Marianne Tare

K+ currents underlying the action of endothelium-derived hyperpolarizing factor in guinea-pig, rat and human blood vessels.

1 Membrane currents attributed to endothelium‐derived hyperpolarizing factor (EDHF) were recorded in short segments of submucosal arterioles of guinea‐pigs using single microelectrode voltage clamp. The functional responses of arterioles and human subcutaneous, rat hepatic and guinea‐pig coronary arteries were also assessed as changes in membrane potential recorded simultaneously with contractile activity. 2 The current‐voltage (I‐V) relationship for the conductance due to EDHF displayed outward rectification with little voltage dependence. Components of the current were blocked by charybdotoxin (30‐60 nM) and apamin (0.25‐0.50 μM), which also blocked hyperpolarization and prevented EDHF‐induced relaxation. 3 The EDHF‐induced current was insensitive to Ba2+ (20‐100 μM) and/or ouabain (1 μM to 1 mM). 4 In human subcutaneous arteries and guinea‐pig coronary arteries and submucosal arterioles, the EDHF‐induced responses were insensitive to Ba2+ and/or ouabain. Increasing [K+]o to 11‐21 mM evoked depolarization under conditions in which EDHF evoked hyperpolarization. 5 Responses to ACh, sympathetic nerve stimulation and action potentials were indistinguishable between dye‐labelled smooth muscle and endothelial cells in arterioles. Action potentials in identified endothelial cells were always associated with constriction of the arterioles. 6 18β‐Glycyrrhetinic acid (30 μM) and carbenoxolone (100 μM) depolarized endothelial cells by 31 ± 6 mV (n= 7 animals) and 33 ± 4 mV (n= 5), respectively, inhibited action potentials in smooth muscle and endothelial cells and reduced the ACh‐induced hyperpolarization of endothelial cells by 56 and 58 %, respectively. 7 Thus, activation of outwardly rectifying K+ channels underlies the hyperpolarization and relaxation due to EDHF. These channels have properties similar to those of intermediate conductance (IKCa) and small conductance (SKCa) Ca2+‐activated K+ channels. Strong electrical coupling between endothelial and smooth muscle cells implies that these two layers function as a single electrical syncytium. The non‐specific effects of glycyrrhetinic acid precludes its use as an indicator of the involvement of gap junctions in EDHF‐attributed responses. These conclusions are likely to apply to a variety of blood vessels including those of humans.

Stretch revealed three components in the hyperpolarization of guinea‐pig coronary artery in response to acetylcholine.

1. Membrane potential was recorded with intracellular microelectrodes from the smooth muscle of coronary arteries of guinea‐pigs, and the responses to endothelium‐derived relaxants were studied under a variety of conditions. 2. Stimulation of the endothelium with brief applications of acetylcholine or substance P evoked concentration‐dependent hyperpolarizations that were complex in nature. A transient component, which is likely to result from endothelium‐derived hyperpolarizing factor (EDHF), was followed by a slow component that resulted from the production of nitric oxide (NO) and a prostaglandin. 3. The ability of exogenous and endogenous NO and prostacyclin to hyperpolarize the membrane depended upon the smooth muscle being under stretch. Unstretched preparations responded to acetylcholine with only the transient component of hyperpolarization; NO and prostacyclin were without effect. 4. In stretched preparations exogenous NO and prostacyclin, and its synthetic analogue methyl prostacyclin (Iloprost), evoked hyperpolarization, and the slow component of the response induced by acetylcholine appeared. The amplitudes of these responses reached maximum when the tissues were stretched to the equivalent of approximately 50 mmHg. 5. From a resting membrane potential of ‐61 +/‐ 0.6 mV, exogenous NO and Iloprost hyperpolarized the smooth muscle to around ‐80 mV. The EC50 values for NO‐ and Iloprost‐induced hyperpolarization were 2.6 x 10(‐6) and 1.3 x 10(‐8) M, respectively. 6. Coronary arterial smooth muscles from rats, rabbits and sheep also hyperpolarized in response to exogenous NO, although their sensitivities were less than those of preparations obtained from guinea‐pigs. Iloprost hyperpolarized tissues from rabbits and sheep but not those obtained from rats. 7. It is concluded that the endothelial lining of coronary arteries can release three factors, EDHF, NO and prostacyclin, all of which can hyperpolarize the membrane of the smooth muscle. The relative proportions and significance of each factor depends on the amount of stretch, on the artery and on the species of animal.

