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1. The effect of iodination on the distribution of peptide hormones into the aqueous two-phase dextran–polyethylene glycol system and on the solubility of these hormones in aqueous polyethylene glycol and in water was assessed. Hormones that were studied included insulin, glucagon and parathyroid hormone. 2. The partition coefficient of native insulin in the dextran–polyethylene glycol system showed a minimum (about 1) near the isoelectric point of the hormone (pH 5). Partial iodination of insulin (one atom per molecule) caused little change in the distribution of the hormone. More extensive iodination markedly decreased the partition coefficient in the region of the isoelectric point and displaced the pH value at which the partition coefficient was a minimum towards lower values. 3. The solubility of native insulin in aqueous polyethylene glycol and in water showed a pH-dependence similar to that observed for the distribution in the dextran–polyethylene glycol system. Iodination of insulin decreased the solubility of the hormone in polyethylene glycol and in water in parallel, and decreased the pH value at which solubility was a minimum. The changes in solubility correlated with the degree of iodination and accounted for the changes in distribution observed at high concentrations of insulin. 4. Comparable effects of iodination on distribution and solubility were also observed with glucagon. 5. At concentrations of insulin below its maximum solubility, serum proteins caused a decrease in the partition coefficient of iodinated hormone, but not of native hormone. These effects correlated with the degree of iodination and resulted from a co-precipitation of iodinated insulin with serum proteins.

The influence of bradykinin-binding antibodies on the hypotensive and the oxytocic responses to bradykinin was investigated. Bradykinin-binding antibodies were obtained from rabbits injected with bradykinin coupled to ovalbumin. Pre- and post-immunization sera were tested for their capacity to bind intrinsically labeled 14C-bradykinin. Six injected rabbits produced bradykinin-binding antibodies. The binding ofa radio-iodotyrosine analog of bradykinin to antiserum was inhibited by unlabeled bradykinin or kallidin, but was unaffected by oxytocin, vasopressin or angiotensin. Incubation of bradykinin with antiserum resulted in an inhibition of its hypotensive activity in the rat. Bradykinin-induced hypotension was also abolished in rats injected with antiserum. Pre-immunization serum was ineffective in both respects. Bradykinin binding antibodies also inhibited the oxytocic activity of bradykinin on the isolated rat's uterus. The inhibitory effects became evident after dialysis of the antiserum. Undialyzed antisera contained an oxytocic substance which masked the inhibition of bradykinin activity. Inhibition of pharmacological effects of bradykinin by antibradykinin was reversed by heat inactivation of antibodies. Bradykinin-binding antibodies not only inhibited bradykinin activity, but also protected the peptide against kininases.

An antiserum to human follicle-stimulating hormone (FSH) prepared with highly purified FSH obtained from frozen pituitary glands has been examined with respect to its avidity. It was found that FSH contained in two human pituitary preparations and in a postmenopausal urinary extract was bound to a significantly greater degree by this antiserum than by an FSH antiserum distributed by the NIAMDD.

A simple radioreceptor-assay technique for human follicle stimulating hormone (hFSH) with use of rat testicular slice and enzymatically 125I-labeled hFSH was described. It was shown that 125I-hFSH bound to the testicular slice (30mg) was approximately 2% of the total amount added to the tube (2ng/tube), half of which was displaceable in the presence of 1μg/tube of unlabeled hFSH. Although only small amount of 125I-hFSH was bound specifically to the tissue, the binding was not inhibited by other peptide hormones tested. The testicular receptor for hFSH had high affinity constant (2.5×109M-1) and hormonal specificity, suggesting a possibility of the physiological application of this radioreceptor assay for the measurement of hFSH in body fluids.

Three well characterized placental protein hormones —human chorionic thyrotropin, human chorionic gonadotropin and human placental lactogen—and their use as fetal monitors of the fetoplacental unit are reviewed.

