETA-NONOate) and to the K+-channel opener pinacidil were similar in diabetics and controls, indicating intact endothelium-independent vasodilator mechanisms. The contribution of endothelium-derived hyperpolarizing factor to vasodilation induced by acetylcholine was greatest in the smallest arterioles. In diabetic rats, the response to acetylcholine was increasingly impared as vessel size decreased. Defective vasodilation in diabetic kidneys was rapidly normalized by 5-methyltetrahydrofolate. CONCLUSION-INTERPRETATION: Endothelium-derived hyperpolarizing factor-mediated vasodilation is impaired in the renal microcirculation of diabetic rats, in particular in the smallest arteries. Treatment with folate restores the impaired endothelial function in diabetes.

Regulatory mechanisms in the retinal and choroidal circulation.

The retina receives its nutrients from two separate circulations: retinal and choroidal circulation. This short overview describes the determinants in the regulation of these circulations. Retinal circulation is characterized by a low blood flow while flow in the choroid is high. The choroidal circulation is mainly controlled by sympathetic innervation and is not autoregulated. Retinal circulation lacks autonomic innervation, shows an efficient autoregulation and is mainly influenced by local factors. Local mediators released by endothelial cells and surrounding retinal tissue also have a substantial role in the regulation of retinal circulation.

A retinal-derived relaxing factor mediates the hypoxic vasodilation of retinal arteries.

PURPOSE: To investigate the mechanisms involved in hypoxic vasodilation using an in vitro setup. METHODS: Retinal arteries with and without retinal tissue were mounted on a wire myograph. The segments were contracted with prostaglandin (PG)F(2alpha) (30 microM) or 120 mM K(+). Hypoxia was induced by replacement of O(2) by N(2) in the gas used to bubble the Krebs-Ringer bicarbonate organ bath solution. RESULTS: Hypoxia induced complete relaxation of preparations with adherent retinal tissue contracted with PGF(2alpha). Preparations without retinal tissue were not affected by the change in oxygenation. When the retinal arteries were contracted with 120 mM K(+), hypoxia no longer induced relaxation of the preparation with adherent retinal tissue. The presence of an NO-synthase inhibitor (L-NA, 0.1 mM), a cyclooxygenase inhibitor (indomethacin, 50 microM), or an adenosine receptor antagonist (8-sulfophenyltheophylline, 1 mM) did not affect hypoxic vasodilation. Excitatory amino acids and lactate had no or only a limited effect on the PGF(2alpha)-induced contraction and are therefore unlikely mediators of hypoxic vasodilation. HCl (10 mM) reduced the pH to 6.1 +/- 0.08 (n = 4) and induced a pronounced but transient relaxation of the retinal artery contracted with PGF(2alpha) or 120 mM K(+), whereas hypoxia induced relaxation of the retinal artery contracted with PGF(2alpha) only in the presence of adherent retinal tissue. CONCLUSIONS: Adherent retinal tissue mediates the hypoxic vasodilatation of bovine retinal arteries in vitro. Neither NO, prostanoids, adenosine, excitatory amino acids lactate or changes in pH seem to be involved in this hypoxic response.

Endothelial dysfunction in diabetes.

Endothelial dysfunction plays a key role in the pathogenesis of diabetic vascular disease. The endothelium controls the tone of the underlying vascular smooth muscle through the production of vasodilator mediators. The endothelium-derived relaxing factors (EDRF) comprise nitric oxide (NO), prostacyclin, and a still elusive endothelium-derived hyperpolarizing factor (EDHF). Impaired endothelium-dependent vasodilation has been demonstrated in various vascular beds of different animal models of diabetes and in humans with type 1 and 2 diabetes. Several mechanisms of endothelial dysfunction have been reported, including impaired signal transduction or substrate availibility, impaired release of EDRF, increased destruction of EDRF, enhanced release of endothelium-derived constricting factors and decreased sensitivity of the vascular smooth muscle to EDRF. The principal mediators of hyperglycaemia-induced endothelial dysfunction may be activation of protein kinase C, increased activity of the polyol pathway, non-enzymatic glycation and oxidative stress. Correction of these pathways, as well as administration of ACE inhibitors and folate, has been shown to improve endothelium-dependent vasodilation in diabetes. Since the mechanisms of endothelial dysfunction appear to differ according to the diabetic model and the vascular bed under study, it is important to select clinically relevant models for future research of endothelial dysfunction.

Pressure-induced myogenic responses in isolated bovine retinal arteries.

PURPOSE: To investigate whether a pressure-induced myogenic vasoconstriction can be demonstrated in isolated bovine retinal arteries and to determine the cellular mechanisms involved. METHODS: Isolated bovine retinal arteries were mounted on a pressure myograph without flow and exposed to stepwise increases in intraluminal pressure. Changes in internal diameter were monitored continuously using an inverted microscope video system. RESULTS: Bovine retinal arteries showed myogenic tone at pressures higher than 10 mm Hg. This pressure-induced contraction was absent in calcium-free Krebs-Ringer bicarbonate solution. Inhibition of L-type voltage-operated calcium channels with nifedipine (1 microM) suppressed the myogenic contraction. After depolarization of the vascular smooth muscle cells with a K+ 120 mM solution, a pressure-induced contraction was still observed, indicating that besides stimulation of voltage-operated calcium channels, depolarization-independent mechanisms contribute to the pressure-induced myogenic vasoconstriction. CONCLUSIONS: Isolated bovine retinal arteries spontaneously contract when exposed to raised intraluminal pressure. This response depends on extracellular calcium and is blocked by nifedipine. In addition, depolarization-independent mechanisms seem to be involved.

