Role of nitric oxide and prostacyclin as vasoactive hormones released by the endothelium

The endothelium lines the luminal surface of every blood vessel, allowing it contact with circulating blood elements, as well as the underlying vascular smooth muscle layer. In healthy vessels, the endothelium expresses constitutive forms of nitric oxide synthase (NOSIII) and cyclo-oxygenase (COX-1), which produce the vasoactive hormones NO and prostacyclin, respectively. Both NO and prostacyclin relax blood vessels and inhibit platelet activation. The actions of prostacyclin are mediated by cell surface prostacyclin (IP) receptors and/or intracellular peroxisome proliferator-activated receptors (PPAR) beta. The actions of NO are mediated predominately by activation of intracellular guanylyl cyclase, leading to the formation of cGMP. In platelets, the actions of NO and prostacyclin are synergistic, but in vessels their actions are additive. In diseased vessels, inducible forms of NOS (NOSII) and cyclo-oxygeanse (COX-2) are expressed in vascular smooth muscle, resulting in the release of large amounts of NO, prostacyclin and prostaglandin E2. The relative contribution of NOSII and COX-2 to vascular inflammation is still debated, but is likely to result in both protective and damaging responses. The relative contribution of constitutive forms of NOS and COX, as well as interactions between IP, PPAR beta and guanylyl cyclase pathways in vessels and platelets, is discussed.     

Nitric oxide (NO) primarily accounts for endothelium-dependent component of beta-adrenoceptor-activated smooth muscle relaxation of mouse aorta in response to isoprenaline.

Isoprenaline is known to produce vascular relaxation through activation of beta-adrenoceptors. In recent years, beta-adrenoceptor-activated vascular relaxation has been the focus of pharmacological study in terms of both the receptor subtypes and the intracellular signaling mechanisms which trigger smooth muscle mechanical functions. In addition, the possible contribution of the endothelium to beta-adrenoceptor-activated relaxation of vascular beds has provoked considerable discussion, with consensus still to be established. In the present study, we examined the effects of isoprenaline on isolated mouse aortic smooth muscles to determine whether the presence of the endothelium plays a substantial role in the relaxation it produces. A possible role for nitric oxide (NO) as a primary endothelium-derived factor released in response to isoprenaline was also elucidated pharmaco-mechanically. In isolated thoracic and abdominal aortae pre-contracted with phenylephrine (3 x 10(-7)-10(-6) M), isoprenaline elicited relaxation in a concentration-dependent fashion (10(-9)-10(-5) M). In endothelium-denuded preparations, isoprenaline-elicited relaxation was reduced to 40-50% of the response obtained in endothelium-intact preparations. In the preparations treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 3 x 10(-4) M; an NO synthase inhibitor) or 1H-[1,2,4]-oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ, 10(-5) M; a soluble guanylyl cyclase inhibitor), isoprenaline-elicited relaxation was attenuated almost to the same degree as the response in endothelium-denuded preparations. The degree of endothelium-dependency in isoprenaline-elicited relaxation was largely diminished when treated with propranolol (3 x 10(-6) M). The present findings indicate that isoprenaline substantially relaxes the mouse aorta with both endothelium-dependent and -independent mechanisms. The endothelium-dependent component seems to correspond to about 50% of the isoprenaline-elicited relaxation, and is almost entirely due to endothelium-derived NO. Activation of propranolol (3 x 10(-6) M)-inhibitable beta-adrenoceptors seems to be primarily responsible for the NO-mediated endothelium-dependent pathway in isoprenaline-elicited relaxant response of mouse aorta.     

MaxiK channel mediates beta2-adrenoceptor-activated relaxation to isoprenaline through cAMP-dependent and -independent mechanisms in guinea-pig tracheal smooth muscle.

