Inhibitory effect of tetrabutylammonium ions on endothelium/nitric oxide-mediated vasorelaxation

Apart from the well-described K+ channel blocking effects in vascular smooth muscle cells, monovalent quaternary ammonium ions may also interact with endothelial cells in the endothelium-intact mammalian arteries. The present study was aimed to examine the effect of tetrabutylammonium ions on endothelium-dependent and -independent relaxation in the rat isolated aortic rings. Pretreatment with tetrabutylammonium concentration dependently reduced the endothelium-dependent relaxation induced by acetylcholine, cyclopiazonic acid and ionomycin. Tetrabutylammonium also inhibited endothelium-independent relaxation induced by hydroxylamine or nitroprusside. Pretreatment of endothelium-denuded rings with tetrabutylammonium did not affect relaxation induced by NS1619 or by diltiazem. In contrast, tetrabutylammonium significantly reduced the pinacidil- or cromakalim-induced relaxation. Tetrabutylammonium also inhibited the acetylcholine- but not nitroprusside-induced increase of tissue content of cyclic GMP in the aortic rings. The present study indicates that tetrabutylammonium ions could inhibit endothelial and exogenous nitric oxide-mediated aortic relaxation while it had no effect on relaxation induced by activation of Ca2+-activated K+ channels (by NS1619) or by inhibition of voltage-gated Ca2+ channels (by diltiazem). The inhibitory effect on pinacidil- and cromakalim-induced relaxation suggests that tetrabutylammonium ions also inhibit ATP-sensitive K+ channels in aortic smooth muscle cells.     

A longitudinal study of radiation-induced changes in tumor vasculature by contrast-enhanced magnetic resonance imaging

Dynamic contrast-enhanced MRI with two different-sized contrast agents, Gd-DTPA and Gadomer-17, was used to study the effects of radiation on the pharmacokinetics of the paramagnetic enhancement of water relaxation in the rat R3230 AC adenocarcinoma tumor model. The kinetics of enhancement was analyzed by a two-compartment pharmacokinetic model to derive parameters related to vascular volume (V(b)) and permeability (K(2)). Rats implanted with tumors were divided into two groups; one received 5 Gy and the other received 20 Gy (137)Cs gamma rays. Sequential dynamic contrast-enhanced MRI studies were performed, one before irradiation, one at day 1 after irradiation, and another at day 3 after irradiation, to investigate the effect of the radiation dose and the changes that occurred over time. The analysis was performed on a pixel-by-pixel basis to study the heterogeneity within the tumor. The pixel distribution profiles of V(b) and K(2) from each tumor were obtained to assess the regional radiation-induced effects on vascular volume and permeability. No significant change in vascular volume was detected with either Gd-DTPA or Gadomer-17 after irradiation of the tumor; however, a small dependence of K(2) on the radiation dose was observed. After low-dose (5 Gy) irradiation, the mean value of K(2) decreased by 46% at day 1 compared to the baseline, presumably due to cell swelling, and decreased further by 67% from the baseline on day 3. When the dose was increased to 20 Gy, the mean value of K(2) measured with Gadomer-17 did not show any significant changes at either day 1 or day 3 after irradiation. The value of K(2) measured with Gd-DTPA did not show any significant changes after either the low or the high radiation dose.     

Divergent actions of chronic insulin treatment in vivo versus acute treatment ex vivo on diabetic-induced endothelial dysfunction

Streptozotocin-induced diabetes is associated with hyperglycemia and hypoinsulinemia. The role of insulin in diabetes-induced endothelial dysfunction is unknown. In the present study, we evaluated the effect of chronic insulin treatment in vivo versus acute insulin administration ex vivo on endothelium-dependent relaxation in aortic rings of the streptozotocin-induced diabetic rat. Relaxation to acetylcholine (but not A23187) was impaired in diabetic compared to control rings. This defect was prevented by chronic insulin treatment but was not reversed by acute insulin administration ex vivo. Thus, endothelial dysfunction in the streptozotocin-induced diabetic rat is specific for the chronic diabetic state and not to vascular toxicity of streptozotocin. Nevertheless, it is apparent that insulin at a physiological concentration does not cause an acute direct effect on facilitating endothelium-dependent relaxation in diabetic blood vessels.     

