Kirsty M. McCulloch

5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats.

1 We investigated 5‐hydroxytryptamine (5‐HT)‐receptor mediated vasoconstriction in the main, first branch and resistance pulmonary arteries removed from control and pulmonary hypertensive rats. Contractile responses to 5‐HT, 5‐carboxamidotryptamine (5‐CT, non‐selective 5‐HT1 agonist), and sumatriptan (5‐HT1D‐like receptor agonist) were studied. The effects of methiothepin (non‐selective 5‐HT1+2‐receptor antagonist) and ketanserin (5‐HT2A receptor antagonist) and GR55562 (a novel selective 5‐HT1D receptor antagonist) on 5‐HT‐mediated responses were also studied. Basal levels of adenosine 3′:5′‐cyclic monophosphate ([cyclic AMP]i) and guanosine 3′:5′‐cyclic monophosphate ([cyclic GMP]i) were determined and we assessed the degree of inherent tone in the vessels under study. 2 5‐HT was most potent in the resistance arteries. pEC50 values were 5.6 ± 0.1, 5.3 ± 0.1, 5.0 ± 0.2 in the resistance arteries, pulmonary branch and main pulmonary artery, respectively (n = at least 5 from 5 animals). The sensitivity to, and maximum response of, 5‐HT was increased in all the arteries removed from the chronic hypoxic (CH) rats. In CH rats the pEC50 values were 5.9 ± 0.2, 6.3 ± 0.2, 6.4 ± 0.2 and the increase in the maximum response was 35%, 51% and 41% in the resistance arteries, pulmonary branch and main pulmonary artery, respectively. Sumatriptan did not contract any vessel from the control rats whilst 5‐CT did contract the resistance arteries. In the CH rats, however, they both contracted the resistance arteries (responses to sumatriptan were small) (pEC50: 5‐CT; 5.4 ± 0.2) and the pulmonary artery branches (pEC50: sumatriptan, 5.4 ± 0.2; 5‐CT, 5.4 ± 0.2). 5‐CT also caused a contraction in the main pulmonary artery (pEC50: 6.0 ± 0.3). 3 Ketanserin (1 nM‐1 μm) caused a competitive antagonism of the 5‐HT response in all vessels tested. In control rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 8.3, 7.8 and 9.2, respectively. Methiothepin (1 nM‐1 μm) inhibited responses to 5‐HT in the first branch (estimated pKb value: 7.8) and main pulmonary artery. In CH rats, the estimated pKb values for ketanserin in resistance arteries, pulmonary branches and main pulmonary artery were 7.7, 8.3 and 9.6, respectively. Methiothepin also inhibited contractions to 5‐HT in the pulmonary artery branch and main pulmonary artery with estimated pKb values of 7 and 9.5, respectively. In control animals, GR55562 had no effect on responses to 5‐HT in any of the vessels tested. In the CH rats the estimated pKb values for GR55562 were 6.5, 7.8 and 7.0 in the pulmonary resistance arteries, first branch and main pulmonary artery, respectively. 4 Large pulmonary arteries from controls demonstrated inherent tone and this was increased three fold in the CH rats. The resistance arteries from controls demonstrated little inherent tone though this was enhanced in those from the CH rats. 5 [Cyclic AMP]i was 259 ± 23 pmol mg−1 protein in the pulmonary artery branches removed from control rats and decreased to 192 ± 11 pml mg−1 protein in the CH rats (P < 0.01, n = 8). [Cyclic GMP]i also decreased in the pulmonary artery branches (from 550 ± 15, control to 462 ± 31 pmol mg−1 protein in CH vessels, n = 8, P < 0.01) and in the main pulmonary arteries (from 566 ± 33, control to 370 ± 25 pmol mg−1 protein in CH vessels, n = 8, P < 0.001). No changes in either [cyclic AMP]i or [cyclic GMP]i were observed in the resistance arteries. 6 The results suggest that the increased vasoconstrictor response to 5‐HT in CH rat pulmonary arteries is due to an increase in 5‐HT2A‐receptor mediated contraction combined with an increase in r5‐HT1B‐like receptor‐mediated contraction. An increase in vascular tone and decreased levels of [cyclic GMP]i in the large pulmonary arteries may contribute to the observed increase in activity of r5‐HT1B‐like receptors.

Endothelin ETA- and ETB-receptor-mediated vasoconstriction in rat pulmonary arteries and arterioles.

