Effect of in vivo fetal infusion of dexamethasone at 0.75 GA on fetal ovine resistance artery responses to ET-1.

At 110-111 days gestation, instrumented fetal sheep were administered saline or dexamethasone (2.2 microgram. kg(-1). h(-1) iv) for 48 h. Measurement of fetal blood pressure showed a greater increase in dexamethasone-treated (n = 6) compared with control (n = 5) fetuses (7.3 +/- 2.3 vs. 0.6 +/- 2.3 mmHg, P < 0.05). Fetuses were delivered by cesarean section, and the femoral muscle and brain were obtained under halothane anesthesia. Femoral and middle cerebral arteries (approximately 320-micrometer internal diameter) were evaluated using wire myography. Sensitivity to KCl (2.5-125 mM) and the magnitude of the maximal vasoconstriction to 125 mM K(+) were similar in femoral and middle cerebral arteries from dexamethasone-treated vs. control fetuses. Acetylcholine-induced vasorelaxation was similar in femoral arteries from control and dexamethasone-treated fetuses. Middle cerebral arteries did not relax to acetylcholine. Sensitivity to endothelin-1 (ET-1; 0.1 pM-0.1 microM) and magnitude of the ET-1-induced vasoconstriction were greater in femoral arteries from dexamethasone-treated vs. control fetuses (P < 0.05). Autoradiographical studies with receptor-specific ligands demonstrated increased ET(A)-receptor binding, the principal receptor subtype, in femoral muscle vessels (P < 0.001) but decreased ET(A)-receptor binding in middle cerebral arteries (P < 0.01) from dexamethasone-treated compared with control fetuses. Relatively little ET(B)-receptor binding was evident in all tissues examined. We conclude that hyperreactivity to ET-1, due to increased ET(A)-receptor binding, may be involved in the dexamethasone-induced increase in peripheral vascular resistance in fetal sheep in vivo.

Developmental changes in reactivity of small femoral arteries in the fetal and postnatal baboon.

OBJECTIVE: We evaluated in vitro responsiveness of small arteries (internal diameter, 300 microm) from the femoral vascular bed of normal fetal (0.75-1.0 gestation) and neonatal (43-46 days) baboons to investigate whether the transition from fetal to neonatal life was associated with functional alterations in vasoconstrictor and vasodilator responses. STUDY DESIGN: The maximum response and sensitivity to potassium and to the constrictor agonists norepinephrine and U46619 (a thromboxane mimetic) were studied by in vitro myography. Vasodilator responses to the endothelium-dependent dilators acetylcholine and bradykinin were also investigated. RESULTS: The maximum response to norepinephrine and U46619 and to potassium increased with gestational age, whereas the sensitivity to these vasoconstrictors was similar in all groups studied. In contrast, acetylcholine- and bradykinin-induced relaxation (median effective concentration and maximum response) did not change with age. CONCLUSION: Receptor-mediated responses to a catecholamine, a prostanoid, and 2 endotheliumdependent vasodilators are similar in the fetal and neonatal baboon. The increase in maximal constriction with development, which is probably associated with growth or maturation of vascular smooth muscle, is likely to be a functionally important aspect in the development of cardiovascular function.

Development of fetal vascular responses to endothelin-1 and acetylcholine in the sheep.

Responses to K(+), endothelin-1 (ET-1), and acetylcholine (ACh) of isolated adrenal, femoral, middle cerebral, and renal arteries from fetal [110--145 days gestational age (dGA, term approximately 148 dGA)] and 0- to 24-h newborn (NB) lambs were evaluated using the technique of wire myography. Responses at distinct developmental ages for each vascular bed were compared. In all arteries sensitivity to K(+)-induced vasoconstriction was similar at all fetal age points examined. In contrast, sensitivity to ET-1 increased with increasing fetal age in arteries from all vascular beds. The magnitude of the maximal vasoconstriction was positively correlated with GA for K(+) in adrenal, femoral, and cerebral arteries and for ET-1 in femoral, cerebral, and renal arteries. Cerebral arteries showed a greater sensitivity when compared with the other systemic arteries to K(+) and ET-1 at all fetal ages and to K(+) in NB. ACh evoked relaxatory responses in fetal and NB femoral and adrenal arteries. However, renal arteries relaxed comparatively less in response to ACh, and no vasodilation was noted in middle cerebral arteries at any age points examined. For femoral arteries ACh-induced vasorelaxation decreased with increasing GA but was restored in arteries from NB lambs. In summary, the responsiveness of isolated resistance arteries varies with developmental age in the fetal and perinatal sheep and these effects are both agonist and vascular bed specific. The augmented sensitivity in response to ET-1 of middle cerebral compared with other systemic arteries may reflect the importance of cerebral blood flow control during this critical developmental period.

