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The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

Experiments were conducted to determine the ability of the newly discovered ETA receptor antagonist, BQ-123, to inhibit the pressor responses to several members of the human endothelin peptide family in anesthetized rats. Big ET-1 produced a greater increase in mean arterial pressure compared to ET-1 (67 ± 10% vs. 44 ± 4%, p < 0.05; both at 1 nmol/kg i.v.). Prior administration of BQ-123 (10 mg/kg i.v.) was more effective at inhibiting the response to Big ET-1 compared to ET-1. Following ETA receptor blockade, ET-1 produced a small, but significant, increase in mean arterial pressure (15 ± 2%) while Big ET-1 had no effect (5 ± 2%). Big ET-3 and ET-3 at 1 nmol/kg i.v. produced similar increases in mean arterial pressure, although less than that produced by either Big ET-1 or ET-1 (p < 0.05). ETA receptor antagonism blocked 64% of the pressor response to both Big ET-3 and ET-3. We conclude that blockade of pressor responses to each of the endothelin peptides suggests that the increases in arterial pressure ...

The importance of glycosylation to endothelin (ET) receptors was studied in cultured Swiss 3T3 fibroblasts. ET stimulated phosphatidylinositol hydrolysis by 270% in control cells. This stimulatory effect was decreased to < 130% in tunicamycin (TU)-treated cells. TU inhibited protein glycosylation, but did not effect DNA synthesis, cell morphology, or cell numbers. ET-1 binding was greatly reduced in TU-treated cells. In membranes prepared from control cells, [125]ET-1 binding was saturable, and appeared as a straight line in Scatchard analysis with values of 0.7 pmol/mg and 0.9 nM for Bmax and KD, respectively. [125]ET-1 binding to membranes from TU-treated cells was not saturable. Scatchard analysis showed a biphasic line. Values of Bmax, and KD for the higher affinity site were 0.17 pmol/mg and 0.12 nM, respectively. The low affinity site exhibited a much weaker affinity for ET with an estimated KD value of 6.8 nM. Cross-linking studies revealed that the receptor in control membranes was a protein with ...

Experiments were conducted to determine the effect of surgical preparation and infusion of plasma and/or saline on circulating immunoreactive endothelin (irET) concentrations. Male Sprague-Dawley rats anesthetized with Inactin were placed on a surgical heating table for about 2 h before collecting blood via puncture of the abdominal aorta. The plasma concentration of irET was 8.1 ± 0.9 pg/ml in rats that did not undergo surgical preparation or receive any further treatment. Standard surgical preparation as for renal clearance experiments (catheters in the trachea, femoral artery, femoral vein, and bladder) resulted in a dramatic rise in irET to 17.7 ± 3.0 pg/ml (p < 0.05). Infusion of plasma (5 ml/kg over 20 min), but not saline, resulted in an additional increase to 32.6 • 3.5 pg/ml (p < 0.05). These results clearly demonstrate that standard whole-animal techniques such as surgery and plasma infusion produce significant changes in circulating endothelin which may affect a variety of physiological and pha...

We have recently reported that administration of the ANF analog A68828 improves renal function in an acute model of postischemic acute renal failure. The current investigation examined the question whether a short-term infusion of A68828 can attenuate the long-term decrement in renal function following an ischemic event. Acute renal failure was induced in male Sprague-Dawley rats (200-250 g) by complete occlusion of both renal arteries for 30 min. During the initial 60 min following ischemia, vehicle (0.1% BSA in saline), A68828 (10 micrograms/kg/min); dopamine (10 micrograms/kg/min); A68828 (10 micrograms/kg/min) plus dopamine (10 micrograms/kg/min); or ANF[1-28] (0.5 microgram/kg/min) were infused intravenously. In vehicle-treated animals, a very large increase in plasma creatinine was observed, with peak levels at 2 days postischemia (5.5 +/- 1.2 mg/dL). A68828 alone, A68828 with dopamine, or ANF[1-28] infusion attenuated the rise in plasma creatinine levels by approximately 50% on each day of the study; dopamine alone was no different from vehicle-treated controls. Gross histological examination of kidneys on the fourth day postischemia revealed that significantly less damage occurred only in the group treated with A68828 alone. These results indicate that infusion of a reduced-size analog of ANF for a brief period in the postischemic kidney improves renal function and lessens tissue damage as evaluated several days after the ischemic event. Furthermore, dopamine infusion provides no discernable beneficial effect.