Involvement of Na(+)/Ca(2+) exchanger in endothelial NO production and endothelium-dependent relaxation

Endothelial nitric oxide (NO) synthase (eNOS) is controlled by Ca(2+)/calmodulin and caveolin-1 in caveolae. It has been recently suggested that Na(+)/Ca(2+) exchanger (NCX), also expressed in endothelial caveolae, is involved in eNOS activation. To investigate the role played by NCX in NO synthesis, we assessed the effects of Na(+) loading (induced by monensin) on rat aortic rings and cultured porcine aortic endothelial cells. Effect of monensin was evaluated by endothelium-dependent relaxation of rat aortic rings in response to acetylcholine and by real-time measurement of NO release from cultured endothelial cells stimulated by A-23187 and bradykinin. Na(+) loading shifted the acetylcholine concentration-response curve to the left. These effects were prevented by pretreatment with the NCX inhibitors benzamil and KB-R7943. Monensin potentiated Ca(2+)-dependent NO release in cultured cells, whereas benzamil and KB-R7943 totally blocked Na(+) loading-induced NO release. These findings confirm the key role of NCX in reverse mode on Ca(2+)-dependent NO production and endothelium-dependent relaxation.     

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca²⁺-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.     

Modulation of endothelial and smooth muscle function by bed rest and hypoenergetic, low-fat nutrition.

Prolonged microgravity alters the regulation of the peripheral vasculature. The influence of reduced food intake, as often observed in astronauts, on vascular function is unclear. In a randomized, four-phase, crossover study, the effect of simulated microgravity (13 days of bed rest), energetic restriction (-25%, fat reduced), and their combination on endothelium-dependent and -independent vasodilation was compared with ambulatory control conditions. Using venous occlusion plethysmography, cumulative intra-arterial dose-response curves to endothelium-dependent (acetylcholine) and -independent (sodium nitroprusside) vasodilators were constructed in 10 healthy male volunteers before and on day 13 of each of the four intervention periods. Bed rest combined with normoenergetic nutrition impaired the dose-response to acetylcholine (ANOVA, P = 0.004) but not to sodium nitroprusside, whereas hypoenergetic diet under ambulatory conditions improved responses to acetylcholine (P = 0.044) and sodium nitroprusside (P < 0.001). When bed rest was combined with hypoenergetic diet, acetylcholine responses did not change. Similarly, under control conditions, no change was observed. Individual changes in the total cholesterol-to-HDL ratio were correlated with changes in endothelial and vascular smooth muscle relaxation. In conclusion, short-term bed rest impairs endothelium-dependent arterial relaxation in humans. A hypoenergetic, low-fat diet modulates serum lipids, improves endothelium-dependent and -independent relaxation, and may antagonize the unfavorable effects of simulated microgravity on endothelial function.     

Effect of tumor necrosis factor on hypoxic pulmonary vasoconstriction.

The effects of tumor necrosis factor (TNF) on hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent relaxation were examined in a blood-perfused rat lung preparation. Lungs from TNF-treated rats (0.26 mg/kg iv 12 h before experimentation) had a significantly greater HPV and a reduced vasorelaxant response to the endothelium-dependent vasodilator acetylcholine (ACh) but a similar vasorelaxant response to the endothelium-independent vasodilator nitroprusside compared with lungs from control rats (pretreated with 0.1 ml saline iv). Pentoxifylline (20 mg/kg iv and ip 20 min before administration of TNF) had no detectable effect on either HPV or ACh-induced relaxation but completely negated the augmentation on HPV and the inhibiting action on ACh-induced relaxation caused by TNF. The TNF effect on ACh relaxation was unaffected by pretreatment with L-arginine. These results indicate that TNF induces endothelial dysfunction and enhances HPV, effects that are inhibited by pentoxifylline.     