Over the past few years the field of endocrinology has witnessed the continued rapid development of sensitive, efficient techniques for the isolation and structural analysis of peptide hormones, along with the refinement of methods for peptide synthesis. As a consequence, our knowledge of the chemistry of parathyroid hormone has advanced to the stage where it may be applied effectively to the investigation of a far-reaching spectrum of questions relating to hormone biosynthesis, mode of action and peripheral metabolism. The opening section of this chapter, by tracing the evolution of current methods in hormone isolation, covers in fact the greater portion of the 50year history of parathyroid hormone chemistry. This culminates in the structural analyses of bovine and porcine parathyroid hormones, representing work completed for the most part within the past five years, and the significant though still incomplete sequence studies of human parathyroid hormone, with its special problems of scarce supply. Synthetic parathyroid peptides are introduced in the succeeding section, which outlines the results of extensive recent work correlating hormone structure with biological activity. The chapter concludes with examples of how this cumulative information, combined with present-day chemical methodology, has been deployed successfully in the investigation of two pertinent aspects of parathyroid pathophysiology: pro-parathyroid hormone and circulating products of hormone metabolism.

This study was carried out to discern the effects of peptide hormone LH-RH on ovulation in the ayu, Plecoglossus altivelis. Total ovary weight and GSI were significantly greater in the fish treated with LH-RH than in the control. Ovulation was clearly observed in the fish treated with 200μg LH-RH within 2 days after a single injection. In the ventral portion of the proximal pars distalis of the pituitary the content of the basophil (gonadotroph) mostly disappeared for the 2 day period. Furthermore, in the LH-RH-treated fish body weight increased by several percent. Water and sodium content were also greater than in the control. This LH-RH, by causing the release of endogenous gonadotropin, induces the same sequence of ovarian events as does the treatment of exogenous gonadotropin.

The biosynthesis of calcitonin was studied using radioimmunochemical methods and suspensions of calcitonin-producing cells derived from trout ultimobranchial glands. [ 14 C]leucine was incorporated into cell proteins in a linear fashion for up to 36 hrs. Acid-extracted cellular radioactivity could be precipitated by trichloroacetic acid and calcitonin antiserum. Chromatography of the cell extracts revealed two distinct peaks of radio-immunoassayable and immunoprecipitable calcitonin activity. One peak coeluted with radioiodinated calcitonin, the other as a higher molecular weight species. The relative incorporation of [ 14 C]leucine into the higher and lower molecular weight peaks during “pulse-chase” experiments was consistent with a precursor-product relationship between them.

Two closely related polypeptides termed thymin I and II were isolated from bovine thymus by their effect in causing a delayed impairment of neuromuscular transmission in vivo. Purified thymin also induced bone marrow cells to develop the characteristics of intrathymic lymphocytes and it is probable that thymin is a lymphopoietic differentiating hormone of the thymus with secondary neuromuscular effects.

The polypeptide hormone glucagon can react with the nucleophiles; glycinamide, taurine or ethylenediamine in the presence of 1-ethyl-3-(3-dimethylaminopropylcarbodiimide). The number of carboxyl groups which are modified depend on the concentration of guanidine hydrochloride in the reaction media. These results demonstrate an additional property which glucagon possesses in common with larger globular proteins and suggests that the hormone has a specific, folded structure in dilute aqueous solution. In the absence of guanidine hydrochloride only one taurine residue is incorporated into the terminal carboxyl group of the peptide. In 7 M guanidine hydrochloride all four of the carboxyl groups react with glycinamide or taurine while only two and a half residues of ethylenediamine are incorporated. All of these derivatives and glucagon have identical circular dichroism spectra in dilute aqueous solution. The taurine modified derivative has greatly enhanced solubility compared with glucagon but still associates to structures of higher helical content. Both of the taurine derivatives of glucagon have the ability to stimulate the adenyl cyclase of rat liver membranes but at concentrations several fold higher than is needed for the native hormone. It is suggested that each carboxyl group contributes to the binding of the hormone to the specific membrane receptor sites.

