Hypertension is a major risk factor in the development of cardiovascular disease. Adenovirus gene transfer of endothelin-1 (Ad.CMV.ET-1) in rats produced significant (5-fold) increases in plasma ET-1 and systemic blood pressure (46%) 4 days after viral administration, compared with β-galactosidase (Ad.CMV.β-gal) injected as control. The density ( B max ) of the ET receptor ET A measured in aortas was reduced significantly by more than 50% to 17±2fmol·mg -1 of protein for the Ad.CMV.ET-1 group compared with 39±6fmol·mg -1 of protein for the control. There was no change in the density of the smaller population of the ET B sub-type. In agreement, the ratio of ET A mRNA to cyclophilin mRNA (a housekeeping gene) measured by Northern analysis was reduced in Ad.CMV.ET-1 rats compared with controls. The ratio of mRNA encoding the ET B sub-type did not change. ET-1 vasoconstriction was significantly reduced ( P <0.05) in aortas from Ad.CMV.ET-1-treated rats [pD 2 = 8.67±0.14 (where pD 2 is -log 10 EC 50 ); n = 11] versus the control (pD 2 = 9.11±0.06; n = 14) but there was no significant difference in the potency of two other vasoconstrictors tested (noradrenaline and Arg-vasopressin), indicating this was a specific effect on ET receptors. There was no change in the affinity of ET-1 binding to either receptor sub-type in the experimental group compared with the control, demonstrating that the attenuation in the constrictor response is the result of the reduced density of receptors rather than a change in affinity. The results show that ET A (but not ET B ) receptors are modulated in this experimental model of hypertension and provide further evidence for selective blockade of the ET A receptor as a therapeutic strategy.

Blood pressure (BP) and heart rate (HR) in endothelin-3 (ET-3) null (-/-) knockout mice and ET A receptor (-/-) mice were measured using the servo null pressure measuring technique under halothane anaesthesia. In infant ET-3 (-/-) mice (2–3 weeks old), mean BP and HR were 55±2mmHg and 436±30beats/min respectively. These values were not different from those in age-matched wild-type mice (53±3mmHg and 430±18beats/min respectively). Baroreflex sensitivity, which was calculated as the slope of the relationship between systolic BP and RR interval on an ECG, was also similar in ET-3 (-/-) mice (0.84±0.20ms/mmHg) and wild-type mice (1.07±0.38ms/mmHg). ET A receptor (-/-) mice were obtained by caesarean section on the expected day of delivery and tracheotomized, so that they would live for more than 24h. Mean BP and HR in ET A receptor (-/-) mice were 15±1mmHg and 333±6beats/min respectively. These values were not different from those in age-matched, similarly treated wild-type mice (16±3mmHg and 308±10beats/min respectively). Baroreflex sensitivity in the newborn ET A receptor (-/-) mice (0.45±0.15ms/mmHg) and wild-type mice (0.31±0.06ms/mmHg) were very low compared with the values in infant wild-type mice, but not different between the mutant mice and their littermates. Moreover, HR in awake ET A receptor (-/-) mice (396±13beats/min) was not different from that in wild-type mice (409±13beats/min). These results show that the ET A receptor and ET-3 are not involved in cardiovascular regulation, at least during the very early life of the mice. A possible involvement of the ET A receptor in BP regulation, if any, seems to occur at later times and/or in some pathological settings.

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five TM insect cells in suspension culture. Both the monomeric (85kDa) and dimeric (170kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11mg of ECE-1a monomer and 6mg of dimer were obtained from l litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the K m and k cat values for the monomer were 2.2 µ M and 1.6min -1 respectively, while those of the dimer were 1.4 µ M and 4.9min -1 respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Trypanosoma cruzi is the aetiological agent of Chagas' disease, an important cause of chronic cardiomyopathy. We previously demonstrated a role for endothelin-1 (ET-1) in the pathogenesis of chagasic heart disease. In order to explore further the significance of ET-1 in chagasic heart disease, we infected ET-1 (flox/flox); α-MHC-Cre(+) (ET-1KO) mice, in which the ET-1 gene has been deleted from cardiac myocytes, with 10 4 T. cruzi (Brazil strain) trypomastigotes. As controls, we used ET-1 (flox/flox);Cre(-) (FLOX) and C57BL/6×129sv (WT) mice. All mice survived and were evaluated 150–160 days post-infection. Cardiac magnetic resonance imaging revealed a significant increase in right ventricular internal diameter in all infected animals except ET-1KO mice (control WT, 1.6±0.10mm; infected WT, 2.8±0.15mm; control FLOX, 2.04±0.02mm; infected FLOX, 2.76±0.28mm). There was no significant difference in right ventricular internal diameter between infected and uninfected ET-1KO mice (control ET-1KO, 1.83±0.11mm; infected ET-1KO, 2.14±0.20mm). In another series of experiments, transthoracic echocardiography was performed on uninfected as well as infected ET-1KO mice, and uninfected and infected FLOX mice. Both infected groups had an increased left ventricular end-diastolic diameter and reduced fractional shortening. In addition, relative wall thickness was also decreased in infected animals. However, the magnitude of these changes was less in infected ET-1KO mice. These data provide further support for a role for ET-1 in the pathogenesis of chronic chagasic heart disease, and indicate that cardiac myocytes are an important source of ET-1 in this disease.