Publisher Summary Studies of receptor binding generally employ highly radioactive ligand derivatives; however, knowledge of the biological activity of the radioactive derivative is essential for a complete interpretation of the binding data. This chapter describes the relation between biological activity and binding, physicochemical detection and assay of receptors, solubilization of membrane receptors, provides examples of membrane-localized receptors, affinity techniques, polyvalent hormone derivatives, and receptor studies, and theoretical mechanisms of hormone–receptor binding and action. The biological response toward many neurotransmitters and hormones, such as insulin, acetylcholine, and epinephrine is rapid both in onset and in the termination of action on removal of these agents; therefore, it may be anticipated that the initial hormone–receptor interaction will also be reversible. The chapter shows that progress has been made toward understanding the way by which hormones, via interactions with cell–surface receptors, modulate overall cellular function. This interaction is only the first in a presumably complicated series of protein–protein interactions within the plane of the cell membrane; the mechanism, whereby the ultimate signal is transmitted to the internal cellular milieu is undoubtedly an equally intricate process. The chapter is considered to be useful in delineating the challenge that the elucidation of these processes presents and in providing a basis and incentive for more discoveries in this field.

Membrane receptors and hormone action.

Adrenocorticotrophic hormone (adrenocorticotropin, corticotropin, ACTH, FIGURE 1 ) carries vital biologic information from the anterior lobe of the pituitary to the adrenal cortex and other parts of the mammalian body. Practical interest in ACTH has been stimulated by its unique and valuable therapeutic properties. The field of medicinal application has been considerably broadened by the commercial availability of highly purified, synthetic hormone derivatives, e.g., ACTH1-24-tetracosipeptide.1 It now encompasses the treatment of ailments like pituitary insufficiencies, special forms of arthritis, allergies, multiple sclerosis, acute encephalitis and myelitis, tuberculous meningitis, childhood epilepsies, brain edema, acute polyradiculoneuritis, temporal arteritis, herpes zoster, idiopathic paresis of the nervus facialis, myasthenia gravis, etc. The pharmaceutical chemist strives to develop molecules with still improved properties, especially with simpler applicability, longer duration of action, and less antigenicity. The physiologist, the pharmacologist, and the biologist are intrigued by the possibilities offered by ACTH and numerous analogues and derivatives for investigation into the mechanism of hormone action, the precise roles of cyclic AMP and other mediators, and the validity of modern receptor theory. Biochemists are investigating the biosynthetic paths leading to ACTH; molecular biologists and biophysicists are interested in the oneand threedimensional organization of the hormonal information in the ACTH molecule (which is reminiscent of our written language) and the molecular mechanism of its read-out by receptor mo1ecules.2-16 In this short introduction, I would like to remind the participants of this symposium of some fundamental facts and problems on the molecular level that might be of use during the coming, more specialized discussions. ACTH is a linear nonatriacontapeptide with species diflerences in the COOH-terminal two-thirds of the molecule. The amino acid sequences of ACTH from four mammalian species were elucidated between 1954 and 1961 by the three groups of Bell, Li, and Lerner. In 1972, it was discovered by my former co-workers in Basel that the proposed sequences contained minor errors. The corrected primary structures are shown in TABLE 1 . They are taken from the most recent paper of Riniker,17 which refers to the earlier literature in detail. The four structures are virtually identical, except for changes in positions 31 and 3 3 . Dogfish (Squalus acanthias) ACTHIS is equally long, but differs in 11 positions from the human hormone. The replacements are all conservative of the general biologic and physico-chemical properties.

ACTH: a short introductory review.

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ∼55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [3H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K+ and dibutyryl cyclic AMP) is impaired, but after a 6–12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control.

https://rupress.org/jcb/article-pdf/59/2/276/1386581/276.pdf

Hormone secretion by cells dissociated from rat anterior pituitaries

Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein.

ACTH like peptides and opiate receptors in the rat brain. Structure-activity studies

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2871%2980520-3

Isolated adrenal cells: Log dose response curves for steroidogenesis induced by ACTH1–24, ACTH1–10, ACTH4–10 and ACTH5–10

A new approach to the structure-activity relationship for ACTH analogs using isolated adrenal cortex cells.

SummarySuspensions of isolated adrenal cortex cells of the rat respond to the addition of ACTH1-24 and related peptides with production of cAMP and corticosterone. A concentration of ACTH1-24 which results in less than 20% of maximum cAMP production induces near-maximum corticosterone production; a concentration which results in 1-3% cAMP production induces 50% of maximum corticosterone production. ACTH4-23(NH2) and ACTH5-24 are less potent than AC TH1-24, but induce the same maximum corticosterone production (Bmax) as ACTH1-24; ACTH6-24 induces a maximum equal to 0.4 of Bmax; ACTH7-23(NH2) is inactive. AC TH4-23(NH2), ACTH5-24 and ACTH6-24 induce 0.86, 0.45 and 0.01 of the maximum cAMP production characteristic of AC TH1-24; respectively; ACTH7-23(NH2) is inactive. ACTH7-23(NH2) is a competitive antagonist and shifts the ACTH1-24 log concentration curves for cyclic AMP and for corticostemne to the same degree. These observations are discussed in terms of a dual receptor model and in terms of a single sit...

Isolated Adrenal Cortex Cells: ACTH4-23 (NH2), ACTH5-24, ACTH6-24 and ACTH7-23 (NH2); Cyclic AMP and Corticosterone Production

Isolated adrenal cortex cells in suspension: stimulation and inhibition of steroidogenesis by analogues of ACTH.

Publisher Summary Suspensions of cells, freshly isolated from the organism, offer a number of advantages for the study of hormone action. They can be prepared within a few hours by methods that demand attention to detail but present no great technical difficulties. Isolated cells in suspension are in immediate contact with hormones, substrates, and oxygen. In contrast, cells in a slice or fragment of tissue are separated from these substances by diffusion barriers. Accumulated evidences on sensitivity of response and on secretory capacity strongly suggest that isolated cells in suspension closely reflect the functional activity of cells in vivo . The intact cell is examined within the confines of a controlled environment, free from the complexities and uncertainties imposed by whole animal studies. Finally, isolated cells provide a set of references against which to judge the physiological significance of observations made at a subcellular level.

Steven Seelig

Papers

Isolated adrenal cells: Log dose response curves for steroidogenesis induced by ACTH1–24, ACTH1–10, ACTH4–10 and ACTH5–10

A new approach to the structure-activity relationship for ACTH analogs using isolated adrenal cortex cells.

Isolated Adrenal Cortex Cells: ACTH4-23 (NH2), ACTH5-24, ACTH6-24 and ACTH7-23 (NH2); Cyclic AMP and Corticosterone Production

Isolated adrenal cortex cells in suspension: stimulation and inhibition of steroidogenesis by analogues of ACTH.

Isolation of adrenal cortex cells--hormone responses.