Summary: We investigated the endothelin (ET) receptors involved in the vasoconstrictor responses to ET-1 in rat pulmonary arteries and arterioles and the effect of endo-thelium removal, nitric oxide (NO) synthase inhibition, and hypoxia on ET-1-induced responses in the arteries. In isolated rat pulmonary artery rings (2–3 mm ID) prepared from the pulmonary artery branch before its entry into the lung, ET-1-induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were competitively antagonized by the ETA receptor antagonist FR 139317, and the ETB-receptor agonist sarafotoxin S6c (SXS6c) was a very weak vasoconstrictor in these vessels, inducing maximum contractions only 9% of those of ET-1. In contrast, in rat intrapulmonary resistance arteries (100–150 μm ID), SXS6c induced FR 139317-resistant contractions, and these vessels were more sensitive to SXS6c than to ET-1. SXS6c produced maximum contractions 92% those of ET-1, suggesting that ET-1-induced contractions were mediated by the ETB receptor in these resistance vessels. In the larger pulmonary arteries, the NO synthase inhibitor L-N ω ni-troarginine methyl ester (L-NAME) (100 μM) potentiated responses to ET-1, an effect that was reversed by FR 139317. Endothelium removal also potentiated response to ET-1, and L-NAME had no effect on ET-1 responses in endothelium-denuded vessels, suggesting that in these vessels the ETA receptor-mediated responses to ET-1 are normally suppressed by endothelium-derived NO. Hypoxia did not affect the sensitivity of the vessels to ET-1, but did increase the ability of FR 139317 to antagonise these responses. L-NAME did not affect responses to SXS6c in pulmonary resistance vessels.

EndothelinB receptor-mediated contraction in human pulmonary resistance arteries.

1 Using wire myography, we have examined the endothelin (ET) receptor subtypes mediating vasoconstriction to ET peptides in human pulmonary resistance arteries (150–200 μm, i.d.). 2 Cumulative concentration‐response curves to ET‐1, sarafotoxin 6c (SX6c) and ET‐3 were constructed in the presence and absence of the selective antagonists FR 139317 (ETA‐selective), BMS 182874 (ETA‐selective) and BQ‐788 (ETB‐selective). 3 All agonists induced concentration‐dependent contractions. However, the response curves to ET‐1 were biphasic in nature. The first component demonstrated a shallow slope up to 1 nM ET‐1. Above 1 nM ET‐1 the response curve was markedly steeper. Maximum responses to ET‐3 and SX6c were the same as those to 1 nM ET‐1 and 30% of those to 0.1 μm ET‐1. The order of potency, taking 0.3 μm as a maximum concentration was SX6c > > ET‐3 > ET‐1 (pEC50 values of: 10.75 ± 0.27, 9.05 ± 0.19, 8.32 ± 0.08 respectively). Taking 1 nM ET‐1 as a maximum, the EC50 for ET‐1 was 10.08 ± 0.13 and therefore ET‐1 was equipotent to ET‐3 and SX6c over the first component of the response curve. 4 Responses to ET‐1 up to 1 nM were resistant to the effects of the ETA receptor antagonists, FR 139317 and BMS 182874 but were inhibited by the ETB receptor antagonist, BQ‐788. Conversely, responses to ET‐1 over 1 nM were inhibited by the ETA receptor antagonists, FR 139317 and BMS 182874 but unaffected by the ETB receptor antagonist, BQ‐788. 5 The results suggest that at concentrations up to 1 nM, responses to ET‐1 are mediated via the ETB receptor, whilst the responses to higher concentrations are mediated by ETA receptors.

Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat.