The effect of peptide hormones and other small intestinal secretory stimulants on ion and water transport by ascending and descending rat colon was investigated in vitro using a muscle-stripped everted open sac preparation. Net water flux was measured gravimetrically at 30min intervals for 150 min, each sac serving as its own control. Water flux rate was constant over the entire period and equal in ascending (15.6 ± 1.1) and descending (14.9 ± 1.1 μl hr-1 mg-1 of dry weight) colon. Both segments responded identically to all test substances. Neither glucagon 10-5 m nor pentagastrin 10-5 m, singly or in combination, had a significant effect on net water flux. In contrast, theophylline 10-2 M and dibutyryl cyclic AMP 10-3 M reduced net flux significantly by 23% (P

The serum triiodothyronine (T3) and thyroxine (T4) concentration of healthy persons remains almost constant for at least ten months. Fresh serum and serum frozen for two years were compared. The mean difference in T3 concentration is negligible, only 3 ng/100 ml. The radioimmunoassay has a Between Assay Coefficient of 6.3% and is therefore reliable. Results can be obta

A radioimmunoassay for porcine intestinal calcium-binding protein (CaBP) has been developed. 125 I-labeled CaBP was prepared using chloramine-T. Antisera to porcine CaBP were raised in guinea pigs and rabbits. Antibody-bound CaBP was separated from free CaBP using talc. The immunoassay was highly sensitive, measuring concentrations of CaBP in duodenal extracts as low as 1 ng/ml with good reproducibility. This is sufficiently sensitive to measure CaBP in small amounts of duodenal tissue obtained by peroral capsule biopsy. The method was specific for CaBP, showing no reactivity toward other duodenal proteins or several peptide hormones. Measurements of CaBP by immunoassay showed a good correlation with measurements of CaBP-specific calcium-binding. The immunodilution curves obtained when assaying pig renal cortical extracts against pure duodenal CaBP were parallel when tested with six different antisera, demonstrating a high degree of immunochemical similarity between kidney and duodenal CaBP. Clearly, the radioimmunoassay possesses distinct advantages for the study of the biologic role of CaBP.

The brains of 14-month-old White Leghorn cocks were homogenized, fractionated by differential centrifugation, and used as enzymatic materials. When [4-14C]pregnenolone was incubated with the brain homogenate, the substrate was recovered without conversion. After incubation of [4-14C] progesterone with the brain homogenate, it was converted to 5α-pregnanedione and 3α-hydroxy-5α-pregnan-20-one. When [4-14C]17α-hydroxyprogesterone was incubated with the brain homogenate, it was metabolized to androstenedione, 3α, 17α-dihydroxy-5α-pregnan-20-one and 17α,20α-dihydroxy-4-pregnen-3-one. [4-14C] Androstenedione was metabolized to 5α-androstanedione, testosterone, androsterone, epiandrosterone and 5α-androstanediol; and [4-14C]testosterone was metabolized to androstenedione, 5β-dihydrotestosterone and 5β-androstanediol in the chicken brain. The enzymatic pattern in the brain of cocks was characterized by high activities of the 17β-hydroxysteroid dehydrogenase. Δ4-5β- and Δ5-5β-hydrogenase which acted on both C19- and C21-steroids, but was lacking in Δ5-3β-hydroxysteroid dehydrogenase associated with the Δ5a-Δ4isomerase, and 17α-hydroxylase.

