Bioactive GH-like immunoglobulins G in active acromegaly: response to long-term treatment with bromocriptine.

In acromegaly, certain forms of circulating immunoreactive hGH are not true GH but IgGs which possess GH biological activity (bioactive GH-like IgGs). In this study, we tested the effect of bromocriptine on circulating bioactive GH-like IgGs in an acromegalic woman. Increasing doses of oral bromocriptine (2.5, 5.0 and 7.5 mg/day) were administered (for 2, 8 and 6 months respectively). TRH tests were performed before treatment and at the end of treatment with each dose. The patient was without detectable pituitary or extra-pituitary tumour by magnetic resonance imaging. Her serum contained bioactive GH-like IgGs equivalent to 240 mU/l of hGH and elevated insulin-like growth factor I (IGF-I; 9500 U/l). Basal hGH was 12.8 mU/l and increased to 220 mU/l 15 min after TRH (200 micrograms, i.v.). In addition, in the basal samples of each test we measured total IgGs (radial immunodiffusion), bioactive GH-like IgGs (isolated by Sephadex and protein A affinity chromatography and assayed using the Nb2 cell assay) and IGF-I(RIA). Bromocriptine treatment gradually reduced serum levels of bioactive GH-like IgGs and IGF-I, with significant falls observed first at 10 months of treatment. Bioactive GH-like IgGs were 240, 240, 36.0 and < 0.124 mU/l and IGF-I levels were 9500, 8700, 4000 and 3100 U/l at 0, 2, 10 and 16 months of treatment, respectively. In contrast, IR-hGH response to TRH decreased after 2 months of treatment to 89 mU/l and to 49.2 mU/l at the end of the study while basal IR-hGH remained between 13 and 8.4 mU/l. Basal PRL fell to almost undetectable levels. Bromocriptine treatment decreased the GH response to TRH and the serum concentration of bioactive GH-like IgGs and IGF-I. The striking similarity between the pattern of decrease of serum bioactive GH-like IgGs and IGF-I supports the presence of an immuno component in our patient's acromegaly.

New perspectives on the actions of sulphonylureas and hyperglycaemic sulphonamides on the pancreatic beta-cell.

Sulphonylureas and the hyperglycaemic sulphonamide diazoxide are commonly employed in the therapy of non-insulin-dependent (Type 2) diabetes mellitus and insulinoma, respectively. Stimulatory effects of sulphonylureas on insulin secretion and the inhibitory action of diazoxide are thought to be primarily mediated through modulation of the activity of ATP-sensitive K+ channels (K(+)-ATP channels) in the beta-cell plasma membrane. Certain sulphonylureas are known to be internalised by the pancreatic B-cell. Recent studies suggest that these drugs and diazoxide can influence insulin secretion from electropermeabilized beta-cells in which K(+)-ATP channels and other plasma membrane ion channels are inoperative. This observation suggests that sulphonylureas and diazoxide interact with intracellular sites in the pancreatic B-cell which are directly involved in the regulation of the final stages of exocytosis.

Growth hormone (GH) receptor antibodies with GH-like activity occur spontaneously in acromegaly.

Previous studies in our laboratory have identified a portion of big-big GH as actually being anti-GH receptor immunoglobulins. We now report the isolation of two types of anti-GH receptor antibodies from the serum of active acromegalic patients. One of them (patient A) interferes with the human GH RIA, thus overestimating the real plasma GH values. The other type of immunoglobulin G (IgG; patient B) was detected in an acromegalic patient with almost normal immunoreactive GH level. The main aim of the present study was to explore whether these anti-GH receptor IgGs possess GH-like biological activity. The IgGs of both patients were isolated by chromatography on Sephadex G-100 and then on protein-A-Sepharose. In the bioassay, cultured Nb2 lymphoma cells were incubated with hGH standards and serial dilutions of the purified IgGs, and cell proliferation was used as a measure of biological activity. The IgGs of both patients showed GH-like bioactivities, which, when calculated as equivalents of human GH, correspond to approximately 260 and 120 micrograms/L, respectively. The results suggest that biologically active anti-GH receptor antibodies may contribute in the pathology of some cases of acromegaly.

[G proteins]. / Proteínas G.