Sulodexide improves endothelial dysfunction in streptozotocin-induced diabetes in rats

Diabetes mellitus is associated with many complications including retinopathy, nephropathy, neuropathy and angiopathy. Increased cardiovascular risk is accompanied with diabetes-induced endothelial dysfunction. Pharmacological agents with endothelium-protective effects may decrease cardiovascular complications. In present study sulodexide (glycosaminoglycans composed from heparin-like and dermatan fractions) was chosen to evaluate its protective properties on endothelial dysfunction in diabetes. Effect of sulodexide treatment (SLX, 100 UI/kg/day, i.p.) in 5 and 10 weeks lasting streptozotocin-induced diabetes (30 mg/kg/day, i.p. administered for three consecutive days) was investigated. Animals were divided into four groups: control (injected with saline solution), control-treated with sulodexide (SLX), diabetic (DM) and diabetic-treated with sulodexide (DM+SLX). The pre-prandial and postprandial plasma glucose levels, number of circulating endothelial cells (EC) and acetylcholine-induced relaxation of isolated aorta and mesenteric artery were evaluated. Streptozotocin elicited hyperglycemia irrespective of SLX treatment. Streptozotocin-induced diabetes enhanced the number of circulating endothelial cells compared to controls. SLX treatment decreased the number of EC in 10-week diabetes. Acetylcholine-induced relaxation of mesenteric arteries was significantly impaired in 5 and 10-week diabetes. SLX administration improved relaxation to acetylcholine in 5 and 10-week diabetes. Diabetes impaired acetylcholine-induced relaxation of rat aorta irrespective of SLX treatment. Our results demonstrate that SLX treatment lowers the number of circulating endothelial cells and improves endothelium-dependent relaxation in small arteries. These findings suggest endothelium-protective effect of sulodexide in streptozotocin-induced diabetes.     

Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome

Recent studies implicate channels of the transient receptor potential vanilloid family (e.g., TRPV1) in regulating vascular tone; however, little is known about these channels in the coronary circulation. Furthermore, it is unclear whether metabolic syndrome alters the function and/or expression of TRPV1. We tested the hypothesis that TRPV1 mediates coronary vasodilation through endothelium-dependent mechanisms that are impaired by the metabolic syndrome. Studies were conducted on coronary arteries from lean and obese male Ossabaw miniature swine. In lean pigs, capsaicin, a TRPV1 agonist, relaxed arteries in a dose-dependent manner (EC50 = 116 +/- 41 nM). Capsaicin-induced relaxation was blocked by the TRPV1 antagonist capsazepine, endothelial denudation, inhibition of nitric oxide synthase, and K+ channel antagonists. Capsaicin-induced relaxation was impaired in rings from pigs with metabolic syndrome (91 +/- 4% vs. 51 +/- 10% relaxation at 100 microM). TRPV1 immunoreactivity was prominent in coronary endothelial cells. TRPV1 protein expression was decreased 40 +/- 11% in obese pigs. Capsaicin (100 microM) elicited divalent cation influx that was abolished in endothelial cells from obese pigs. These data indicate that TRPV1 channels are functionally expressed in the coronary circulation and mediate endothelium-dependent vasodilation through a mechanism involving nitric oxide and K+ channels. Impaired capsaicin-induced vasodilation in the metabolic syndrome is associated with decreased expression of TRPV1 and cation influx.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombomodulin as a marker of radiation-induced endothelial cell injury.

Cultured confluent human umbilical vein endothelial cells were irradiated in vitro with 60Co gamma rays at doses from 0 to 50 Gy. After irradiation thrombomodulin was measured at different times over 6 days in the supernatants of endothelial cell culture medium, on the surface of the cells, and within the cells. At 24 h after irradiation, an increase in the release of thrombomodulin from irradiated endothelial cells and an increase in the number of molecules and the activity of thrombomodulin on the surface of the cells were observed; these reactions were dependent on radiation dose. The capacity of the cells to produce and release thrombomodulin was decreased from 2 to 6 days after exposure to 60Co gamma rays. Our data indicate that radiation can injure endothelial cells, and that thrombomodulin may be used as a marker of radiation-induced injury in endothelial cells. The interrelationship between the dysfunction of irradiated endothelial cells and the pathological mechanisms of acute radiation disease is also discussed.     