The interaction of 125 I-labeled glucagon with isolated fat cell membranes of the rat has been studied. Specific binding of 125 I-labeled glucagon to membranes is a saturable process with respect to glucagon. The maximal binding capacity of the membranes is about 0.4 pmole of glucagon per mg of protein. Binding is readily displaced by low concentrations of native glucagon, but not by a large excess of other peptide hormones which affect lipolysis. Fragments 1–27 and 1–23 of glucagon compete for binding, but about thirty and thirty thousand times greater concentrations, respectively, are necessary to achieve effects similar to those observed with native glucagon. The 125 I-labeled glucagon eluted from the glucagon-fat cell membrane complex retains the ability of unused glucagon to interact with liver cell membranes. Glucagon in the incubation medium containing fat cell membranes is rapidly inactivated and undergoes marked physical changes. Inactivation is blocked by certain polypeptides, such as ACTH and bacitracin, and by 1.10 phenantroline. These compounds increase glucagon binding to membranes when subsaturating concentrations of hormone are used. The rate constants of glucagon-membrane association (1.6 × 10 6 M −1 sec −1 ) and dissociation (2.4 × 10 −4 sec −1 ) have been measured independently, and the dissociation constant based on these rate constants (1.5 × 10 −10 M) is similar to that obtained from equilibrium data (2.4 × 10 −10 M). High ionic strength and membrane digestions by trypsin and phospholipase A causes a virtually complete loss of glucagon binding. Fractionation of fat cell homogenates reveals that most of the glucagon-binding activity of such homogenates is present in the plasma membrane of the cell. The similarities between the glucagon-binding properties of fat cell membranes and those of liver cell membranes suggest that the glucagon receptors of liver and of adipose tissue may be closely related structures.

An original series of 62 endocrine tumours was reduced to 46 which could be regarded, on morphological grounds, as carcinoids or carcinoid-islet cell tumours. In sixteen of the 46 we were unable to demonstrate the presence of any hormonal peptide but the remainder comprised 10 gastrinomas, 6 insulinomas, 5 calcitoninomas, 4 corticotrophinomas, 1 vipoma and 4 tumours producing more than one hormone. By cytochemical and ultrastructural criteria all 46 tumours possessed the characteristic properties of the cells of the APUD series. They were therefore classified as apudomas (endocrine neurolophomas) arising from stem cells having a common origin in the neuroectodermal cells of the neural crest. We recommend a reappraisal of the morphological criteria for diagnosis of carcinoids and its replacement by a functional one which compels awareness of their true potential or actual nature.

The mechanism of action of many hormones is thought to be based on the interaction between the hormone and the membranes of target cells. We have examined the interaction of AII** with membranes from smooth muscle of guinea pig ileum [l] through binding studies with the radioactive hormone and monitoring of conformational changes at the membranes with the spin label technique [2,3]. 2.

Publisher Summary This chapter discusses the molecular coding of memory. The color discrimination experiments revealed a high degree of stimulus specificity within the same sensory modality. The most remarkable specificity has been found in maze learning in which injection of extracts of brain taken from mice trained to run a maze caused significantly faster learning but only when the recipients were tested in a maze identical With the one in which the donors were trained. It would be premature to assume absolute behavioral specificity because cross-reactions can occur even in the most exquisitely specific immune reactions. It also remains to be proven that each peptide produced in the brain corresponds to a given learned behavior. Many observations suggest that the behavior-inducing peptides are not species specific. Rat scotophobin was found to be active in mice and several species of fish, but it is probable that dark avoidance training in goldfish produces a different, perhaps related, peptide. These facts are analogous to those observed with peptide hormones. There is general agreement on memory being stored in the brain in terms of specific neuronal circuits, created by the formation of new synaptic connections at the time of learning.