G proteins play a central role in the mechanism of action of most hormones and neurotransmitters. They act as signal transducers between membrane receptors activated by extracellular stimuli on the one hand and intracellular effectors which control the concentrations of cytosolic messenger molecules (cAMP, cGMP, inositol phosphates, Ca2+) on the other. G proteins form a highly conserved family of membrane-associated proteins composed of alpha, beta and subunits. The alpha subunit, which is unique for each G protein, determines its biological activity and binds GDP and GTP. A number of diseases are already known to involve structural and/or quantitative changes of G proteins in plasma membranes. Interestingly, proteins encoded by some oncogenes show a high degree of homology with G proteins, which suggests that certain malignancies may be caused by alterations of transmembrane signaling.

Effects of tunicamycin on growth hormone binding in rat adipocytes.

Digestion of covalently linked [125I]human (h) GH-receptor complexes with neuraminidase or endoglycosidase F reduced the mass of the principal hormone receptor complex from about 130 kilodaltons (kDa) to 120 and 110 kDa, respectively, suggesting that about 20% of the mass of the GH receptor of rat adipocytes consists of N-linked sialocarbohydrates. Incubation of adipocytes with tunicamycin, an inhibitor of N-linked glycosylation, decreased the incorporation of [35S]methionine into membrane glycoproteins by more than 50% in 4 h and decreased specific binding of [125I]hGH by about 70% after 8 h. Decreased binding and incorporation of [35S]methionine were seen only after a lag time of about 2 h. Cross-linking of [125I] hGH to cells that had been treated with tunicamycin resulted in the appearance of a new labeled species of hormone-receptor complex with an apparent mass of about 110 kDa. This band appeared after a delay of about 3 h and reached approximately equal prominence with the 130 kDa band at 5 h. By 8 h, the 110 kDa complex was the predominant band in radioautograms, but some of the 130 kDa species remained. Scatchard analysis of binding data in tunicamycin-treated adipocytes indicated that decreased binding of [125I]hGH resulted from a 3- to 4-fold decrease in affinity accompanied by only a small (30%) decline in receptor number. Tunicamycin did not affect the rate of receptor turnover in cells that were also treated with cycloheximide to block protein synthesis, but receptor turnover decelerated with increasing time of incubation. Treatment with tunicamycin for 8 h markedly slowed the rate at which specifically bound [125I]hGH disappeared from adipocytes, suggesting that N-linked carbohydrates may play some role in internalization and processing of labeled hormone. We conclude that 1) N-linked carbohydrates contribute about 20 kDa to the apparent mass of the GH receptor of rat adipocytes; 2) N-linked glycosylation is not required for GH receptors to be inserted into the adipocyte membrane in the proper orientation and to retain their ability to recognize and bind GH; 3) N-linked sugar chains are required for maintenance of a normal high affinity of receptors for GH; 4) N-linked carbohydrates are necessary for normal rates of internalization and processing of bound hGH.

Certain large forms of circulating immunoreactive human growth hormone are in fact immunoglobulins.

To explain frequent discordances between serum GH levels and clinical manifestation of acromegaly, we investigated the possibility that certain immunoglobulins G (IgGs) might be responsible for the displacement of [125I]human (h) GH in the hGH RIA. We incubated dilute sera from seven active acromegalics (basal immunoreactive hGH, 22-313 micrograms/L) with rat adipocyte plasma membranes adsorbed on polystyrene plates. IgGs that bound to GH receptor sites in the absence and presence of 250 nM hGH (for nonspecific binding) were detected using anti-hIgG (Fc-specific) antibody conjugated with alkaline phosphatase. In this system two of the seven sera studied tested positive for IgGs against GH-binding sites (serum 4 in 1:400 dilution, and serum 7 in 1:10 dilution). We studied further the serum with the highest titer. On Sephadex G-100, most of the GH-like immunoreactivity (assayed by RIA) present in serum 4 coeluted with IgGs (assayed by immunodiffusion) as a high mol wt (greater than or equal to 150 kDa) component. To confirm its IgG nature, this material was then adsorbed on protein-A-Sepharose and eluted with 0.1 M sodium citrate, pH 3.0. The protein-A-purified IgGs from serum 4 bound specifically to GH receptor sites in adipocyte membranes and displaced [125I]hGH in the hGH RIA. In contrast, IgGs purified from another acromegalic patient (313 micrograms/L hGH) repeatedly tested negative in the membrane binding assay and hGH RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Different growth hormone-receptor interactions mediate insulin-like and lipolytic responses of rat adipose tissue.