Pulmonary responses to 5-hydroxytryptamine and endothelin-1 in a rabbit model of left ventricular dysfunction.

OBJECTIVE: To determine whether pulmonary hypertension developed in a coronary artery-ligated rabbit model of left ventricular dysfunction (LVD) and to examine the effects of i.v. 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) on pulmonary arterial pressure (PAP). METHODS: Eight weeks after experimental coronary artery ligation or sham operation, ejection fractions were assessed by echocardiography. The rabbits were later anaesthetised and pulmonary arterial pressure was measured via a catheter inserted into the pulmonary artery via the right external jugular vein. 5-HT (1-400 micrograms/kg) and ET-1 (0.001-4 nmol/kg) were administered i.v. RESULTS: Ejection fraction was significantly decreased from 76.6 +/- 1.4% in sham-operated to 42.2 +/- 1.3% in coronary artery-ligated rabbits (n = 9 in each group; P < 0.001), consistent with LVD. Baseline mean pulmonary arterial pressure was significantly increased in the coronary artery-ligated group compared to the shams, (16.5 +/- 0.5 vs. 11.5 +/- 0.8 mmHg; P < 0.001). A significant degree of right ventricular hypertrophy was found in the coronary artery-ligated rabbits (0.70 +/- 0.04 g/kg final body weight (f.b.wt.), n = 8 cf. 0.48 +/- 0.02 g/kg f.b.wt. in sham-operated controls, n = 8; P < 0.001). There was a significant increase in the percentage of muscularised pulmonary vessels adjacent to alveolar ducts and alveoli < 60 microns i.d. in the LVD rabbits compared with their sham-operated controls (8.5 +/- 0.4 cf. 20 +/- 0.5%; P < 0.0005). 5-HT produced a greater response in the coronary artery-ligated rabbits (a maximum increase of 8.7 +/- 1.0 mmHg in mean pulmonary artery pressure vs. 4.6 +/- 1.5 mmHg for sham-operated controls; P < 0.05). ET-1 did not have any effect on pulmonary arterial pressure in either group. CONCLUSION: In the rabbit, LVD secondary to coronary artery ligation, causes right ventricular hypertrophy, pulmonary vascular remodelling, and an increased PAP consistent with the onset of pulmonary hypertension (PHT). The greater PAP response to i.v. 5-HT in the PHT group supports the hypothesis that this substance could be involved in the development of PHT. A role for ET-1 cannot be excluded, despite its lack of effect on PAP when intravenously administered in either group.

Heterogeneity of endothelin-B receptors in rabbit pulmonary resistance arteries.

We have previously shown that endothelin-B (ETB) receptors mediate contraction in human and rat pulmonary resistance arteries (PRAs). Here we characterize the endothelin (ET) receptors in rabbits PRAs. PRAs (approximately 150 microns i.d.) were studied using wire myography. Vasoconstrictor effects to ET-1, ET-3, and the ETB-selective agonist sarafotoxin S6c (S6c) were studied in the presence and absence of the ETA receptor antagonist FR139317, the ETB-selective antagonist BQ788, and the mixed ETA/ETB antagonist SB209670. The effect of SB209670 was also studied in human PRAs (approximately 250 microns i.d.). Competitive ET-1 binding studies were also carried out on rabbit small pulmonary artery homogenates. The potencies of the agonists were in the following order: S6c > ET-3 = ET-1. Concentration-response curves (CRCs) to ET-1 were biphasic, with a gradual slope up to approximately 1 nM and a steeper component at higher concentrations of ET-1. Neither FR139317 (1 microM) nor BQ788 (1 microM) inhibited responses to ET-1. BQ788 inhibited S6c- and ET-3 induced contractions with pKb values of 6.8 +/- 0.1 and 6.3 +/- 0.2, respectively. SB209670 inhibited responses to ET-1 in the higher concentration component of the CRC and inhibited responses to S6c in a competitive fashion. pKb values for ET-1 and S6c were 6.8 +/- 0.2 and 7.5 +/- 1, respectively. SB209670 (0.1-1 microM) totally abolished responses to ET-1 in human PRAs. The binding assay established two ET binding sites in rabbit PRAs, one low affinity (Ki = 480 pM) and one high affinity (Ki = 64 fM). The study provides evidence for a heterogeneous population of ETB-like receptors in pulmonary resistance arteries, including an atypical ETB receptor sensitive to SB209670.

EndothelinB receptors in rabbit pulmonary resistance arteries: effect of left ventricular dysfunction.