The existence of polypeptide hormone receptors in the human placenta was evaluated by studying the specific binding of 125-I-labeled insulin, human growth hormone (hGH), human pro lac tin (hPRL) and glucagon to a defined placental membrane fraction. Only insulin showed specific binding to placental membranes. The binding of 125-I-insulin was time and temperature dependent. Its dissociation from the membrane was first order with a half time, at 24° C, of twenty minutes. Specific binding was readily observed at a concentration of 5 × 10−11 (7.5 μU./ml.). Inhibition of 125-I-insulin binding by unlabeled insulin was 30 per cent at 10−9 M, and >90 per cent at 10−9 M. Desalanine insulin was equally effective in inhibiting the binding of 125-I-insulin. Proinsulin was about twenty times less effective, and desoctapeptide insulin was even less effective. Structurally unrelated polypeptide hormones were without significant inhibitory effect. Binding sites of relatively high affinity (K1 = 4.2 × 108 M−1) and low capacity could be distinguished from those of lower affinity (K2 = 0.7 × 108M−1) and higher capacity. Insulin degrading activity was shown to be present in the placental membranes. Under standard binding assay conditions, less than 20 per cent of the 125-I-insulin present was degraded. The 125-I-insulin could be eluted from the membranes and appeared intact by several criteria. Specific binding was augmented by Ca++ and other divalent cations but was unaffected by high sodium chloride concentrations. Binding was greatly reduced by pretreatment of membranes with proteolytic enzymes. Incubation with phospholipase C, RNase and neur-aminidase had relatively little effect on binding. Insulin binding was unaffected by maternal diabetes but was reduced in membranes from early gestational placentas. There was considerable species variation in insulin binding, with the placental membranes of the guinea pig and monkey having the highest and those of the rat having the lowest binding. The characteristics of the insulin binding sites in the human placenta are similar to those in established insulin target tissues.

Experiments were performed to determine the mechanism for the effect s of mercuric chloride on the reproductive system of the hamster. Tissue levels of mercury were determined in animals which were treated with daily sc doses of saline 1 mg of mercuric chloride or 1 mg of mercuric chloride and 50 mg of N-acetyl-DL-penicillamine (NAP) throughout 1 4-day estrous cycle. The relations between concentations of mercury in several organs were found to be kidney>liver>anterior pituitary>ovary> blood>uterus>hypthalamus>cerebral cortex. Animals which were injected with mercuric chloride and NAP had significantly less (p less than .001) mercury than animals treated with mercuric chloride alone in all tisses except the cerebral cortex. Tissues from a nimals that were injected daily with 12 mcCi of mercuric chloride-203 an d 1 mg of mercuric chloride were prepared for radioautography. In the ovary mercury was more concentrated in the corpora lutea than the folli cles of interstitium. Mercury was also found lining the sinusoids of the pituitary and in some of the neurons of the arcuate nucleus of the hypothalamus. It has been suggested that the neurons in the region of the arcuate nucleus and median eminence synthesize and store the gonadotropin-releasing hormones of FSH-RH LH-RH and PIF which are released by the neural stimuli originating in the anterior hypothalamus. Mercury may have interfered with the synthesis and release of FSH-RH LH-RH and PIF from these neutrons. The possibility exists that other releasing hormones also may be affected by damage to the arcuate region of the hypothalamus. When hamsters were given a tota l of 3 or 4 mg of mercuric chloride during the 1st cycle 60% of the animals did not ovulate by Day 1 of the 3rd cycle.(AUTHORS MODIFIED)

Secretion of hydrochloric acid by the stomach has captured the attention and research efforts of some of the most distinguished investigators in the history of gastrointestinal physiology. Enormous interest in hydrochloric acid secretion by the stomach has been retained both because of the extraordinary sequence of physiologi­ cal events that permits the stomach to concentrate hydrogen ions by more than one millionfold in gastric juice and also because of the direct clinical implications of abnormalities in rates of gastric acid secretion. A variety of common, unusual, and rare clinical syndromes and diseases are associated with disturbances in rates of acid secretion by the stomach. In several of these diseases disordered gastric acid secre­ tion appears to be of crucial importance in the initiation and/or perpetuation of the pathological process. The precise mechanism(s) by which acid secretion participates in the pathogenesis of some of these diseases, e.g. peptic ulcer, remains to be clarified in completeness. It is in relation to the control of gastric acid secretion that we direct our attention to a substance about which an enormous amount of information has been recently accumulated and which appears to play a most important role in stimulating gastric acid secretion-the polypeptide hormone gastrin.

