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Biomedical subjects

R I Gregerman

Publications and source records attributed to R I Gregerman.

At least 37 records · Page 2Linked to original sources
PubMed1985

Suppression of thyrotropin in the low-thyroxine state of severe nonthyroidal illness.

In a prospective study, we assessed the role of thyrotropin in the development of the low-thyroxine state that is associated with severe illness. We measured the serum thyrotropin and thyroid hormone concentrations longitudinally in 35 patients with hematopoietic cancer or aplastic anemia who were treated by bone-marrow transplantation. In 19 patients thyroxine declined sharply after bone-marrow transplantation and was associated with a reduction of the serum thyrotropin in the 17 patients tested, often to levels below the normal range. The serum triiodothyronine level, free thyroxine index, and free thyroxine level also declined in these patients. In the patients who recovered, clinical improvement was accompanied by the return of thyrotropin and thyroid hormone concentrations to their pretreatment ranges. These and related findings suggest that the low-thyroxine state of severe illness is the result of several events, one of which is failure of the normal negative-feedback control of the pituitary-thyroid axis due to illness-associated, decreased secretion of thyrotropin. The notion that such patients are "euthyroid" must be questioned, but the possible value of thyroid hormone-replacement therapy in these circumstances remains to be determined.

Adolescent↗
PubMed1985

Ectopic beta-adrenergic receptors coupled to adenylate cyclase in human adrenocortical carcinomas.

The adenylate cyclase of an adrenocortical carcinoma of the rat is activated not only by ACTH but also by beta-adrenergic agonists, which bind to ectopic beta-adrenergic receptors not present in normal rat adrenal cortex. Previous reports examining possible beta-adrenergic control of adenylate cyclase in human adrenocortical carcinomas failed to demonstrate beta-adrenergic receptor-linked enzyme activity. We studied six human adrenal carcinomas and normal adrenal cortex from three subjects for beta-adrenergic agonist-sensitive adenylate cyclase and beta-adrenergic binding sites. Three of the six carcinomas had adenylate cyclase responses to both ACTH and beta-agonists. Two tumors were ACTH responsive but not beta-agonist responsive; one tumor responded to beta-agonists but not to ACTH. Adenylate cyclase activity of normal adrenal cortex from three subjects was stimulated by ACTH but not by beta-agonists. In membrane preparations from three tumors with beta-agonist-sensitive adenylate cyclase, the radiolabeled beta-adrenergic antagonist [125I]pindolol bound specifically and with high affinity (Kd = 38-83 pM) to a single class of binding sites which showed saturation with ligand concentration, reversibility of binding, pharmacological specificity, and stereospecificity. Normal cortex and one tumor without beta-adrenergic agonist-sensitive adenylate cyclase had no specific binding of [125I]pindolol. These results indicate that malignant transformation of adrenal cortex in man is frequently but not invariably associated with the appearance of ectopic beta-adrenergic receptors functionally linked to adenylate cyclase. Loss of ACTH-responsive adenylate cyclase may also occur simultaneously with the development of beta-adrenergic receptor-linked adenylate cyclase.

Adenylyl Cyclases↗
PubMed1984

Stimulation of human fat cell adenylate cyclase by GDP and guanosine 5'-O-(2-thiodiphosphate).

GDP regulation of basal and receptor-mediated catecholamine-sensitive human fat cell adenylate cyclase was studied using purified plasma membrane preparations and assay conditions selected to minimize conversion of GDP to GTP. Under ordinary assay conditions (low NaCl concentration) and with App(NH)p as substrate to prevent GDP conversion to GTP, basal enzyme activity was stimulated up to 2-fold by GDP (0.1 mM) while addition of epinephrine (0.1 mM) eliminated stimulation by GDP and reduced basal adenylate cyclase activity. With ATP as substrate, the enzyme was not responsive to hormone in the absence of guanyl nucleotides and GDP augmentation of basal activity was small (0-1.5-fold) while stimulatory effects of epinephrine and isoproterenol were minimally but definitely exhibited (1.5-fold over basal). Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a GDP analog resistant to phosphorylation and hydrolysis and an antagonist of GTP, stimulated enzyme activity more than did GDP but did not promote epinephrine action. Rather, inhibition of GDP beta S-stimulated adenylate cyclase activity was seen with both epinephrine and isoproterenol and also with GTP. In the presence of NaCl (200 mM), which alone produced 2-3-fold increase in basal enzyme activity, GDP (0.1 mM) and GDP beta S (50 microM) produced 8- and 15-fold increases of activity, respectively. Addition of UDP, to prevent possible conversion of GDP to GTP, had no effect on NaCl-enhanced activation by GDP. The results indicate that the human fat cell adenylate cyclase system is unique in responding to GDP and its analog GDP beta S by stimulation in the absence of hormone but suggest that as in other systems catecholamine-mediated stimulation is normally dependent on GTP. Salts (Na+) appear to stimulate the enzyme by facilitating the interaction of the guanyl nucleotide regulatory protein (N8) with the catalytic unit.