We examined the contribution of large-conductance, Ca(2+)-sensitive K+ (MaxiK) channel to beta2-adrenoceptor-activated relaxation to isoprenaline in guinea-pig tracheal smooth muscle focusing on the role for cAMP in the coupling between beta2-adrenoceptor and MaxiK channel. Isoprenaline-elicited relaxation was confirmed to be mediated through beta2-type of adrenoceptor since the response was antagonized in a competitive fashion by a beta2-selective adrenoceptor antagonist butoxamine with a pA2 value of 6.56. Isoprenaline-induced relaxation was significantly potentiated by a selective inhibitor of cyclic AMP-specific phosphodiesterase, Ro-20-1724 (0.1-1 microM). cAMP-dependent mediation of MaxiK channel in the relaxant response to isoprenaline was evidenced since the potentiated response to isoprenaline by the presence of Ro-20-1724 (1 microM) was inhibited by the channel selective blocker, iberiotoxin (IbTx, 100 nM). This concept was supported by the finding that the relaxation to a membrane permeable cAMP analogue, 8-bromo-cAMP (1 mM), was susceptible to the inhibition by IbTx. On the other hand, isoprenaline-induced relaxation was not practically diminished by an adenylyl cyclase inhibitor SQ 22,536 (100 microM). However, isoprenaline-induced relaxation in the presence of SQ 22,536 was suppressed by IbTx. Characteristics of isoprenaline-induced relaxant response, i.e., impervious to SQ 22,536 but susceptible to IbTx, were practically mimicked by cholera toxin (CTX, 5 microg/ml), an activator of adenylyl cyclase coupled-heterotrimeric guanine nucleotide-binding regulatory protein Gs. These findings indicate that in guinea-pig tracheal smooth muscle: 1) MaxiK channel substantially mediates beta2-adrenoceptor-activated relaxation; 2) both cAMP-dependent and -independent mechanisms underlie the functional coupling between beta2-adrenoceptor and MaxiK channel to induce muscle relaxation; and 3) direct regulation of MaxiK channel by Gs operates in cAMP-independent coupling between beta2-adrenoceptor and this ion channel.     

Smooth muscle dilatation in the human uterine artery induced by substance P, vasoactive intestinal polypeptide, calcitonin gene-related peptide and atrial natriuretic peptide: relation to endothelium-derived relaxing substances.

The relaxatory influences of substance P (SP), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and atrial natriuretic peptide (ANP) were investigated in human uterine arteries precontracted by noradrenaline in vitro. SP, VIP, CGRP and ANP all relaxed isolated uterine arteries with intact endothelium. When tested on vessels devoid of their endothelium VIP and SP had no effect on smooth muscular tone, while ANP and CGRP still induced unchanged vasodilatation. These results suggest an involvement of an endothelium-derived relaxing substance in the mechanisms by which VIP and SP induce relaxation of the isolated human uterine artery. On the other hand, ANP and CGRP seem to act on the same vessel preparation in vitro independently of the vascular endothelium. Both addition of noradrenaline and exchange of sodium against potassium in the organ chambers resulted in smooth muscle contraction irrespective of the integrity of the endothelium.     

The role of protease-activated receptors on the intracellular calcium ion dynamics of vascular smooth muscles, with special reference to cerebral arterioles

Protease-activated receptors (PARs) mediate cellular responses to various proteases in numerous cell types, including smooth muscles and the endothelium of blood vessels. To clarify whether the stimulation of PARs induces responses in smooth muscle cells of cerebral arterioles, intracellular Ca2+([Ca2+]i) dynamics and nitric oxide (NO) production during PARs stimulation were investigated in the rat cerebral arterioles by real-time confocal microscopy, since [Ca2+]i and NO are both key factors in the maintenance of strain in blood vessels. Testicular arterioles were also investigated for comparison. In smooth muscle cells of small cerebral arterioles (< 50 microm in diameter), thrombin and PAR1-activating peptide (AP) induced an increase in [Ca2+]i and contraction. The response to PAR1 activation was caused by Ca2+ mobilization from intracellular Ca2+ stores. Trypsin and PAR2-AP induced a decrease in [Ca2+]i in the cells which was considered to be mediated by endothelium-derived NO and/or by promoting a Ca2+ sequestration mechanism. PAR3- and 4-AP had little effect. In contrast to small cerebral arterioles, [Ca2+]i dynamics in smooth muscle cells of large cerebral arterioles (< 150 microm in diameter) or testicular arterioles remained unchanged during PARs activation. The effects of PARs activation on the [Ca2+]i dynamics and the contraction/relaxation of cerebral arterioles are also discussed in relation to the role of proteases in the regional tissue circulation of the brain.     