Uteroplacental insufficiency causes a nephron deficit, modest renal insufficiency but no hypertension with ageing in female rats

In rats, uteroplacental insufficiency induced by uterine vessel ligation restricts fetal growth and impairs mammary development compromising postnatal growth. In male offspring, this results in a nephron deficit and hypertension which can be reversed by improving lactation and postnatal growth. Here, growth, blood pressure and nephron endowment in female offspring from mothers which underwent bilateral uterine vessel ligation (Restricted) on day 18 of pregnancy were examined. Sham surgery (Control) and a reduced litter group (Reduced at birth to 5, equivalent to Restricted group) were used as controls. Offspring (Control, Reduced, Restricted) were cross‐fostered on postnatal day 1 onto a Control (normal lactation) or Restricted (impaired lactation) mother. Restricted‐on‐Restricted offspring were born small but were of similar weight to Control‐on‐Control by postnatal day 35. Blood pressure was not different between groups at 8, 12 or 20 weeks of age. Glomerular number was reduced in Restricted‐on‐Restricted offspring at 6 months without glomerular hypertrophy. Cross‐fostering a Restricted pup onto a Control dam resulted in a glomerular number intermediate between Control‐on‐Control and Restricted‐on‐Restricted. Blood pressure, along with renal function, morphology and mRNA expression, was examined in Control‐on‐Control and Restricted‐on‐Restricted females at 18 months. Restricted‐on‐Restricted offspring did not become hypertensive but developed glomerular hypertrophy by 18 months. They had elevated plasma creatinine and alterations in renal mRNA expression of transforming growth factor‐β1, collagen IV (α1) and matrix matelloproteinase‐9. This suggests that perinatally growth restricted female offspring may be susceptible to onset of renal injury and renal insufficiency with ageing in the absence of concomitant hypertension.

Intrauterine growth restriction delays cardiomyocyte maturation and alters coronary artery function in the fetal sheep

There is now extensive evidence suggesting that intrauterine perturbations are linked with an increased risk of developing cardiovascular disease. Human epidemiological studies, supported by animal models, have demonstrated an association between low birth weight, a marker of intrauterine growth restriction (IUGR), and adult cardiovascular disease. However, little is known of the early influence of IUGR on the fetal heart and vessels. The aim of this study was to determine the effects of late gestational IUGR on coronary artery function and cardiomyocyte maturation in the fetus. IUGR was induced by placental embolization in fetal sheep from 110 to 130 days of pregnancy (D110–130); term ∼D147; control fetuses received saline. At necropsy (D130), wire and pressure myography was used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in small branches of left descending coronary arteries. Myocardium was dissociated for histological analysis of cardiomyocytes. At D130, IUGR fetuses (2.7 ± 0.1 kg) were 28% lighter than controls (3.7 ± 0.3 kg; P= 0.02). Coronary arteries from IUGR fetuses had enhanced responsiveness to the vasoconstrictors, angiotensin II and the thromboxane analogue U46619, than controls (P < 0.01). Endothelium‐dependent and ‐independent relaxations were not different between groups. Coronary arteries of IUGR fetuses were more compliant (P= 0.02) than those of controls. The incidence of cardiomyocyte binucleation was lower in the left ventricles of IUGR fetuses (P= 0.02), suggestive of retarded cardiomyocyte maturation. We conclude that late gestational IUGR alters the reactivity and mechanical wall properties of coronary arteries and cardiomyocyte maturation in fetal sheep, which could have lifelong implications for cardiovascular function.