Ionic permeability of sarcoplasmic reticulum membranes damaged by x-rays

As early as 1 and 24 h following single local X-irradiation (0.21 C/kg) of rabbit hindlimbs an increase was noted in the permeability of skeletal muscle sarcoplasmic reticulum membranes for Ca2+, K+ and Na+. The effect was maximum 1 h after irradiation and more pronounced for K+ and Na+ than Ca2+.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

High K(+)-induced membrane depolarization attenuates endothelium-dependent pulmonary vasodilation

Impairment of endothelium-dependent pulmonary vasodilation has been implicated in the development of pulmonary hypertension. Pulmonary vascular smooth muscle cells and endothelial cells communicate electrically through gap junctions; thus, membrane depolarization in smooth muscle cells would depolarize endothelial cells. In this study, we examined the effect of prolonged membrane depolarization induced by high K(+) on the endothelium-dependent pulmonary vasodilation. Isometric tension was measured in isolated pulmonary arteries (PA) from Sprague-Dawley rats, and membrane potential was measured in single PA smooth muscle cells. Increase in extracellular K(+) concentration from 4.7 to 25 mM significantly depolarized PA smooth muscle cells. The 25 mM K(+)-mediated depolarization was characterized by an initial transient depolarization (5-15 s) followed by a sustained depolarization that could last for up to 3 h. In endothelium-intact PA rings, ACh (2 microM), levcromakalim (10 microM), and nitroprusside (10 microM) reversibly inhibited the 25 mM K(+)-mediated contraction. Functional removal of endothelium abolished the ACh-mediated relaxation but had no effect on the levcromakalim- or the nitroprusside-mediated pulmonary vasodilation. Prolonged ( approximately 3 h) membrane depolarization by 25 mM K(+) significantly inhibited the ACh-mediated PA relaxation (-55 +/- 4 vs. -29 +/- 2%, P < 0.001), negligibly affected the levcromakalim-mediated pulmonary vasodilation (-92 +/- 4 vs. -95 +/- 5%), and slightly but significantly increased the nitroprusside-mediated PA relaxation (-80 +/- 2 vs. 90 +/- 3%, P < 0. 05). These data indicate that membrane depolarization by prolonged exposure to high K(+) concentration selectively inhibited endothelium-dependent pulmonary vasodilation, suggesting that membrane depolarization plays a role in the impairment of pulmonary endothelial function in pulmonary hypertension.     

Male-female differences in the relative contribution of endothelial vasodilators released by rat tail artery

Several different vasodilator substances can be released by vascular endothelium in response to mechanical stimuli and vasoactive agents. The purpose of this study was to determine whether there is a male-female difference in the relative contributions of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent vasodilation. Perfusion pressure was measured in isolated tail arteries from male and female rats. Vasodilators released by mechanical shear stress were assessed by constricting the artery with methoxamine; acetylcholine was applied to induce receptor-mediated vasodilation. We used an inhibitor of NO synthase, N(G)-monomethyl-L-arginine acetate (L-NMMA), and elevated levels of K(+) (27 mM) to reveal the relative contributions of NO and EDHF, respectively. Indomethacin was present in all experiments to block prostanoid production. The results indicate that NO was the primary vasodilator released by male tail arteries in response to both mechanical stress and acetylcholine (the L-NMMA-sensitive component of the combined L-NMMA/K(+) effect was 83 +/- 8% and 101 +/- 4%, respectively). However female tail arteries appeared to utilize both NO and EDHF for vascular relaxation (e.g., L-NMMA sensitivity: 58 +/- 9%; K+-sensitivity: 42 +/- 9% in mechanical stress experiments). These findings suggest endothelial regulation differs between males and females.     