Epoxyeicosatrienoic acids and endothelium-dependent responses

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are produced by the vascular endothelium in response to agonists such as bradykinin and acetylcholine or physical stimuli such as shear stress or cyclic stretch. In the vasculature, the EETs have biological actions that are involved in the regulation of vascular tone, hemostasis, and inflammation. In preconstricted arteries in vitro, EETs activate calcium-activated potassium channels on vascular smooth muscle and the endothelium causing membrane hyperpolarization and relaxation. These effects are observed in a variety of arteries from experimental animals and humans; however, this is not a universal finding in all arteries. The mechanism of EET action may vary. In some arteries, EETs are released from the endothelium and are transferred to the smooth muscle where they cause potassium channel activation, hyperpolarization, and relaxation through a guanine nucleotide binding protein-coupled mechanism or transient receptor potential (TRP) channel activation. In other arteries, EETs activate TRP channels on the endothelium to cause endothelial hyperpolarization that is transferred to the smooth muscle by gap junctions or potassium ion. Some arteries use a combination of mechanisms. Acetylcholine and bradykinin increase blood flow in dogs and humans that is inhibited by potassium channel blockers and cytochrome P450 inhibitors. Thus, the EETs are endothelium-derived hyperpolarizing factors mediating a portion of the relaxations to acetylcholine, bradykinin, shear stress, and cyclic stretch and regulate vascular tone in vitro and in vivo.     

L—精氨酸,牛磺酸对大鼠血管损伤后血管平滑肌细胞增生的影响

在大鼠血管内皮剥脱模型上，观察了Ｌ－精氨酸，牛磺酸及Ｌ－精氨酸＋牛磺酸对血管损伤诱导的血管平滑肌细胞增生的影响。结果发现，内皮剥脱可致ＶＳＭＣ增生，同膜增厚；应用Ｌ－精氨酸，牛磺酸及Ｌ－精氨酸＋牛磺酸治疗后，剥脱组ＶＳＭＣ增生明显受抑制，且Ｌ－精氨酸＋牛磺酸对ＶＳＭＣ的抑制效应强于单纯应用Ｌ－精氨酸或牛磺酸组；Ｌ－精氨酸及Ｌ－精氨酸＿＋牛磺酸治疗后，血管对乙酰胆碱的内皮依赖性舒张，血ｃＧＭＰ含量明     

Beta1-adrenoceptor-mediated relaxation with isoprenaline and the role of MaxiK channels in guinea-pig esophageal smooth muscle.

The possible functional coupling between beta1-adrenoceptor and MaxiK channels which results in smooth muscle relaxation was examined in the guinea-pig esophageal muscularis mucosae. Isoprenaline-elicited relaxation of esophageal smooth muscle was confirmed to be mediated through beta1-adrenoceptors as the response was competitively antagonized by a beta1-selective antagonist atenolol with a pA2 value of 7.01. Iberiotoxin (IbTx, 10(-7) M), a selective MaxiK channel inhibitor, substantially diminished the relaxant response to isoprenaline. The extent of the MaxiK channel contribution to the relaxant response was 15-40% of the control response when estimated as the E50%-Emax responses to isoprenaline. The relaxation to isoprenaline was also attenuated by high-KCl (80 mM) to the same degree as the relaxant response generated in the presence of IbTx, and thus the estimated extent of the K+ channel contribution was 10-40%. These findings indicate that beta1-adrenoceptors are substantially coupled with MaxiK channels to produce relaxation of esophageal smooth muscle in the guinea-pig. Although MaxiK channels account for the contribution of K+ channels to the beta1-adrenoceptor-mediated relaxation in this smooth muscle preparation, their contribution seems to be less when compared to the beta2-adrenoceptor-mediated relaxation of tracheal smooth muscle.     