Adenosine Triphosphate↗
PubMed1984

Partial purification from rat and pig liver of cytosolic stimulators of hormone-sensitive adenylate cyclase: quantitation and resolution of two components.

Procedures were carried out to isolate from liver cytosol the protein activators of hormone-sensitive adenylate cyclase. A method for quantifying amounts of activator protein was used to monitor recovery after each isolation step. The activator proteins were precipitable by ammonium sulfate (30-60% saturation) and partially recoverable from the precipitate. On gel filtration of cytosol, stimulatory activity for glucagon-sensitive adenylate cyclase was recovered in two peaks representing proteins with molecular weights of 49,000 and 25,500. Exposure to GTP-Sepharose reduced liver cytosol's content of stimulatory factors for glucagon-sensitive adenylate cyclase by up to 70%. However, soluble protein adenylate cyclase activators distinct from GTP could not be subsequently eluted from the affinity matrix. Purification efforts were thwarted by factor instability and large losses during simple and conventional steps despite the use of a variety of protein stabilizers and protease inhibitors. If the problem of stimulator instability can be overcome, large-scale purification should be possible using pig liver as a starting material.

Adenylyl Cyclases↗
PubMed1983

Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes.

The mechanism of action of forskolin stimulation of adenylate cyclase was investigated by examining its effects on the enzyme's Mg2+ activated catalytic unit (C) from bovine sperm, both preceding and following complementation with human erythrocyte membranes as a source of guanine nucleotide regulatory protein (N). Prior to complementation, sperm C was not activated by either NaF (10 mM) or 5'-guanylyl-beta-gamma-imidodiphosphate (Gpp(NH)p, 10 microM), suggesting that functional N was not present in this preparation. Forskolin (100 microM) was also without effect on C. Following complementation of the sperm membranes with those of erythrocytes, Mg2+-dependent sensitivity to forskolin, NaF, and Gpp(NH)p was imparted to C. Our findings are incompatible with the current hypothesis that forskolin stimulates adenylate cyclase by direct activation of C. Rather, the data suggest that the activation process occurs through an effect on N or by augmentation of the interaction between the components of the adenylate cyclase complex.

Adenylyl Cyclases↗
PubMed1982

Pepstatin inhibition of proteinase-free human renin.

The kinetics of inhibition of proteinase-free human renin and of pepsin by the carboxyl proteinase inhibitor pepstatin have been examined. Inhibition was of the tight-binding type with both enzymes. Inhibition of crude human renin was of the classical, freely reversible type, but most of the renin-like activity of the preparation was due to contaminating proteinases which could be separated chromatographically from the renin. These results clarify previous kinetic studies of pepstatin inhibition of human renin.

Animals↗
PubMed1982

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites.

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.

Adipose Tissue↗
PubMed1981

Proteinase activity of human renin preparations: the International Reference Preparation (Renin Standard 68/356).

1. Several commonly used preparations of human renin, including the International Reference Preparation (Renin Standard 68/356), were examined for the presence of contaminating proteinase activity by using a 14C-glycinated bovine haemoglobin substrate assay. 2. All of the human renin preparations tested cleaved haemoglobin even in the presence o di-isopropylfluorophosphate and ethylenediaminetetra-acetate (EDTA). For a given amount of renin activity, varying amounts of proteinase activity were seen. The pH optimum also varied between preparations. 3. Small peptide inhibitors of human renin were not able to inhibit the proteinase activity. Furthermore a diazoacyl reagent and pepstatin, both potent inhibitors of aspartic acid-active site proteinases, were only partially inhibitory. 4. These and other observations suggest that the proteinase activity of the human renin preparations is not due to renin itself, but to contaminating proteinases of different types. Since these enzymes may produce angiotensin I or peptides which may interfere in renin assays, crude preparations of renin which contain proteinase activity, including the International Reference Preparation, should be used with caution or replaced by proteinase-free human renin which can be easily prepared by use of suitable affinity chromatography.

Chromatography, Affinity↗
PubMed1981

Salts promote activation of fat cell adenylate cyclase by GTP: special role for sodium ion.

The effects of GTP on adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of human and rat fat cell membranes ("ghosts" and purified membranes) were examined in the absence and presence of added inorganic salts. With human ghosts GTP alone (0.1 mM) inhibited enzyme activity by 40% at 30 degrees C and had no significant effect at 37 degrees C. At both temperatures Na+ salts of Cl-, N3-, and SO2-(4) stimulated activity (up to 4-fold basal activity for 200 mM NaN3), with maximal effects at salt concentrations of 100-200 mM. Over the same concentration range these salts also allowed temperature-dependent stimulation by GTP. GTP increased the maximal activity produced by salt alone by about 2-fold at 30 degrees C and about 4-fold at 37 degrees C. Na+ (added as Cl-) was much more effective than other alkali metal cations in promoting activation by GTP. Na+ salts allowed activation of the human enzyme by the GTP analog 5'-guanylyl imidodiphosphate and also promoted stimulation of rat fat cell adenylate cyclase by both nucleotides. In time course studies of human and rat fat cell ghosts, GTP appeared to sustain an initial high rate of salt-stimulated activity, which in the absence of nucleotide subsequently fell to a lower rate, suggesting that salts might activate adenylate cyclase by promoting the stimulatory effect of endogenous membrane-bound GTP. However, with purified human fat cell membranes and a GTP-free system, salts were still stimulatory and promoted activation by added GTP. These results differ from those of previous reports in other systems in which Na+ has promoted only inhibitory effects in GTP regulation of adenylate cyclase.