We examined the endothelin (ET) receptors mediating contractions to ET‐1, ET‐3 and sarafotoxin S6c (SX6c) in rat pulmonary resistance arteries by use of peptide and non‐peptide ET receptor antagonists. Changes induced by pulmonary hypertension were examined in the chronically hypoxic rat. The effect of the mixed ETA/ETB receptor antagonist SB 209670 on endothelin‐mediated contraction was also examined in human pulmonary resistance arteries. In rat vessels, the order of potency for the endothelin agonists was SX6c=ET‐3>ET‐1 (pEC50 values in control rats: 9.12±0.10, 8.76±0.14 and 8.12±0.04, respectively). Maximum contractions induced by ET‐3 and ET‐1 were increased in vessels from chronically hypoxic rats. The ETA receptor antagonist FR 139317 (1 μM) had no effect on the potency of ET‐1 in any vessel studied but abolished the increased response to ET‐1 in the chronically hypoxic vessels. The ETA receptor antagonist BMS 182874 (1 μM) increased the potency of ET‐1 in control rat vessels without effecting potency in the pulmonary hypertensive rat vessels. Bosentan (non‐peptide mixed ETA/ETB receptor antagonist) increased the potency of ET‐1 in control rat vessels but was without effect in the pulmonary hypertensive rat vessels. Bosentan (1 μM) inhibited responses to SX6c in control and chronically hypoxic rat vessels with pKb values of 5.84 and 6.11, respectively. The ETB receptor antagonist BQ‐788 (1 μM) did not inhibit responses to ET‐1 in any vessel tested but did inhibit responses to both SX6c and ET‐3 (pKb values in control and chronically hypoxic rat vessels respectively: SX6c 7.15 and 7.22; ET‐3: 6.68 and 6.89). BQ‐788 (1 μM) added with BMS 182874 (10 μM) did not inhibit responses to ET‐1 in control vessels but caused a significant inhibition of responses to ET‐1 in chronically hypoxic preparations. SB 209670 inhibited responses to ET‐1 in both control and chronically hypoxic vessels with pKb values of 7.36 and 7.39, respectively. SB 209670 (0.1 and 1 μM) virtually abolished responses to ET‐1 in the human pulmonary resistance artery. In conclusion, in rat pulmonary resistance arteries, vasoconstrictions induced by ET‐1, SX6c and ET‐3 are mediated predominantly by activation of an ETB–like receptor. However, lack of effect of some antagonists on ET‐1 induced vasoconstriction suggests that ET‐1 stimulates an atypical ETB receptor. The increase in potency of ET‐1 in the presence of some antagonists suggests the presence of an inhibitory ETA‐like receptor. The influence of this is reduced, or absent, in the chronically hypoxic rats. Increased responses to ET‐1 are observed in the chronically hypoxic rat and may be mediated by increased activation of ETA receptors. SB 209670 is unique in its potency against responses to ET‐1 in both control and chronically hypoxic rats, as well as human, isolated pulmonary resistance arteries.

Effects of pulmonary hypertension on vasoconstrictor responses to endothelin-1 and sarafotoxin S6C and on inherent tone in rat pulmonary arteries.

Vasoconstrictor responses to endothelin-1 (ET-1) and the ETB receptor agonist sarafotoxin S6c (SXS6c) were investigated in the main pulmonary artery and pulmonary artery branch removed from rats previously exposed to 10% O2 [chronic hypoxic (CH) rats] or room air (control rats) for 2 weeks. The effects of nitric oxide synthase (NOS) inhibition with L-Nomega-nitroarginine methyl ester (L-NAME) (100 microM) on ET receptor-induced responses in these arteries were also investigated. In control rats, in rings of main pulmonary arteries and pulmonary artery branches. ET-1 induced vasoconstrictor responses. These responses were mediated by the ETA receptor as they were antagonized by the ETA receptor antagonist FR 139317 whereas SXS6c did not vasoconstrict. Chronic hypoxia had no effect on the sensitivity of the main pulmonary arteries to ET-1, whereas small vasoconstrictor responses to SCS6c were evident. ET-1 was more potent in the CH rat pulmonary artery branches than in controls. SXS6c also caused vasoconstriction with a maximum response 30% of that to ET-1 in both endothelium-intact and endothelium-denuded vessels. L-NAME increased the sensitivity to ET-1 in the CH rat main pulmonary arteries and increased the responses to low concentrations of ET-1 in the control rat main pulmonary arteries but did not affect any ET-1 responses in any other vessels. It did disclose responses to SXS6c in control rat main pulmonary arteries. L-NAME itself increased vascular tone to a greater extent in CH rat pulmonary arteries than in controls. In preconstricted pulmonary arteries, however, relaxations to acetylcholine (ACh) were diminished in the CH rats as compared with their controls. All pulmonary artery branches, denuded of their vascular endothelium, relaxed to sodium nitroprusside (SNP) and therefore exhibited endogenous vascular tone. This effect was greatest in the pulmonary artery branches from the CH rats. The results suggest that rat large pulmonary artery responses to ET-1 are normally mediated by ETA receptors. Pulmonary hypertension can potentiate ETA receptor-mediated vasoconstriction and facilitate ETB receptor-mediated vasoconstriction. Endogenous NO may normally suppress ETA receptor-mediated responses in rat main pulmonary arteries. Rat pulmonary arteries exhibit endogenous tone, which is increased by exposure to chronic hypoxia.