Endothelial potassium channels, endothelium‐dependent hyperpolarization and the regulation of vascular tone in health and disease

1. The elusive nature of endothelium‐derived hyperpolarizing factor (EDHF) has hampered detailed study of the ionic mechanisms that underlie the EDHF hyperpolarization and relaxation. Most studies have relied on a pharmacological approach in which interpretations of results can be confounded by limited specificity of action of the drugs used. Nevertheless, small‐, intermediate‐ and large‐conductance Ca2+‐activated K+ channels (SKCa, IKCa and BKCa, respectively) have been implicated, with inward rectifier K+ channels (KIR) and Na+/K+‐ATPase also suggested by some studies.

A role for nitroxyl (HNO) as an endothelium-derived relaxing and hyperpolarizing factor in resistance arteries

Background and purpose:  Nitroxyl (HNO) is emerging as an important regulator of vascular tone as it is potentially produced endogenously and dilates conduit and resistance arteries. This study investigates the contribution of endogenous HNO to endothelium‐dependent relaxation and hyperpolarization in resistance arteries.

Vitamin D insufficiency is associated with impaired vascular endothelial and smooth muscle function and hypertension in young rats

Non‐Technical Summary  Adverse environments during early life are linked with an increased risk of cardiovascular disease. There is an alarming increase in the prevalence of vitamin D (VitD) deficiency in women of reproductive age. We show that male and female rat offspring that were exposed to VitD deficiency in the womb and early life have high blood pressure. The arteries from VitD deficient offspring have an impaired ability to relax due to deficiencies in the production of two important factors, nitric oxide and endothelium‐derived hyperpolarizing factor. VitD deficient female offspring have an additional impairment in the nitric oxide signalling pathway in the arterial muscle. The findings of this study are particularly relevant for women intending to become pregnant. Ensuring VitD sufficiency before and during pregnancy in women will reduce the burden of cardiovascular disease risk in their offspring.

Role of membrane potential in endothelium‐dependent relaxation of guinea‐pig coronary arterial smooth muscle.

1. Membrane potential and tension were measured simultaneously in ring segments of main coronary artery of guinea‐pigs. The synthetic thromboxane A2 analogue U46619 depolarized the tissues from ‐58 +/‐ 2 to ‐40 +/‐ 1 mV and increased tension by 12 +/‐ 1 mN mm‐1. Nitric oxide (NO) and Iloprost, the stable analogue of prostacyclin, evoked hyperpolarization and relaxation. 2. The concentration of NO required to evoke half‐maximal hyperpolarization (EC50 of 2 x 10(‐5) M) was 40‐fold higher than that which was required to induce relaxation (EC50 of 5 x 10(‐7) M). The EC50 for Iloprost‐induced hyperpolarization (3 x 10(‐8) M) was similar to that for relaxation (4 x 10(‐8) M). 3. Glibenclamide (10(‐6) M) abolished the hyperpolarization in response to both NO and Iloprost but was without effect on the amplitudes of the relaxations over the complete concentration‐response curves. 4. Acetylcholine evoked concentration‐dependent hyperpolarization and relaxation in the presence of N omega‐nitro‐L‐arginine methyl ester (NAME; 10(‐5) M) and indomethacin (10(‐6) M), and these responses were attributed to endothelium‐derived hyperpolarizing factor (EDHF). The hyperpolarization produced by EDHF always preceded relaxation, and relaxation never occurred at concentrations of acetylcholine that were insufficient to evoke hyperpolarization. 5. The concentration‐hyperpolarization and concentration‐relaxation curves in response to acetylcholine were not affected by glibenclamide or barium (1‐3 mM) but were shifted to the right 4‐ and 5‐fold, respectively, by 1 mM tetraethylammonium. The hyperpolarization and relaxation evoked by acetylcholine were also reduced in a parallel manner when the potassium concentration in the superfusate was increased. 6. Hyperpolarizing current steps, applied to spiral strips of coronary artery denuded of endothelium and depolarized and constricted with U46619, caused relaxation. The relationship between hyperpolarization and relaxation evoked electronically was similar to that which was due to EDHF in intact tissues stimulated with acetylcholine. 7. It is concluded that the ability of NO or Iloprost to relax guinea‐pig coronary artery does not depend upon hyperpolarization of the smooth muscle. In contrast, hyperpolarization is likely to play a major, if not the only, role in the relaxation in response to EDHF in this tissue.