The GH receptor in adipocytes is a glycoprotein that has a half-life of less than 1 h. After 2 h of treatment with the alkaloid swainsonine, which interferes with carbohydrate processing, virtually all of the GH receptors on the surface of adipocytes are replaced with receptors whose carbohydrate side-chains are incomplete. We examined the effects of swainsonine on the responsiveness of adipose tissue to GH to determine whether these receptors, which bind GH normally, retain biological competence. In the concentration range of 100-300 ng/ml human (h) GH rapidly evokes insulin-like responses in adipose tissue or adipocytes that have been deprived of GH for at least 3 h. hGH, at concentrations ranging from 1-10 ng/ml, also increases lipolysis after a delay of at least 2 h. Pretreatment with 50 micrograms/ml swainsonine failed to influence insulin-like responsiveness to hGH, as judged by increased glucose oxidation, but nearly completely abolished the lipolytic response. Pretreatment with swainsonine, however, did not reduce lipolysis in response to isoproterenol, suggesting that signal transmission rather than the lipolytic apparatus per se had been affected. To determine whether the same receptors mediate lipolytic and insulin-like responses, the binding properties of hGH were compared to those of Da1, a chemically modified form of hGH, whose insulin-like potency is reduced relative to its lipolytic potency. Da1 and hGH were equipotent in promoting lipolysis and had an ED50 of about 3 ng/ml, but hGH was at least 6 times as potent as Da1 in promoting glucose oxidation (ED50 of 65 vs. 400 ng/ml). Scatchard plots of both Da1 and hGH binding data were linear, consistent with a single class of binding sites whose affinity for hGH was about 3.5 times higher for hGH than Da1. hGH and Da1 both produced half-maximal stimulation of glucose oxidation when about 90% of the GH receptors were occupied. In contrast, half-maximal lipolysis was produced by Da1 when 8% of GH receptors were occupied, but 21% occupancy was required for a similar effect of hGH. If a subclass of GH receptors mediates lipolysis, it is likely to comprise 10% or less of the total receptor population.

Purification of the pancreatic cholecystokinin receptor.

We have previously shown that the pancreatic cholecystokinin (CCK) receptor can be solubilized in 1% digitonin. In this study, digitonin-solubilized CCK receptors from rat pancreas were purified using sequential affinity chromatography on ricin-II agarose and on AffiGel-CCK. Electrophoresis of the radioiodinated purified receptors on SDS-polyacrylamide gels followed by autoradiography revealed two proteins: a major band of Mr = 80,000-90,000, and a minor band of Mr = 55,000. Through the purification procedure, the receptors preserved their agonist specificity (CCK-8 less than CCK-33 less than desulfated CCK-8 less than CCK-4) and binding affinity. Scatchard transformations of binding data for the purified receptor preparation were best fit by linear plots compatible with a single class of binding sites with Kd = 9.4 nM. The estimated purification was about 80,000 fold and consistent with the expected Bmax for a pure Mr = 80,000 protein binding one CCK molecule. This two-step purification procedure opens the possibility for molecular studies of the CCK receptor.

Characterization of covalently cross-linked pancreatic somatostatin receptors.

The receptor for somatostatin present in rat pancreatic plasma membranes was characterized by affinity labeling with [125I-Tyr11]somatostatin utilizing three different heterobifunctional cross-linking agents: N-5-azido-2-nitrobenzoyloxy-succinimide, N-succinimidyl 6-(4-azido 2'-nitrophenylamine)hexanoate, and N-hydroxysuccinimidyl 4-azido-benzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a broad band of Mr = 92,000 when any of the three cross-linkers was used; N-succinimidyl 6-(4-azido 2'-nitrophenylamine), however, was most efficient. Labeling of the Mr = 92,000 protein band was not affected by reducing agents but was sensitive to somatostatin and guanine nucleotides, particularly GTP gamma S, at concentrations which reduced binding to the receptor. The affinity-labeled protein could be solubilized completely with Zwittergent 3-12, partially with Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and poorly with Zwittergent 3-08 and digitonin. When exposed to agarose-coupled lectins, the detergent solubilized, labeled Mr = 92,000 protein was completely adsorbed to wheat germ agglutinin, partially to ricin communis II, and not at all to concanavalin A or lotus or lentil lectin. The Mr = 92,000 protein bound to wheat germ agglutinin-agarose was not eluted by N-acetylglucosamine but was by triacetylchitotriose, providing a considerable purification of the somatostatin receptor. These data allow us to conclude that the somatostatin receptor is a monomeric glycoprotein with an Mr = 90,000 binding subunit which probably contains a polymeric arrangement of N-acetylglucosamine residues.