The activity of ornithine decarboxylase is increased 20–500-fold in three situations in tadpole hepatic tissue. Two effects are thyroxine-induced. The first is transient and occurs 3–8 hr after the injection of thyroxine; the second occurs several days after the hormone injection; and the third effect is independent of exogenous thyroxine and occurs 2–10 hr after tadpoles which had been kept in water at 5° are transferred to water at 25°C. The latter effect, which is maximal if tadpoles are kept at 5° for 24 hr, is partially inhibited by cycloheximide.

The hypothalamus secretes hormones which in turn affect the release of hormones from the anterior lobe of the pituitary gland. Evidence has so far been adduced for the existence of seven hypothalamic releasing hormones and three inhibiting hormones. These neurohormones are all oligopeptides and occur in only nanogram amounts in the hypothalamus. The study of synthetic analogs has provided valuable information regarding their mechanism of action.

This report provides a detailed analysis of the methods employed in our laboratory for the growth and maintenance of functional monolayer cultures of murine adrenal tumor cells. Radiochemical analysis of the products of incubation of these cells with 3H-pregnenolone, 14C-progesterone, and 14C-sodium acetate has confirmed that the predominant steroids secreted into the medium are 20α dihydroprogesterone and 11β hydroxy, 20α dihydroprogesterone. Chemical and enzymatic procedures for the synthesis of the latter compound and other 20α hydroxylated steroids are described. A fluorometric assay has been developed that permits precise quantitation of steroid output within several minutes' incubation. Consistent Steroidogenic responses to ACTH ranging as high as ten times the basal output have been observed. There is a linear log-dose response to ACTH (or Synacthen, a β1–24 peptide fragment of ACTH) ranging from 10 pg/ml to 1000 pg/ml. A variety of other peptide hormones were found to be inactive. The response to ACTH proceeds linearly from 10 min to 24 hr. These cells provide a viable and highly reproducible system for the in vitro study of ACTH action.

Glucagon-sensitive, insulin-secreting tumors of the Syrian (golden) hamster were homogenized in 1 mm NaHCO3 and subjected to differential centrifugation. The 10,000 x g particles were used for both binding of 125I-glucagon and activation of adenylate cyclase. Glucagon was the only hormone that activated adenylate cyclase. 125I-Glucagon bound to receptors rapidly and was competitively displaced by 1 µg per ml or less of unlabeled hormone; other polypeptide hormone were without effect. The glucagon concentration that produced half-maximal activation of adenylate cyclase was 10 ng per ml and half-maximal displacement of 125I-glucagon was 5 ng per ml; 2–29 glucagon, missing only the NH2-terminal histidine, bound to receptors but did not activate adenylate cyclase. When 2–29 glucagon was added to native glucagon, it blocked activation of adenylate cyclase. Extracts of porcine gut that contained glucagon immunoreactivity also activated adenylate cyclase but were only one-tenth as potent as pancreatic glucagon; 2–29 glucagon inhibited the effect of "gut glucagon."

It is proposed that neuroendocrine cells migrate into the primitive allimentary tract mucosa and are carried with the developing endocrine glands to their final resting places, where they mature into endocrine cells of the anterior pituitary, thyroid, parathyroid, islets of Langerhand, ultimobranchial body and thymus. Neuroendocrine cells in the hypothalamus produce the posterior pituitary hormones, and related cells (argentaffin cells) are present throughout the alimentary tract mucosa and in the various derivatives of the gut—bronchi, salivary glands, pancreatic and biliary ducts, bladder and genitourinary system. Related cells can be found in the adrenal medualla, sympathetic ganglia, paraganglia and glomus or chemoreceptor system. These widely scattered neuroendocrine cells have the capacity to develop into a wide variety of peptide-producing endocrine tumors. If this hypothesis is correct, the syndrome of multiple endocrine adenomatosis may simply be a dysplasia of neural ectoderm. Arguments in support of this theory are given.