Mutation induction by very low dose rate gamma rays in cultured mouse leukemia cells L5178Y

Induction of cell killing and mutation to 6-thioguanine resistance was studied in growing mouse leukemia cells in culture following gamma rays at dose rates of 30 Gy/h, 20 cGy/h, and 6.3 mGy/h, i.e., acute, low dose rate, and very low dose rate irradiation. A marked increase was observed in the cell survival with decreasing dose rate; no reduction in the surviving fraction was detected after irradiation at 6.3 mGy/h until a total dose of 4 Gy. Similarly, the induced mutation frequency decreased after low dose rate irradiation compared to acute irradiation. However, the frequency after irradiation at 6.3 mGy/h was unexpectedly high and remained at a level which was intermediate between acute and low dose rate irradiation. No appreciable changes were observed in the responses to acute gamma rays (in terms of cell killing and mutation induction) in the cells which had experienced very low dose rate irradiation.     

The suppression of metastases and the change in host immune response after low-dose total-body irradiation in tumor-bearing rats.

We have shown that metastasis is suppressed by low-dose total-body irradiation (TBI) in tumor-bearing rats. We have evaluated the immunological effects of low-dose TBI. Total-body irradiation with 0.2 Gy was given 14 days after the implantation of 5 x 10(5) allogenic hepatoma cells (KDH-8) which produce transforming growth factor beta (TGF-beta). On day 21, the splenocytes and tumor-tissue infiltrating lymphocytes were analyzed by FACScan and RT-PCR for the mRNA of the genes that encode tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), TGF-beta, interleukin (IL)-4, IL-10 and IL-6. The same procedure was conducted with untreated rats and with rats that underwent local irradiation with 0.2 Gy. The low-dose TBI significantly decreased the incidence of lung and lymph node metastasis (P < 0.01), whereas the same dose of local irradiation had no effect on the incidence of metastasis. The proportion of CD8+ cells in splenocytes increased in the low-dose TBI group (P < 0.01) compared to the locally irradiated and the untreated groups. The tumor-tissue infiltrating lymphocytes were also significantly increased after low-dose TBI (P < 0.01). The FACScan analysis revealed that 72% of the tumor-tissue infiltrating lymphocytes were CD8+. In both spleen and tumor tissue after low-dose TBI, mRNA expression of the genes that encode IFN-gamma and TNF-alpha increased, while that of the Tgfb gene decreased. There was no expression of the mRNAs of the Il4, Il6 and Il10 genes. CD8+ cells and the cytokine network may play an important role in the antitumor effect of low-dose TBI.     

Involvement of endothelium/nitric oxide in vasorelaxation induced by purified green tea (-)epicatechin

The present study investigated the involvement of endothelial nitric oxide in relaxation induced by purified green tea (-)epicatechin in rat isolated mesenteric arteries. (-)Epicatechin caused both endothelium-dependent and -independent relaxation. NG-Nitro-L-arginine methyl ester (L-NAME, 100 microM) and methylene blue (10 microM) significantly attenuated (-)epicatechin-induced relaxation in endothelium-intact tissues. L-Arginine (1 mM) partially antagonized the effect of L-NAME. (-)Epicatechin-induced relaxation was inhibited by Rp-guanosine 3',5'-cyclic monophosphothioate triethylamine. In contrast, indomethacin and glibenclamide had no effect. (-)Epicatechin (100 microM) significantly increased the tissue content of cyclic GMP and NG-nitro-L-arginine (100 microM) or removal of the endothelium abolished this increase. (-)Epicatechin (100 microM) induced an increase in intracellular Ca2+ levels in cultured human umbilical vein endothelial cells. Iberiotoxin at 100 nM attenuated (-)epicatechin-induced relaxation in endothelium-intact arteries and this effect was absent in the presence of 100 microM L-NAME. In summary, (-)epicatechin-induced endothelium-dependent relaxation is primarily mediated by nitric oxide and partially through nitric oxide-dependent activation of iberiotoxin-sensitive K+ channels. In addition, there may be a causal link between increased Ca2+ levels and nitric oxide release in response to (-)epicatechin.