The endothelin (ET) receptor that mediates vasoconstriction of the isolated rabbit pulmonary resistance artery was characterized using selective ET receptor agonists and antagonists. We also examined changes in ET-induced vasoconstriction brought about by left ventricular dysfunction using the rabbit coronary ligation model. The rank order of potency for contraction was sarafotoxin S6c (S6c) > ET-1 = ET-3, which is characteristic of an ETB-like receptor. The combined ETA/ETB receptor antagonist SB209670 (1 microM) antagonized responses to ET-1 and S6c with estimated pKb values of 6.8 +/- 0.2 and 7.8 +/- 0.2, respectively. BQ788 (1 microM) antagonized responses to S6c and ET-3 (but not ET-1) with estimated pKb values of 7.1 +/- 0.2 and 6.6 +/- 0.1, respectively. The ETA receptor antagonist FR139317 (1 microM), either alone or in combination with BQ788, did not inhibit responses to ET-1. The profile of the ET-1 response was not altered by left ventricular dysfunction. In control rabbits, the inhibitor of nitric oxide synthase N omega-nitro-L-arginine methyl ester (100 microM) had no significant effect on the potency of either ET-1 or S6c. In the coronary-ligated rabbits, however, it significantly increased the potency (10-15-fold) of both ET-1 and S6c. These results suggest that the ET receptor that mediates contraction in rabbit pulmonary resistance arteries has the characteristics of an ETB-like receptor. The responses to ET-1 are not altered by LVD but may be modified by increased release of nitric oxide.

Effect of novel mixed ETA/ETB antagonists on responses to ET-1 in human small muscular pulmonary arteries.

Selective ETA and ETB receptor antagonists do not fully inhibit responses to ET-1 in human small pulmonary arteries. Here, we have compared the influence of the novel mixed ETA/ETB antagonists SB/217242, SB/234551 and SB/209670 on ET-1-mediated vasoconstriction in these vessels. ET-1 was a potent vasoconstrictor (pEC50: 8.14+/-0.05, n=5) and the concentration-response curve to ET-1 was biphasic in nature. All three mixed antagonists (1 microM) inhibited the responses to ET-1, abolishing the biphasic form of the concentration response curve. The order of potency was SB 209670>SB 234551>/=SB 217242 with estimated pKb values of 8.0+/-0.20, 6.8+/-0.17 and 6. 6+/-0.11 respectively (n=3-5).

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 microns, 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 microM ET-1. The order of potency, taking 0.3 microM 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.

Bronchodilator and pre-protective effects of urodilatin in bovine bronchi in vitro: comparison with atrial natriuretic peptide.

1. This study examined the activity and mechanisms of action of urodilatin in bovine bronchi. For comparison, the ability of urodilatin to evoke bronchodilatation or protect against subsequent challenge was compared to that of the closely related peptide alpha-human atrial natriuretic peptide (ANP). 2. Urodilatin reversed methacholine-evoked contraction in a concentration-dependent manner in bovine bronchi. In the absence of any attempt to prevent degradation by neutral endopeptidases, urodilatin was more potent than ANP in this tissue. 3. The bronchodilator properties of urodilatin were significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon (3.68 x 10(-5) M). This provides evidence for at least partial degradation of urodilatin by neutral endopeptidases. With phosphoramidon present, urodilatin and ANP were equipotent. 4. In the presence of phosphoramidon (3.68 x 10(-5) M), pre-incubation with urodilatin (10(-6) M) had a protective effect against subsequent methacholine-induced contraction. This action of urodilatin was quantitatively similar to that of ANP in the presence of this endopeptidase inhibitor. 5. The actions of urodilatin appear to involve ATP-sensitive K+ channels since tolbutamide (10(-6) - 10(-5) M) significantly attenuated the relaxations induced by this peptide. 6. Small conductance Ca(2+)-activated K+ channels seem likewise to be implicated in the actions of urodilatin since blockade of these channels with apamin (10(-7) - 10(-6) M) resulted in a marked attenuation of urodilatin-evoked responses. 7. The presence of charybdotoxin (10-9 M-10-M) had no significant effect on subsequent responses tourodilatin suggesting that large conductance Ca2+-activated K+ channels are not involved in the relaxations evoked by this peptide.8. In the presence of phosphoramidon (3.68 x 10-5 M), urodilatin (10-6 M) evoked elevation of cyclic GMP levels within bovine bronchial tissue. Levels of cyclic GMP increased significantly within 5-10 s in response to this peptide and preceded the initiation of relaxant responses. Maximum increases in cyclic GMP levels were reached within 5 min; the time required for maximal relaxation evoked by this peptide.9. In conclusion, urodilatin, like ANP reversed and protected against, subsequent methacholine-induced bronchoconstriction; an action enhanced by the presence of phosphoramidon (3.68 x 1O-5 M).Associated with these actions of urodilatin was a rise in cyclic GMP levels as well as the opening of ATP-sensitive K+ and small conductance Ca2+-activated K+ channels.