The fraction F3 obtained after gelfiltration of a pineal extract on Sephadex G-25 (Fine) which inhibitsin vitro the secretion of FSH of the anterior hypophysis has been chromatographed on the weak cation-exchanger Amberlite IRC-50, XE-64. This method which was very useful as a purification step of the Sephadex G-25 fraction F2 and has been used with success to isolate the peptide hormones of the neurohypophysis, does appear to be unsuitable for further purification of a pineal fraction with inhibiting activity.

A method has been developed for separation and partial purification of four protein hormones from acetone-dried human pituitary glands. The major portion of growth hormone, follicle-stimulating hormone, luteinizing hormone and thyroid-stimulating hormone activities are each obtained in a separate fraction. Although the yields of the individual hormones are similar to those obtained by other investigators, the present procedure is simpler and a cleaner separation of the hormones has been achieved. Methods for further purification of each fraction are indicated including preparation of luteinizing hormone of very high potency (equivalent to 5·3mg. of standard NIH–LH–S3/mg.).

The widespread distribution of certain steroids and amino acid derivatives with hormonal properties is considered evidence in support of the dictum that “it is not the hormones that change, but rather the uses to which they are put.” However, analyses of the distributions, biological activities, immunological cross-reactivities, and sequences of amino acids of five representative peptide and protein hormones or groups of hormones—lactogenic hormone, growth hormone, the corticotropin-MSH-β lipotropin family, insulin, and the neurohypophysial hormones—support a concept of change and of molecular evolution of these polypeptidic molecules. When analyzed in terms of the genetic code, the amino acid interchanges which have been revealed by determination of sequences of amino acids can, most often, be explained by single base mutations in the appropriate codons. In two instances where two base mutations within a single codon are required, intermediate replacements of amino acid have been suggested; one of these would lead to a 2-ALA-β MSH, and the other to a 4-PRO, 8-ILE oxytocin.

THE currently accepted hypothesis of the mechanism of action of peptide hormones affecting smooth muscle tone includes an initial obligatory step of binding to specific receptors in the cell plasma membranes. We have shown the presence of specific angiotensin receptors in microsomal membranes of rabbit aorta1. Here we report the isolation of a membrane fraction derived from the plasma membrane possessing specific binding sites for angiotensin, and the structural requirements of the angiotensin molecule for its binding.

A large number of hormones, of diverse molecular structure, evoke characteristic responses in target cells via the intermediary 3′,5′-AMP, the specificity of hormone action upon cell type being achieved by selective stimulation of adenyl cyclase. In the fat cells of rat adipose tissue, adenyl cyclase is stimulated by a number of hormones of disparate molecular structure, posing the question whether this cell type posesses multiple cyclase systems with distinctive specificities for individual hormones, or a single cyclase with broad specificity to a variety of hormones. Studies of the stimulatory effects of adenocorticotropin, glucagon, and epinephrine upon the adenyl cyclase of the rat fat cell “ghosts” (plasma membrane sacs) have shown that distinctive selectivity sites for each of these hormones can be differentiated. The β-adrenergic blocking agent Kö 592 abolished the stimulatory effect of epinephrine without influencing adenocorticotropin or glucagon; Ca was required for adenocorticotropin action, but not for glucagon or epinephrine. Dose-response curves show that the affinity of hormones to the cyclase system was in the order: glucagon > adenocorticotropin ≫ epinephrine; the magnitude of cyclase activation by maximal doses of hormones had a reversed order. Combinations of maximal doses of hormones failed to produce additive stimulation. The results show that in the membrane of the fat cell a single catalytic unit of adenyl cyclase is coupled to distinctive selectivity sites for three lipolytic hormones.

Changes in the skin color of mammals, including man, have been brought about by the administration of certain hormones (1, 2, 3). The manner in which the hormones act at the cellular level is not yet clear. In this report, the effects of four hormones on normal adult guinea pig melanocytes in tissue culture are described. The hormones are alpha melanocyte-stimulat-ing hormone (a-MSH), acetylcholine, norepinephrine, and melatonin.