Functional integrity of endothelium determines Ca2+ channel availability in smooth muscle: involvement of nitric oxide

Endothelium regulates smooth muscle contractility in part via nitric oxide (NO). We tested the hypothesis that endothelial dysfunction, either produced by injury or simulated pharmacologically by reducing the bioavailability of NO, results in elevated Ca2+ channel availability (ngmax=maximum conductance/cell capacitance) in smooth muscle cells isolated from the vessel. Using basilar arteries of normotensive Wistar rats, we measured ngmax in smooth muscle cells from control vessels, from vessels in which endothelium was injured using Na fluoroscene plus light, and from vessels in which the bioavailability of NO was reduced by pretreatment with the NO scavenger 1H-imidazol-1 -yloxy,2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide , potassium salt (C-PTIO), or the endothelial nitric oxide synthase (eNOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Values of ngmax in these four groups of cells were 0.28+/-0.02 nS/pF (n=22), 0.51+/-0.05 nS/pF (n=15), 0.430+/-.03 nS/pF (n=12), and 0.47+/-0.04 nS/pF (n=14) (P<0.05, ANOVA), respectively. To determine whether larger currents associated with endothelial dysfunction exhibit altered sensitivity to exogenous NO, we quantified the response to various concentrations of NO donor, Na nitroprusside (SNP), in 37 cells from control vessels and 33 cells from vessels pretreated with L-NAME. SNP exhibited identical potency (half-maximum values, 18.7 and 21.1 nM) but greater apparent efficacy (maximum fractional block, 0.82 versus 0.63) in down-regulating Ca2+ channel currents in cells isolated from vessels with dysfunctional endothelium. Our results are consistent with a direct influence of endogenous NO on Ca2+ channel availability in smooth muscle cells, and indicate that Ca2+ channel availability in isolated smooth muscle cells may be a sensitive measure of the functional integrity of the endothelium in the parent vessel.     

Endothelial dysfunction and vascular disease – a 30th anniversary update

The endothelium can evoke relaxations of the underlying vascular smooth muscle, by releasing vasodilator substances. The best‐characterized endothelium‐derived relaxing factor (EDRF) is nitric oxide (NO) which activates soluble guanylyl cyclase in the vascular smooth muscle cells, with the production of cyclic guanosine monophosphate (cGMP) initiating relaxation. The endothelial cells also evoke hyperpolarization of the cell membrane of vascular smooth muscle (endothelium‐dependent hyperpolarizations, EDH‐mediated responses). As regards the latter, hydrogen peroxide (H₂O₂) now appears to play a dominant role. Endothelium‐dependent relaxations involve both pertussis toxin‐sensitive G_i (e.g. responses to α₂‐adrenergic agonists, serotonin, and thrombin) and pertussis toxin‐insensitive G_q (e.g. adenosine diphosphate and bradykinin) coupling proteins. New stimulators (e.g. insulin, adiponectin) of the release of EDRFs have emerged. In recent years, evidence has also accumulated, confirming that the release of NO by the endothelial cell can chronically be upregulated (e.g. by oestrogens, exercise and dietary factors) and downregulated (e.g. oxidative stress, smoking, pollution and oxidized low‐density lipoproteins) and that it is reduced with ageing and in the course of vascular disease (e.g. diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis toxin‐sensitive pathway for NO release which favours vasospasm, thrombosis, penetration of macrophages, cellular growth and the inflammatory reaction leading to atherosclerosis. In addition to the release of NO (and EDH, in particular those due to H₂O₂), endothelial cells also can evoke contraction of the underlying vascular smooth muscle cells by releasing endothelium‐derived contracting factors. Recent evidence confirms that most endothelium‐dependent acute increases in contractile force are due to the formation of vasoconstrictor prostanoids (endoperoxides and prostacyclin) which activate TP receptors of the vascular smooth muscle cells and that prostacyclin plays a key role in such responses. Endothelium‐dependent contractions are exacerbated when the production of nitric oxide is impaired (e.g. by oxidative stress, ageing, spontaneous hypertension and diabetes). They contribute to the blunting of endothelium‐dependent vasodilatations in aged subjects and essential hypertensive and diabetic patients. In addition, recent data confirm that the release of endothelin‐1 can contribute to endothelial dysfunction and that the peptide appears to be an important contributor to vascular dysfunction. Finally, it has become clear that nitric oxide itself, under certain conditions (e.g. hypoxia), can cause biased activation of soluble guanylyl cyclase leading to the production of cyclic inosine monophosphate (cIMP) rather than cGMP and hence causes contraction rather than relaxation of the underlying vascular smooth muscle.     