Do antagonists of pancreatic cholecystokinin receptors interact with central nervous system cholecystokinin receptors?

The abilities of the pancreatic cholecystokinin (CCK) receptor antagonists dibutyryl cyclic GMP, proglumide, benzotript, CBZ-tryptophan, CBZ-cysteine and CCK-27-32-amide to inhibit CCK binding to its receptor in the pancreas and brain of mice and guinea pigs was examined. In both species, the same relative potencies of the antagonists in brain and pancreas was seen except that dibutyryl cyclic GMP was considerably more potent on pancreas than on cerebral cortex CCK receptors. CCK-27-32-amide was the most potent inhibitor for both brain and pancreas but was more potent in the guinea pig than in the mouse. Proglumide, a relatively weak antagonist, was a more potent inhibitor of the guinea pig than of the mouse pancreas receptor. Thus, these data suggest that there are both tissue-specific and species-specific differences in CCK antagonist interactions with the CCK receptor.

Solubilization and characterization of CCK receptors from mouse pancreas.

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.

Effects of PHI on hormonal secretion from perfused rat pancreas.

Effects of the novel gastrointestinal polypeptide PHI with N-terminal histidine, C-terminal isoleucine amide, and 27 amino acids have been studied in isolated perfused rat pancreas. PHI increased the release of insulin, glucagon, and somatostatin. The amounts of these hormones released were strictly dependent on the prevailing glucose concentrations. In the absence of glucose, PHI (1 nmol/liter) stimulated glucagon release. In the presence of 4.4 and 6.7 mmol/liter glucose, the same dose of this peptide stimulated insulin and somatostatin release. In the presence of 16.7 mmol/liter glucose, only insulin secretion was increased by PHI. When arginine was used as a secretagogue, PHI (10 nmol/liter) potentiated secretion of insulin, glucagon, and somatostatin. Thus, PHI may take part in the regulation of the function of the pancreatic A, B, and D cells.

Effects of porcine intestinal heptacosapeptide and vasoactive intestinal polypeptide on insulin and glucagon secretion in rats.

Pure porcine vasoactive intestinal polypeptide (VIP) and porcine intestinal heptacosapeptide (PHI) were infused in fed rats in doses of 1, 10, and 100 pmol/kg . min. The peptides were administered alone or together with glucose (40 mg/kg . min) or arginine (50 mg/kg . min). In the basal state, VIP at a dose of 1 pmol/kg . min and PHI at a dose of 10 pmol/kg . min produced a slight but significant hypoglycemia, whereas 100 pmol/kg . min VIP and PHI had a hyperglycemic effect. VIP at a dose of 10 and 100 pmol/kg . min enhanced the glucose-induced secretion of insulin. PHI exerted such an effect only when administered at the highest dose. When arginine was present as a secretagogue, 10 pmol/kg . min of both VIP and PHI potentiated secretion of insulin and glucagon and elevated the prevailing hyperglycemia. In conclusion, when given as a constant infusion in rats, both PHI and VIP exert a direct hypoglycemic effect and modulate the influence of glucose and arginine on insulin and glucagon secretion. It is as yet unclear to what extent these findings reflect normal physiological events.

Effects of PYY and PP on endocrine pancreas.

Novel intestinal polypeptide YY (PYY) and pancreatic polypeptide (PP) were infused in fed anaesthetized rats. The peptides (10 and 100 pmol/kg X min) were administered during 30 min, alone, together with glucose or together with arginine. Plasma concentrations of glucose, insulin and glucagon were measured. At the dose of 10 pmol/kg X min the peptides had no effect. PP at the dose of 100 pmol/kg X min slightly augmented basal, but had no effect on stimulated insulin and glucagon release. PYY at the dose of 100 pmol/kg X min was without effect on basal insulin and glucagon levels and on glucose-induced insulin release, but exerted an inhibitory effect on arginine-induced secretion of both insulin and glucagon. It is unlikely that PYY and PP can affect secretion of insulin and glucagon via blood circulation. The potential capability of high doses of PP to affect insulin and glucagon secretion suggests that this peptide may exert direct (paracrine) effects on the pancreatic A- and B-cells.

Effects of cholecystokinin, gastric inhibitory polypeptide, and secretin on insulin and glucagon secretion in rats.