Incubation of bovine thyroid slices in the presence of thyroid-stimulating hormone resulted in increased slice content of adenosine 3',5'-phosphate. This effect of thyroid-stimulating hormone was potentiated by the inclusion of 1 mm theophylline in the incubation medium. In the presence or absence of theophylline, adenosine 3',5'-phosphate accumulation was maximally stimulated by 10 munits per ml of thyroid-stimulating hormone, and the highest nucleotide levels were attained by 3 to 6 minutes of incubation. Levels declined slowly following this early peak. The system responded similarly with two thyroid-stimulating hormone preparations of differing purities. Other peptide hormones tested were inactive. Among various chemical agents tested, only epinephrine was found to stimulate adenosine 3',5'-phosphate accumulation. This effect of epinephrine was half-maximal at 0.2 to 0.3 µm, but the magnitude of the response was small and its duration was brief as compared to the action of thyroid-stimulating hormone. The response to epinephrine was antagonized well by dichloroisoproterenol, but poorly by phenoxybenzamine.

Substrate specificity of cathepsin C derived from rat liver, describing polymeric structure and behavior as acidic protein

Summary 1 . Skins of the lizard Anolis carolinensis darken in vitro in response to melanophore stimulating hormone (MSH), a peptide hormone, as well as to catecholamines. These hormones darken Anolis skins by dispersing the subcellular organelle, the melanosome, out into the dendritic processes of the dermal melanophores. 2 . Dibutyryl cyclic AMP and methylxanthines also darken skins. In addition, methylxanthines are synergistic with both catecholamines and MSH in causing skin darkening. These data suggest that the dispersion of melanosomes within melanophores in response to both MSH and catecholamines is mediated by cyclic AMP. 3 . α-Adrenoceptor blocking agents inhibit MSH-induced darkening but potentiate catecholamine-induced darkening. β-Adrenoceptor blocking agents, in contrast, inhibit catecholamine-induced darkening but have no effect on MSH-induced darkening. This selective blockade of one receptor while the functional integrity of the other receptor is maintained suggests that MSH and catecholamines increase cyclic AMP levels through different receptors. 4 . Catecholamines exert their action through β-adrenoceptors; MSH darkens skins through what appears to be a component of the β-adrenoceptor. β-Adrenoceptor stimulation may stimulate adenyl cyclase to increase cyclic AMP levels whereas MSH may inhibit cyclic AMP phosphodiesterase thereby preventing cyclic AMP breakdown.

Decapitation of fetuses in utero rendered rat liver tyrosine transaminase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) occasionally inducible by hydrocortisone. By placing fetal rat liver in organ culture, reproducible induction by the steroid could be obtained. Histological and biochemical studies supported the conclusion that the hepatocytes remained healthy over a 3-day period in culture. The response to hormone was very low in fresh explants for the first 12 hours, but thereafter a 4- to 8-fold elevation in enzyme activity was obtained within 4 to 6 hours. The induced level of the transaminase was maintained unless steroid was removed, and then the activity fell rapidly back toward the control level. The optimal effective concentration of hydrocortisone was 10-6 m, but levels as low as 10-8 m produced a limited response. Only the glucocorticoids and the protein hormones insulin and glucagon were active inducers among a number of hormones tested. Cycloheximide and actinomycin D prevented enzyme induction, but hydrocortisone did not affect either general RNA or protein synthesis. Lactic dehydrogenase activity in explants of fetal liver was unaffected by hydrocortisone. Titrations against antibody showed that the tyrosine transaminases in fetal and adult liver are immunologically identical and proved that the increase in enzyme activity in explants exposed to hydrocortisone was due to an increase in enzyme protein. Immunochemical labeling experiments confirmed that synthesis de novo of enzyme protein is stimulated by hydrocortisone. The results are consistent with the hypothesis that the synthesis of this enzyme is repressed during gestation.