Multiple sites of oxygen sensing and their contributions to hypoxic pulmonary vasoconstriction

Oxygen sensing by the pulmonary vasculature is important for the regulation of vessel tone and the matching of lung perfusion to ventilation. Airways hypoxia is a major stimulus for vasoconstriction, which diverts blood from hypoxic alveoli to better ventilated areas of the lung. Several hypotheses have emerged to explain how pulmonary arteries sense a decrease in oxygen and mediate hypoxic pulmonary vasoconstriction (HPV). They differ mainly in where they place the main site of HPV: in the endothelial or smooth muscle cells of the artery wall. HPV probably results from synergistic actions on both cell types, but it can proceed in the absence of endothelium, suggesting that the primary oxygen sensor is the smooth muscle cell and endothelium-derived agents modulate the muscle response. Several oxygen-sensing targets have been identified in smooth muscle, including potassium channels, Ca(2+) stores in the sarcoplasmic reticulum (SR) and the Ca(2+) sensitivity of the contractile proteins. The evidence for different oxygen-sensing mechanisms in pulmonary vessels is discussed.     

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca²⁺-activated K⁺ channels channels by InsP₃-induced Ca²⁺ release from the endoplasmic reticulum and/or store-operated Ca²⁺ entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.     

Regulation of arterial blood pressure by Akt1-dependent vascular relaxation

Endothelial cell-dependent vascular relaxation plays an important role in the regulation of blood pressure. Here, we show that stimulation of vascular endothelial cells with platelet-derived growth factor (PDGF) results in vascular relaxation through Akt1-dependent activation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. Stimulation of both human umbilical artery endothelial cells and abdominal aortic vessels with PDGF induced NO production. PDGF-dependent production of NO was completely abolished by inhibition of phosphatidylinositol 3-kinase with wortmannin (100 nM). Stimulation of aortic vessels with PDGF resulted in the activation of Akt phosphorylation and eNOS phosphorylation: however, eNOS phosphorylation and production of NO were abolished in aortic vessels of mice lacking Akt1. PDGF strongly induced vascular relaxation in the presence of endothelium, and inhibition of NO production by N-nitro-l-arginine-methyl ester completely blocked PDGF-dependent vascular relaxation. In addition, PDGF-dependent relaxation was completely abolished by inhibition of PI3K with wortmannin (100 nM). Furthermore, vessels from Akt1 heterozygotes showed normal relaxation after PDGF stimulation, whereas vessels from Akt1 knockout littermates did not respond to PDGF stimulation. Finally, administration of PDGF (5 ng/ml) significantly lowered blood pressure in Akt1 heterozygotes, whereas a blood pressure-lowering effect was not observed in Akt1 knockout littermates. These results suggest that Akt1 regulates blood pressure through regulation of vascular relaxation by eNOS phosphorylation and subsequent production of NO.     