Uteroplacental insufficiency programs regional vascular dysfunction and alters arterial stiffness in female offspring

Intrauterine growth restriction caused by uteroplacental insufficiency increases the risk of cardiovascular disease in adulthood. Vascular mechanisms in female offspring are poorly understood. The aim of this study was to investigate the effects of uteroplacental insufficiency on blood pressure, vascular reactivity and arterial stiffness in four vascular beds in female offspring born growth restricted. Uteroplacental insufficiency was induced on day 18 of gestation in Wistar Kyoto rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Controls). Wire and pressure myography were used to test endothelial and smooth muscle function, and passive mechanical wall properties, respectively, in uterine, mesenteric, renal and femoral arteries of 18‐month‐old female offspring. Collagen and elastin fibres were quantified using circular crossed‐polarized light microscopy and quantitative real time polymerase chain reaction. Restricted female offspring were born 10–15% smaller. Restricted females were normotensive, had plasma triglycerides 2‐fold elevated and had uterine endothelial dysfunction, attributed to a 23% reduction in the maximal relaxation produced by endothelium‐derived hyperpolarizing factor. Uterine artery stiffness was increased, with an augmented proportion of thick and decreased proportion of thin collagen fibres. Vascular reactivity and mechanical wall properties were preserved in mesenteric, renal and femoral arteries in growth restricted females. Female offspring born growth restricted have selective uterine artery endothelial dysfunction and increased wall stiffness. The preserved vascular function in other arteries may explain the lack of hypertension in these females. The uterine artery specific dysfunction has potential implications for impaired pregnancy adaptations and a compromised intrauterine environment of the next generation.

Role for endothelium-derived hyperpolarizing factor in vascular tone in rat mesenteric and hindlimb circulations in vivo

The role of endothelium‐derived hyperpolarizing factor (EDHF) in the regulation of blood flow in vivo was examined in the mesenteric and hindlimb circulations of anaesthetized rats. Basal mesenteric conductance decreased from 57 ± 5 to 20 ± 6 μl min−1 mmHg−1 when nitric oxide (NO) production was inhibited, and combined blockade of intermediate‐ and small‐conductance Ca2+‐activated K+ (KCa) channels with charybdotoxin (ChTx) and apamin had no further effect. Basal hindlimb conductance was reduced from 39 ± 3 to 22 ± 2 μl min−1 mmHg−1 by NO synthesis inhibition, with no effect of the KCa channel blockers. Endothelial stimulation with acetylcholine (ACh) infusion directly into the mesenteric bed increased conductance by 20 ± 2 μl min−1 mmHg−1. Blockade of NO synthesis decreased this conductance to 15 ± 1 μl min−1 mmHg−1, leaving the response attributable to EDHF. This was reduced to 2 ± 1 μl min−1 mmHg−1 by ChTx plus apamin but not by iberiotoxin, which selectively blocks large‐conductance KCa channels. Similar results were obtained when bradykinin (BK) was used to stimulate the endothelium. Nitroprusside, which directly relaxes smooth muscle, evoked an increase in conductance that was resistant to all blockers tested. ACh‐induced increases in hindlimb conductance were reduced from 19 ± 1 to 12 ± 1 μl min−1 mmHg−1 by NO synthesis inhibition and further reduced to 2 ± 2 μl min−1 mmHg−1 by ChTx plus apamin. In contrast to NO, ChTx‐ and apamin‐sensitive EDHF appears to contribute little to basal conductance in rat mesenteric and hindlimb beds. However, EDHF accounts for a significant component of the conductance increase during endothelial stimulation by ACh and BK. In these beds, intermediate‐ and small‐conductance KCa channels underpin EDHF‐mediated vasodilatation.