Pure porcine cholecystokinin-33 [the triacontatriapeptide form of cholecystokinin (CCK-33)], gastric inhibitory polypeptide (GIP), and secretin were infused in rats in doses of 1, 10 and 1000 pmol/kg . min. The peptides were administered alone or in combination with glucose (40 mg/kg . min) or arginine (50 mg/kg . min). In the basal state, CCK-33 and GIP produced significant hypoglycemia at all concentrations used, although they elevated insulin levels only at the highest dose. Secretin had no effect. CCK-33 at a dose of 1 pmol/kg . min enhanced the secretion of insulin induced by glucose or arginine. These effects were more pronounced when higher doses of CCK-33 were administered. GIP at a dose of 1 pmol/kg . min had no effect on insulin release. Higher doses of GIP significantly potentiated insulin release stimulated by glucose or arginine. Secretin (100 pmol/kg . min) had no clear-cut effect on glucose-induced insulin secretion, but it slightly enhanced arginine-induced secretion. All hormones investigated, at all doses used, significantly stimulated the arginine-induced secretion of glucagon. We conclude that CCK-33 is a potent stimulatory factor of glucose- and arginine-induced insulin secretion and should therefore be taken into consideration as an incretin candidate. In addition, CCK-33 and GIP modulate glucose homeostasis by affecting glucagon release.

Effect of GIP on the secretion of insulin and somatostatin and the accumulation of cyclic AMP in vitro in the rat.

The effects of gastric inhibitory polypeptide (GIP) on insulin secretion as well as on the intra-islet accumulation of [3H]cyclic AMP were investigated in isolated pancreatic islets of the rat. In the presence of 6.7 mmol/l of glucose, 3.0 and 30 nmol/l of GIP induced both insulin and [3H]cyclic AMP responses, while lower and higher concentrations of the peptide were ineffective. A coupling of the two parameters was also found with regard to interaction between glucose and GIP. Thus while 30 nmol/l of GIP was stimulatory together with 6.7, 16.7 or 33.3 mmol/l of glucose, the peptide stimulated neither insulin release, nor the accumulation of [3H]cyclic AMP in the presence of a low concentration of glucose (3.3 mmol/l). The concomittant release of insulin and somatostatin was studied in the perfused pancreas in order to assess a possible influence by somatostatin on the dose-response pattern for GIP-induced insulin release. In this preparation 1.0 to 10 nmol/l of GIP stimulated insulin and somatostatin secretion; however while these concentrations were equipotent on insulin release, 10 nmol/l of GIP stimulated somatostatin release more than 1 nmol/l, indicating differences in dose-response curves for the GIP-induced stimulation of the two hormones. It is concluded that 1) modulation of GIP-induced insulin release is coupled to changes in cyclc AMP response in the islet, 2) GIP-induced somatostatin secretion may influence the concomittant insulin response.

The interaction of vasoactive intestinal polypeptide (VIP), glucose and arginine on the secretion of insulin, glucagon and somatostatin in the perfused rat pancreas.

Vasoactive Intestinal Polypeptide (VIP) increased the release of insulin, glucagon and somatostatin from the perfused rat pancreas. The amount of these hormones released was dependent upon the prevailing glucose concentration. VIP stimulated glucagon release in the absence of glucose, while insulin and somatostatin release were increased by VIP only in the presence of glucose concentrations of 4.4 mmol/l and above. Glucagon secretion stimulated by arginine in the presence of 4.4 mmol/l glucose was potentiated by VIP. In contrast, VIP did not induce any further increase in the secretion of insulin and somatostatin over that stimulated by arginine. At higher concentrations of glucose (6.7, 16.7, and 33.3 mmol/l) VIP continued to stimulate insulin and somatostatin release, this effect being synergistic on early-phase insulin release. The effects of VIP on islet cells thus depend on the levels of modulating nutrients.

The intestinal calcium and phosphate transport under conditions of experimental hypercalcemia.

The intestinal calcium and phosphate transport under conditions of experimental hypercalcemia. Acta Physiol. Pol., 1977, 28 (2): 127--134. Intestinal calcium and phosphate transport was examined in various segments of small intestine under two differently processed hypercalcemic conditions. The analysis of the serum calcium and phosphate level changes, showed the increasing serum calcium values without any concomitant change in the serum phosphate concentration during the course of the experiment. The observed discrepancy of the intestinal phosphate transport when compared the in vitro and in vivo studies, suggests the presence of phosphate transport regulatory mechanism acting on the intestinal level.