Proteinase-activated receptor 2 activation modulates guinea-pig mesenteric lymphatic vessel pacemaker potential and contractile activity

Lymphatic vessels rhythmically constrict to avoid fluid and protein accumulation in the interstitial space. This activity is critical during inflammation to prevent excessive oedema. Lymphatic pumping is intrinsic to the smooth muscle in the vessel wall and is due to the spontaneous occurrence of action potentials, the pacemaker of which is proposed to be spontaneous transient depolarizations (STDs). This function is highly susceptible to the fluid load and modulated by chemical agents, amongst which inflammatory mediators are important players. Activation of proteinase-activated receptors (PARs) has been involved in inflammation and affects vascular smooth muscle tone. The present study aims to investigate the role of PAR₂, a member of the PAR family, in lymphatic vessel pumping. RT-PCR experiments revealed that PAR₂ message is present in lymphatic vessels of the guinea-pig mesentery. Agonists of PAR₂ such as trypsin and the activating peptide, SLIGRL-NH₂, caused a decrease in the contractile activity of intraluminally perfused lymphatic vessels. Moreover, intracellular microelectrode recordings from isolated vessels revealed that PAR₂ activation hyperpolarized the lymphatic smooth muscle membrane potential and altered STD amplitude and frequency. The decreases in constriction frequency and STD activity as well as the hyperpolarization were dependent on a functional endothelium, not affected by NO synthase or guanylyl-cyclase inhibition, but mimicked by PGE₂ and iloprost and blocked by indomethacin (10 μ m ) and glibenclamide (1 μ m ). These results show that PAR₂ activation alters guinea-pig lymphatic vessel contractile and electrical activity via the production of endothelium-derived cyclo-oxygenase metabolites.     

MaxiK channel-triggered negative feedback system is preserved in the urinary bladder smooth muscle from streptozotocin-induced diabetic rats.

MaxiK channel, the large-conductance Ca2+-sensitive K+ channel, facilitates a negative feedback mechanism to oppose excitation and contraction in various types of smooth muscles including urinary bladder smooth muscle (UBSM). In this study, we investigated how the contribution of MaxiK channel to the regulation of basal UBSM mechanical activity is altered in streptozotocin-induced diabetic rats. Although the urinary bladder preparations from both control and diabetic rats were almost quiescent in their basal mechanical activities, they generated spontaneous rhythmic contractions in response to a MaxiK channel blocker, iberiotoxin (IbTx). The effect of IbTx on the mechanical activity was significantly greater in diabetic rat than in control animal. Similarly, the basal mechanical activity was increased with apamin, an inhibitor for some types of small conductance Ca2+-sensitive K+ channels, and this effect was more pronounced for diabetic rat. However, in both control and diabetic animals, IbTx action was stronger than that of apamin. Diabetes also enhanced the responses to BayK 8644, an L-type Ca2+ channel agonist. The extent of this enhancement in diabetic bladder vs. control was, however, almost the same as that attained with IbTx. Expression levels for MaxiK channel as well as apamin-sensitive K+ channels and L-type Ca2+ channel were not altered by diabetes, when determined as their corresponding mRNA levels. These results indicate that diabetes can potentially increase the basal UBSM mechanical activity. However, in diabetic UBSM, the main negative-feedback system triggered by MaxiK channel is still preserved enough to counteract the possible enhancement of this smooth muscle mechanical activity.     

Studies on the mechanisms underlying beta-adrenoceptor-mediated relaxation of rat abdominal aorta.