Influences of the endothelium and hypoxia on α1- and α2-adrenoceptor-mediated responses in the rabbit isolated pulmonary artery

1 The effects of the inhibitor of nitric oxide synthase, Nω‐nitro‐l‐arginine methylester (l‐NAME, 10−4 m), mechanical disruption of the endothelium and hypoxia on contraction to noradrenaline (α1‐ and α2‐adrenoceptor agonist), phenylephrine (α1‐adrenoceptor agonist) and UK 14304 (α2‐adrenoceptor agonist) were compared in the rabbit isolated pulmonary artery. The effects of the selective antagonists rauwolscine (10−6 m, α2‐adrenoceptors) and prazosin (10−7 m, α1‐adrenoceptors) on the contractions to noradrenaline before and after exposure to l‐NAME were also assessed. 2 Noradrenaline, phenylephrine and UK 14304 all produced concentration‐dependent increases in vascular tone. The responses to noradrenaline were sensitive to both rauwolscine and prazosin (effect of prazosin >> rauwolscine). l‐NAME increased the potency of both noradrenaline and UK 14304, and also the maximum tension achieved. It had no effect on the responses to phenylephrine. After l‐NAME, contractions to noradrenaline, although still sensitivie to both rauwolscine and prazosin, were now more sensitive to inhibition by rauwolscine. 3 Endothelium removal augmented the potency and maximum contractions to noradrenaline, phenylephrine and UK 14304. 4 Hypoxia decreased both the potency of phenylephrine and its maximum contractile response, but increased the maximum response to noradrenaline without effecting responses to UK 14304. 5 In conclusion, in the rabbit pulmonary artery, augmentation of contractile responses to noradrenaline by l‐NAME involves a potentiation of α2‐adrenoceptor‐mediated contraction probably through an effect on the synthesis of endothelium‐derived nitric oxide. Experimental hypoxia had differential effects on all three agonists and did not mimic the effect of nitric oxide synthase inhibition.

Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia

1 The effect of basal tension (transmural tensions 235±29 mg wt (low tension: equivalent to ∼16 mmHg) and 305±34 mg wt (high tension: equivalent to ∼35 mmHg)) on rat pulmonary resistance artery responses to endothelin‐1 (ET‐1) and the selective ETB‐receptor agonist sarafotoxin S6c (S6c) were studied. The effects of nitric oxide synthase inhibition with NΩ‐nitro‐L‐arginine methylester (L‐NAME, 100 μM) on ET receptor‐induced responses, as well as vasodilator responses to acetylcholine (ACh) and S6c, were also investigated. Changes with development of pulmonary hypertension, induced by two weeks of chronic hypoxia, were determined. 2 Control rat preparations showed greatest sensitivity for ET‐1 when put under low tension (pEC50: 8.1±0.1) compared with at the higher tension (pEC50: 7.7±0.1) and there were significant increases in maximum contractile responses to S6c (∼80%) and noradrenaline (∼60%) when put under high tension. 3 In control pulmonary resistance arteries, both ET‐1 and S6c produced potent vasoconstrictor responses. S6c was 12 fold more potent than ET‐1 in vessels set at low tension (S6c pEC50: 9.2±0.1) and 200 fold more potent than ET‐1 when the vessels were set at high tension (S6c pEC50: 9.0±0.1). Chronic hypoxia did not change the potencies of ET‐1 and S6c but did significantly increase the maximum contractile response to ET‐1 by 60% (at low tension) and 130% (at high tension). 4 In control rat vessels, L‐NAME itself caused small increases in vascular tone (5–8 mg wt tension) in 33–56% of vessels. In the chronic hypoxic rats, in vessels set at high tension, L‐NAME‐induced tone was evident in 88% of vessels and had increased to 26.9±6.6 mg wt tension. Vasodilatation to sodium nitroprusside, in non‐preconstricted vessels, was small in control rat vessels (2–6 mg wt tension) but increased significantly to 22.5±8.0 mg wt tension in chronic hypoxic vessels set at the higher tensions. Together, these results indicate an increase in endogenous tone in the vessels from the chronic hypoxic rats which is normally attenuated by nitric oxide production. 5 L‐NAME increased the sensitivity to S6c 10 fold (low tension) and 6 fold (high tension) only in chronic hypoxic rat pulmonary resistance arteries. It had no effect on responses to ET‐1 in any vessel studied. 6 Vasodilatation of pre‐contracted vessels by ACh was markedly greater in the pulmonary resistance arteries from the chronic hypoxic rats (pIC50: 7.12±0.19, maximum: 72.1±0.2.0%) compared to their age‐matched controls (pIC50: 5.77±0.15, maximum: 28.2±2.0%). There was also a 2.5 fold increase in maximum vasodilatation induced by ACh. 7 These results demonstrate that control rat preparations showed greatest sensitivity for ET‐1 when set at the lower tension, equivalent to the pressure expected in vivo (∼16 mmHg). Pulmonary hypertension due to chronic hypoxia potentiated the maximum response to ET‐1. Pulmonary resistance arteries from control animals exhibited little endogenous tone, but exposure to chronic hypoxia increased endogenous inherent tone which is normally attenuated by nitric oxide. Endogenous nitric oxide production may increase in pulmonary resistance arteries from chronic hypoxic rats and attenuate contractile responses to ETB2 receptor stimulation. Relaxation to ACh was increased in pulmonary resistance arteries from chronic hypoxic rats.