EDHF-mediated relaxation in rat gastric small arteries: influence of ouabain/Ba2+ and relation to potassium ions.

In several blood vessels, endothelium-dependent vasorelaxation is in part mediated by an endothelium-derived hyperpolarizing factor (EDHF), the nature of which is as yet unknown. Experiments were performed to investigate whether the recently raised hypothesis that EDHF might be identified as the potassium ion, released by activation of endothelial K(Ca) channels and inducing relaxation by stimulation of Na+/K+-pump and the inward rectifier K+ conductance, might be valid for small rat gastric arteries. EDHF-induced relaxation (assessed as the nitro-L-arginine/indomethacin resistant component of acetylcholine-induced relaxation), but not nitroprus-side-induced relaxation is strongly inhibited in the presence of ouabain (0.5 mM)/Ba2+ (30 microM), ouabain being responsible for the greater part of the inhibition. This inhibition is reversible. Application of increasing concentrations of K+ elicits transient relaxations in some preparations, but in a greater part of the preparations, no or only small relaxations. In membrane potential measurements, it was found that increasing concentrations of extracellular K+ consistently depolarized smooth muscle cells, whereas acetylcholine elicits hyperpolarization. The K(Ca) channel openers NS 1619 and 1-EBIO elicit relaxation effects that are not diminished after removal of the endothelium and are not inhibited by ouabain/Ba2+. It is concluded that EDHF-mediated relaxation is sensitive to inhibition by ouabain/Ba2+, but that the relation of this inhibitory influence to an action of K+ as EDHF is uncertain.

Influence of some phospholipase A2 and cytochrome P450 inhibitors on rat arterial smooth muscle K+ currents.

The hyperpolarizing factor that is liberated by vascular endothelial cells in response to various agonists, and known to induce relaxation by opening of smooth muscle K+ channels, has been suggested to be a product of cytochrome P450 dependent arachidonic acid metabolism. In this study, the direct influence of two phospholipase A2 inhibitors and of five structurally and mechanistically different cytochrome P450 inhibitors on K+ currents in freshly isolated vascular smooth muscle cells from the rat aorta was investigated. On stepping the cell membrane potential from -70 mV to a series of depolarized test potentials, a noisy outward current developed at test potentials > +10 mV, which showed no appreciable inactivation during the voltage pulse. It was largely abolished by 3 mM external tetraethylammonium chloride (TEA), suggesting that it predominantly consisted of current through large-conductance Ca(2+)-activated K+ channels. The phospholipase A2 inhibitor quinacrine considerably inhibited this TEA-sensitive current, while 4-bromophenacylbromide exerted no effect. The cytochrome P450 inhibitors proadifen and miconazole reversibly decreased the amplitude of I(K), while clotrimazole and 1-aminobenzotriazole had no effect. Conversely, 17-octadecynoic acid increased whole-cell I(K). These results show that some phospholipase A2 and cytochrome P450 inhibitors may interfere with K+ channel activation in the rat arterial smooth muscle cell. The relevance of these findings to studies on the involvement of cytochrome P450 dependent metabolism in the generation of the endothelium-derived hyperpolarizing factor in intact arteries is discussed.

Protective effect of cromakalim and diazoxide, and proulcerogenic effect of glibenclamide on indomethacin-induced gastric injury.

We investigated the influences of the K+ channel opening drugs cromakalim and diazoxide and their blocker, glibenclamide, in indomethacin-induced gastric injury in rats. Cromakalim (0.1 and 0.3 mg/kg) and diazoxide (10 and 30 mg/kg) produced dose-dependent gastroprotection at doses that were also effective on the cardiovascular system. Glibenclamide reversed their gastroprotective effects and aggravated indomethacin-induced gastric damage by its own. Cromakalim (10(-9)-10(-5) M) and diazoxide (10(-9)-10(-4) M) relaxed noradrenaline pre-contracted gastric arteries (94.59+/-1.58% and 93.86+/-2.99%, respectively). Their relaxant effects were inhibited by glibenclamide (10(-5) M) but not by indomethacin (10(-5) M) and LG-nitro-L-arginine (10(-4) M). Cromakalim (0.1 and 0.3 mg/kg) did not change gastric mucosal blood flow but increased the gastric mucosal vascular conductance in anaesthetized rats as measured by the hydrogen gas clearance technique. Indomethacin increased myeloperoxidase activity in the gastric mucosa, an effect which was reversed by cromakalim and diazoxide. Glibenclamide abolished their effects on myeloperoxidase activity and, alone, increased this parameter. Additionally, indomethacin caused infiltration of neutrophils which was reduced by cromakalim and diazoxide in a glibenclamide sensitive manner. The effects of cromakalim and diazoxide on mucosal myeloperoxidase activity, neutrophil infiltration and gastric injury correlated with each other. The effects of diazoxide (30 mg/kg) and glibenclamide (10 mg/kg) on blood glucose level were not correlated with their effects on gastric injury. Taken together, K+ channel opening drugs show misoprostol-like protective effects in indomethacin-induced gastric injury which seems to be related to modulation of neutrophil function.