Mechanisms underlying beta-adrenoceptor (beta-AR)-mediated vascular relaxation were studied in the isolated rat abdominal aorta. In the endothelium-denuded helical preparations, a non-selective beta-AR agonist isoprenaline elicited a concentration-dependent relaxation. In the absence of beta-AR antagonists, isoprenaline-induced relaxation was not practically affected by an adenylyl cyclase inhibitor SQ 22,536 (300 microM), but was strongly diminished by high-KCl (80 mM). Isoprenaline-induced relaxation in the presence of SQ 22,536 was significantly diminished by iberiotoxin (IbTx, 0.1 microM), but was not affected by 4-aminopyridine (4-AP, 3 mM). Isoprenaline-induced relaxation was not also affected by SQ 22,536 (300 microM) even in the presence of CGP20712A (a beta(1)-selective antagonist) and ICI-118,551 (a beta(2)-selective antagonist) (0.1 microM for each), but was strongly diminished by high-KCl. By contrast, SQ 22,536-resistant, isoprenaline-induced relaxation in the presence of CGP20712A plus ICI-118,551 was not affected by IbTx (0.1 microM), but was inhibited significantly by 4-AP (3 mM). These results suggest that in rat abdominal aortic smooth muscle: 1) both beta(1)-/beta(2)-AR- and beta(3)-AR-mediated relaxations substantially involve cAMP-independent mechanisms; 2) beta(1)-/beta(2)-AR-mediated, cAMP-independent relaxant mechanisms are partly attributed to the large-conductance, Ca (2+)-sensitive K(+) (MaxiK, BK) channel whereas beta(3)-AR-mediated relaxant mechanisms are attributed to K(v) channel.     

Role of endothelium in the response of the vein wall to magnesium withdrawal

Complete absence of magnesium has a two-fold effect on the arterial tone: direct smooth muscle contraction and relaxation via endothelium-derived relaxing factor (EDRF) release. In the present study performed on a systemic vein we investigated (1) which of these effects dominates following reduction of magnesium concentration from 1.2 mM to 0.8 and 0.4 mM and (2) whether the vessel segments asymmetrically respond when the magnesium concentration is reduced on either the intra- or extraluminal side. The effects of reducing magnesium concentration on both the isometric tension of isolated ring preparations and the diameter of isolated, perfused and superfused feline femoral veins were investigated. In nor-adrenaline-precontracted rings, rapid decreases in the extracellular magnesium concentration from 1.2 mM to 0.8 and 0.4 mM caused relaxation, whereas total omission of magnesium returned the tone to the level of the initial tone induced by noradrenaline. Both in the presence of haemoglobin (5×10^–6M), and in vessels without endothelium, lowering the magnesium concentration caused a dose-dependent elevation of the noradrenaline-induced tone. In perfused and superfused noradrenaline-contracted vein segments, each reduction of extraluminal magnesium concentration caused contraction of the vessels, regardless of whether the endothelium was intact or not. A decrease in intraluminal magnesium concentration did not alter the diameter of the vessel when the endothelium was intact, but caused contraction when the endothelium was disrupted. The results of the present study demonstrate that both the reduction of magnesium concentration or its complete absence cause an EDRF-mediated relaxation and a directly mediated smooth muscle contraction in the femoral vein of the cat. Within the physiological range of extracellular magnesium concentrations, however, the EDRF-mediated relaxation seems to dominate.     

Endothelium-dependent vascular smooth muscle relaxation activated by electrical field stimulation

Electrical field stimulation (EFS) produced relaxation of contracted arteries in the presence of tetrodotoxin. In the present study the contributions of vascular smooth muscle repolarization and endothelial release of nitric oxide to the relaxation response were investigated using isolated rat tail arteries and bovine aortic endothelial cells (BAEC). Intact and endothelium-denuded rings or intact, pressurized artery segments were contracted with either phenylephrine or KCl prior to EFS. Electrical field stimulation induced a small relaxation in denuded, phenylephrine contracted rings that was inhibited by the K⁺ channel blockers glibenclamide and BaCl₂ In intact, phenylephrine-contracted rings, EFS induced significantly larger relaxations that were inhibited by BaCl₂ as well as by _LNAME, an inhibitor of nitric oxide (NO) synthase, and methylene blue. EFS-induced relaxations were completely inhibited when BaCl₂ and _L-NAME or methylene blue were combined. Exposure to Ca2^+- free buffer or diltiazem also inhibited the relaxation while ascorbic acid had no effect. Effluent from electrically stimulated BAEC caused denuded, phenylephrine contracted rings to relax. The ability of the effluent to cause relaxation was almost completely blocked by exposure of the BAEC to _L-NAME or exposure of the recipient vascular smooth muscle to methylene blue; glibenclamide caused partial blockade. Simultaneous measurements of membrane potential and intraluminal pressure showed that EFS-induced membrane repolarization preceded changes in steady-state pressure. It is concluded that (1) the smooth muscle cells possess an endothelium-independent repolarization mechanism, (2) EFS causes endothelial cells of intact arteries to release NO and possibly a hyperpolarizing factor, (3) EFS of BAEC causes release of NO, and (4) EFS-induced relaxation depends on vascular smooth muscle cell membrane repolarization and endothelial cell release of vasoactive substances.     