Influences of the endothelium and hypoxia on neurogenic transmission in the isolated pulmonary artery of the rabbit

1 The effects of nitric oxide (10−6 m), Nω‐nitro‐l‐arginine methylester (l‐NAME, 10−4 m, an inhibitor of nitric oxide synthase), endothelium removal, hypoxia and selective α‐adrenoceptor antagonists on responses to nerve electrical field‐stimulation (EFS) were studied in the rabbit isolated pulmonary artery. 2 EFS induced frequency‐dependent contractions which were abolished by prazosin (α1‐adrenoceptor antagonist) and unaffected by rauwolscine (α2‐adrenoceptor antagonist). EFS‐induced responses were potentiated by l‐NAME and inhibited by nitric oxide. The effect of l‐NAME was reversed by the presence of l‐arginine (2 × 10−4 m), which had no effect on its own. In the presence of l‐NAME, the EFS‐induced responses were reduced by rauwolscine and the residual responses were abolished by prazosin. 3 Removal of the vascular endothelium increased the maximum contractile response to EFS but did not inhibit the ability of l‐NAME to potentiate contractile responses to EFS. 4 Hypoxia inhibited the contractile response to EFS. This effect of hypoxia was also seen in the presence of l‐NAME and in endothelium rubbed preparations. 5 In conclusion, the endothelium modulates EFS‐induced contractions in the rabbit pulmonary artery. The contraction induced by EFS was inhibited by nitric oxide, but potentiated by the nitric oxide‐synthase inhibitor, l‐NAME. The effect of l‐NAME was not mediated solely through the endothelium and revealed involvement of α2‐adrenoceptors in EFS‐induced contraction. Hypoxia inhibited neurogenic responses in rabbit isolated pulmonary arteries.

NOS inhibition potentiates norepinephrine but not sympathetic nerve-mediated co-transmission in resistance arteries.

OBJECTIVE The in vitro interaction between sympathetic nerves and basal nitric oxide release was studied in a resistance artery, since these interact powerfully in large vessels. METHODS The pharmacological interaction between L-NAME and vasoconstriction to field stimulation of sympathetic nerves or exogenous norepinephrine was studied in rabbit cutaneous resistance arteries in wire myographs. RESULTS Relaxation of norepinephrine-induced tone by acetylcholine, but not sodium nitroprusside, was blocked by N omega-nitro-L-arginine methyl ester (L-NAME: 100 microM), indicating that the agonist-induced release of nitric oxide could oppose the vasoconstrictor effect of norepinephrine and confirming that L-NAME had no effect on endothelium-independent vasodilatation. L-NAME increased norepinephrine potency indicating basal NO release. With short bursts of electrical field stimulation purinergic transmission was dominant at low frequencies and adrenergic at high frequencies. L-NAME had no effect on nerve-mediated responses, even after blocking the purinergic component with alpha,beta-methylene ATP (3 microM), suggesting that the influence of spontaneously released nitric oxide does not extend to the vascular smooth muscle cells under adrenergic nervous control. CONCLUSION(S) This resistance artery exhibits a highly effective nitric oxide-mediated vasodilatation to acetylcholine. It has basal release of nitric oxide which antagonises exogenous norepinephrine. However, basal nitric oxide did not influence adrenergic nerve transmission, which contrasts with previous studies of larger arteries and veins. We speculate that in small resistance arteries there may be a spatial limitation to the zones of vascular smooth muscle influenced by the adrenergic nerves and by basal nitric oxide from the endothelium, respectively. The role of endogenous nitric oxide in modulating vascular tone may thus be less in resistance arteries than in conducting arteries or capacitance vessels and purinergic transmission appears to be particularly resistant.