Fred Esch

Physiological roles of somatocrinin and somatostatin in the regulation of growth hormone secretion

Abstract Somatocrinin, a 44 amino acid peptide with potent growth hormone (GH) releasing activity in anesthetized rats, was tested in conscious freely-moving rats. When high doses of 1 to 10 μg were administered (iv) at random times between spontaneous GH pulses, the responses were inconsistent. When similar doses were tested under identical conditions but in rats pretreated with antibodies against somatostatin, all animals demonstrated a marked and immediate increase in plasma GH of 5 to 10 fold. Similarly, a 1 μg dose of somatocrinin was also ineffective in increasing plasma GH when administered to rats subjected to a 72 h fast, a paradigm known to enhance endogenous somatostatin secretion. However, plasma GH increased over 20 fold if rats were pretreated with antibodies against somatostatin. These results demonstrate the dynamic and opposite roles exerted by somatocrinin and somatostatin in regulating GH secretion.

Neuronal nicotinic acetylcholine receptor β‐subunit is coded for by the cDNA clone α4

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for α‐bungarotoxin, which have been purified from rat and chicken brains by immuno‐affinity chromatography, consist of two types of subunits, α and β [1,2]. The β‐subunits form the ACh binding sites [3]. Putative nicotinic AChR subunit cDNAs α3 and α4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR α‐subunit. Here we determine the amino‐terminal amino acid sequence of the rat brain AChR β‐subunit by protein microsequencing to be the same as amino acid residues 27–43 of the protein which could be coded by α4. Further, we present evidence consistent with a subunit stoichiometry of α3β2 for this neuronal nicotinic AChR.

Somatocrinin, growth hormone releasing factor, stimulates secretion of growth hormone in anesthetized rats.

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, invivo, contrary to invitro results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these invivo results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.

characterization of A 40 residue peptide from a human pancreatic tumor with growth hormone releasing activity

Abstract Structural characterization of a 40 amino acid peptide with high intrinsic growth hormone releasing activity isolated from a human pancreatic tumor which had caused acromegaly was accomplished by gas phase sequence analyses of the intact peptide and its carboxy terminal cyanogen bromide digestion fragment. High pressure liquid chromatography of the native peptide and synthetic replicates showed that the molecule possessed a free acid rather than an amidated carboxy terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys- Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly- Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-OH using 1.8 nmoles of material. The structural identity of this material with a previously characterized fragment of a larger growth hormone releasing peptide isolated from a different human tumor is discussed.

Follistatin specifically inhibits pituitary follicle stimulating hormone release invitro

Two forms of purified follistatin, a single-chain polypeptide of mol wt 35,000 (35 Kd) protein, and a related molecule of mol wt 32,000 (32 Kd), which differs from the 35 Kd form in glycosylation or carboxyl terminal truncation, specifically inhibit the release of immunoreactive FSH by primary cultures of rat pituitary cells. Both forms of follistatin and inhibin-A give similar dose-response curves, with identical slopes and maximal effects, suggesting that they may all act through the same mechanism on the pituitary cells. The median effective dose (ED50) of each of the follistatins is 6.2-7.3 ng/ml (1.8 x 10(-10) M), which corresponds to approximately 1/3 of the potency of inhibin. The effect of 35 Kd or 32 Kd follistatin is highly specific for suppressing the release of immunoreactive FSH since there is no demonstrable concomitant effect on the secretion of other pituitary hormones. The effect of follistatins, like that of inhibins, is different from that of the hypothalamic hypophysiotropic factors, requiring greater than or equal to 18 h of incubation in a pituitary monolayer culture system to demonstrate. Coincubation of inhibin and follistatin shows an additive effect in the suppression of FSH release. Pituitary cells exposed to follistatin have significantly less depletion of intracellular FSH (0.01) than those treated with inhibin, indicating that follistatin may act primarily on the suppression of FSH release rather than on both release and synthesis of FSH, as is the case with inhibin.

Polypeptide microsequence analysis with the commercially available gas-phase sequencer.

An evaluation of the commercially available gas-phase sequencer with regard to its sensitivity, ease of operation, and reliability is presented. Techniques for the preparation of ultra-pure reagents and solvents for this new technology are reviewed, as is a highly sensitive reverse-phase liquid chromatography program for the identification and quantitation of phenylthiohydantoin amino acid residues from the new sequenator.

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: Immunoreactivity and sequence homologies

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.

The corticostatic (anti-ACTH) and cytotoxic activity of peptides isolated from fetal, adult and tumor-bearing lung

The possibility that corticosteroids influence lung maturation was suggested in 1968 by Buckingham et al. and supported by studies mounted in humans and in rabbits by Liggins and Howie and by a collaborative study from the N.I.H. To our knowledge no reverse process, i.e. the regulation of adrenal function by the lung, has been postulated. In attempting to isolate ACTH from fetal lung we found peptides which depressed corticosterone production. Eventually, we isolated a group of peptides from rabbit fetal lung which showed potent inhibition of corticosterone production in rat adrenal cell suspensions. Using high-resolution reverse-phase HPLC and high-sensitivity gas-phase sequencing techniques the primary structure of one of these peptides has been determined and it was called corticostatin. The adult rabbit lung contains a large quantity of a very similar peptide, which elutes close to corticostatin but whose amino acid composition differs by a single glycine and possibly a serine. This peptide is also potently corticostatic. We have isolated homologous human peptides but these show no apparent corticostatic activity in rat adrenal cell suspensions. They do, however, show interesting effects on in vitro growth of lung cell lines.

The levels and biologic action of the human neutrophil granule peptide HP-1 in lung tumors

HP-1 is the most abundant human representative of a recently discovered class of neutrophil cystine- and arginine-rich peptides. These peptides have many potentially regulatory activities expressed at nanomolar concentrations. To establish the levels of HP-1 that can accumulate in human lung tumors and nondiseased lung fragments, tissues were extracted for their peptide content. The extracts were purified on reverse phase HPLC, and HP-1 and related peptides were identified by sequence analysis and their concentrations in the tissue quantitated by amino acid analysis. Immunohistochemistry was performed and strongly suggests that HP-1 is confined to granulocytes under most circumstances, and indicates that the levels of HP-1 measured in the tumors reflect the levels obtained when solid tissue is infiltrated by neutrophils. The maximum observed levels were 26 nanomoles per gram wet weight of tissue. Attempts were then made to correlate this level to the cytotoxic potential of HP-1 by performing in vitro cytotoxicity dose-response curves on several cell lines. Most cells were killed at between 1 and 8 microM, and the response depended on the growth conditions of the cells. The levels of HP-1 that accumulate in tumors can exceed the in vitro cytolytic concentrations. The levels are also considerably in excess of those required to exert in vitro regulatory actions.

Human growth hormone releasing factor and somatostatin from two pancreatic tumors: isolation and characterization

Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior.