Article date: November 1991
By: Lesley C. Wilkes, Michael R. Boarder, in Volume 104, Issue 3, pages 750-754
Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK‐N‐MC cell line, derived from a human neuroblastoma.
[125I]‐ET‐1 (2.5 × 10−11m) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 ± 0.04, n = 3) with an apparent Kd of 1.4 ± 0.3 × 10−9m and a Bmax of 3.1 ± 1.0 pmol mg−1 protein. [125I]‐ET‐3 (2.5 × 10−11m), did not specifically bind to SK‐N‐MC cells.
The binding of [125I]‐ET‐1 was competitively inhibited by other ET isoforms, the order of potency being ET‐1 > sarafotoxin S6b > ET‐3.
Association of 1 nm [125I]‐ET‐1 at 37°C reached apparent equilibrium at 60–80 min, with half‐maximal binding being achieved at 12 min.
Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]‐ET‐1 dissociating. The actual amounts of [125I]‐ET‐1 dissociated were similar in both cases.
Incubation of [125I]‐ET‐3 with SK‐N‐MC cells at 37°C for 60 min did not result in significant degradation of this peptide. However, [125I]‐ET‐1 was broken down by incubation with SK‐N‐MC cells, the pattern of degradation of dissociable [125I]‐ET‐1 (and that found in the supernatant) being different from that of non‐dissociable [125I]‐ET‐1.
ET‐1 concentration‐dependently induced an increase in total inositol phosphate accumulation in sub‐confluent (but not in confluent) cultures of SK‐N‐MC cells (EC50 = 6.43 ± 1.9 × 10−10m). ET‐3 was without effect.
These results show that ET‐1 specifically binds to SK‐N‐MC cells with the characteristics of an ETA receptor. Our earlier finding that adrenal chromaffin cells express an ETB receptor indicates the existence of multiple ET receptor types on neuronal cells.
Specific binding sites for synthetic endothelin (ET) isoforms were studied on intact cells of the SK‐N‐MC cell line, derived from a human neuroblastoma.
[125I]‐ET‐1 (2.5 × 10−11m) specifically bound to a single class of binding sites on these cells (Hill coefficient of 1.06 ± 0.04, n = 3) with an apparent Kd of 1.4 ± 0.3 × 10−9m and a Bmax of 3.1 ± 1.0 pmol mg−1 protein. [125I]‐ET‐3 (2.5 × 10−11m), did not specifically bind to SK‐N‐MC cells.
The binding of [125I]‐ET‐1 was competitively inhibited by other ET isoforms, the order of potency being ET‐1 > sarafotoxin S6b > ET‐3.
Association of 1 nm [125I]‐ET‐1 at 37°C reached apparent equilibrium at 60–80 min, with half‐maximal binding being achieved at 12 min.
Dissociation was measured after both 10 min and 60 min of association with 64% and 30% respectively of specifically bound [125I]‐ET‐1 dissociating. The actual amounts of [125I]‐ET‐1 dissociated were similar in both cases.
Incubation of [125I]‐ET‐3 with SK‐N‐MC cells at 37°C for 60 min did not result in significant degradation of this peptide. However, [125I]‐ET‐1 was broken down by incubation with SK‐N‐MC cells, the pattern of degradation of dissociable [125I]‐ET‐1 (and that found in the supernatant) being different from that of non‐dissociable [125I]‐ET‐1.
ET‐1 concentration‐dependently induced an increase in total inositol phosphate accumulation in sub‐confluent (but not in confluent) cultures of SK‐N‐MC cells (EC50 = 6.43 ± 1.9 × 10−10m). ET‐3 was without effect.
These results show that ET‐1 specifically binds to SK‐N‐MC cells with the characteristics of an ETA receptor. Our earlier finding that adrenal chromaffin cells express an ETB receptor indicates the existence of multiple ET receptor types on neuronal cells.
DOI: 10.1111/j.1476-5381.1991.tb12499.x
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