Metabolic control of muscle blood flow during exercise in humans.

During muscle contraction, several mechanisms regulate blood flow to ensure a close coupling between muscle oxygen delivery and metabolic demand. No single factor has been identified to constitute the primary metabolic regulator, yet there are signal transduction pathways between skeletal muscle and the vasculature that induce vasodilation. A link between muscle metabolic events and microvascular control of blood flow is illustrated by local dilation of terminal arterioles during contraction of muscle fibers and conduction of vasodilation upstream. Endothelial-derived vasodilator mechanisms are known to exert control of muscle vasodilation. Adenosine, nitric oxide (NO), prostacyclin (PGI2), and endothelial-derived hyperpolarization factor (EDHF) are possible mediators of muscle vasodilation during exercise. In humans, adenosine has been shown to contribute to functional hyperemia as blood flow is reduced under nonselective adenosine-receptor blockade. No clear role has been demonstrated for either NO or PGI2(2), based on studies employing selective inhibition of these substances individually, suggesting a redundancy of vasodilator mechanisms. This is supported by recent work demonstrating that combined blockade of NOS and PGI2, and NOS and cytochrome P450, both attenuate exercise-induced hyperemia in humans. Combined vasodilator blockade studies offer the potential to uncover important interactions and compensatory vasodilator responses. The signaling pathways that link metabolic events evoked by muscle contraction to vasodilatory signals in the local vascular bed remains an important area of study.     

Effects of nitric oxide donors and inhibitors of nitric oxide signalling on endothelin- and serotonin-induced contractions in human placental arteries

In order to explore the role of nitric oxide (NO) in the control of fetoplacental vascular tone in normal pregnancy we have examined the effects of NO donors on uteroplacental arteries pre-contracted with the vasoconstrictor endothelin-1 (ET-1) or serotonin (5-HT). We have furthermore examined the effects of guanylate cyclase inhibitors on the NO-induced relaxation. Segments of placental arteries (n=102) obtained from 39 placentas immediately after delivery were mounted in organ baths and superfused with Krebs-Ringer solution at 37 degrees C. The vessel segments were exposed to drugs for various intervals and the tension was recorded isometrically and registered on a polygraph. Cyclic guanosine monophosphate (cGMP) analysis was performed after extraction of vessel segments using a specific radioimmunoassay. The placental artery segments responded to ET-1 and 5-HT with a dose-dependent vasoconstriction. After pre-contraction with ET-1 (10(-7) M) or 5-HT (10(-6) M), the vessels relaxed in response to the NO donors glyceryltrinitrate (GTN) (10(-6) M) and S-nitroso-N-acetyl-penicillamine (SNAP) (10(-5) M). In the presence of the non-specific guanylate cyclase inhibitor LY 83583 (10(-6) M), the vessels responded with a small contraction. In the presence of the soluble guanylate cyclase (sGC) inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) the non-treated vessels responded with a relaxation. 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one gave no obvious relaxation in pre-contracted vessels. Addition of 8-Br-cGMP, the cell-permeant analogue of cGMP, with or without pre-contraction had no effect on the vessels. Cyclic guanosine monophosphate analysis showed that GTN treatment caused an increase in cGMP after 12 min. Our results indicate that NO acts as a vasodilator in placental vessels. The cGMP-dependent mechanisms may be involved in NO-induced relaxation but cGMP-independent mechanisms appear also to be involved.