Adenylyl Cyclases↗
PubMed1981

Quantitation of beta adrenergic receptors in rat liver: confounding effect of displaceable but nonstereospecific antagonist binding.

In rat liver membranes three types of ligand binding were seen using [3H]-dihydroalprenolol (DHA) and [125I]-hydroxybenzylpindolol (HYP): binding stereospecifically displaced by beta-adrenergic agonists and antagonists, binding nonstereospecifically displaced by beta-adrenergic antagonists, and binding which was not displaced by beta-adrenergic agonists or antagonists. The magnitude of the nonstereospecific displaceable binding varied with the physiological state of the animal. It was sufficient to prevent the quantitation of the stereospecific displaceable binding in some preparations from young rats but in all preparations of rats greater than 150 g or more than about 6 weeks of age. In adrenalectomized weanling rats 10-30% of the total binding was of nonstereospecific displaceable type while in control rats it comprised up to 60% of the total binding. Addition of 5 X 10(-6) M phentolamine to the assay eliminated a large proportion of the nonstereospecific displaceable binding. When phentolamine was included in the assay, liver membranes from weanling rats stereospecifically bound 30-35% of total binding; membranes from adrenalectomized rats showed stereospecific binding of up to 50 to 80%. Because the amount of displaceable, nonstereospecific binding varied greatly depending on the physiologic state of the animals, stereospecific displacement should be monitored for every type of liver membrane preparation. Furthermore, animal age is an important variable. Using the published antagonist binding methodology (DHA or HYP) in liver membranes, it is not presently possible to quantitate liver beta-adrenergic receptors in normal rats that have reached maturity.

Adrenal Glands↗
PubMed1981

Mechanism of the age-related decrease of epinephrine-stimulated lipolysis in isolated rat adipocytes: beta-adrenergic receptor binding, adenylate cyclase activity, and cyclic AMP accumulation.

beta-adrenergic binding ([3H]dihydroalprenolol), adenylate cyclase activity, and cAMP accumulation were measured in adipocytes to investigate whether the mechanism of decreased hormone-sensitive lipolytic response with age was mediated through membrane-associated events. The dose of epinephrine required for half maximal stimulation of glycerol release (ED50) was significantly lower in 2-month-old rats (0.8 +/- 0.2 microM)than in matur (6- and 12-month-old) rats (5.2 +/- 1.5 and 6.2 +/- 1.5 microM, respectively). In 24-month-old rats the ED50 (0.7 +/- 0.2 microM) was less than in mature rats. maximum rates of hormone-stimulated glycerol release (per 10(6) cells) was highest in the two mature groups and decreased by 50% in the old rats (P less than 0.01). Lipolytic changes were independent of cell size. beta-adreanergic raeceptor number (50-90 thousand sites/call) and affinity (KD 4-5 nM) were the same in each age group. ED50 and maximum level of hormone-stimulated adenylatae cyclase activity did not change with age. The ED50 of cAMP accumulation of young rats was 3 +/- 5 microM compared with 24 +/- 4 and 25 +/- 5 microM in 6- and 12-month-old rats, respectively. In old rats, the ED50 of cAMP accumulation was 2 +/- 1 microM (P less than 0.001 compared with mature rats). Maximally stimulated cAMP levels were the same in old and mature animals. Phosphodiesterase activity in the presence and absence of 10(-5) M isoproterenol did not change with age. The results suggest that age-related decrease of epinephrine-sensitive lipolysis in old rats may be due to alterations of the lipolytic pathway distal to the receptor-adenylate cyclase complex and the generation of cyclic AMP.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Essential role of GTP in epinephrine stimulation of human fat cell adenylate cyclase.

The activity of epinephrine-sensitive adenylate cyclase of human fat cell ghosts is markedly enhanced by the GTP analog 5'-guanylyl-imidodiphosphate (GMP-P(NH)P), but a similar effect of GTP itself has not been heretofore demonstrable. In the present work, comparison of adenylate cyclase activity in the presence of epinephrine alone versus epinephrine plus GTP showed that at 37 degrees C GTP doubled activity (10-min incubation); at 30 degrees C less than half this effect was apparent. However, time course studies at both 30 and 37 degrees C showed that comparisons at a single point in time based on ratios of hormone-stimulated activity to basal or basal plus GTP were misleading, since basal activities were not linear with time and were inhibited by GTP. The inhibitory effect of GTP on basal activity was seen at both temperatures but at 37 degrees C decreased with time so that by 10 min the inhibition was no longer apparent. The time course data showed clearly that epinephrine alone did not stimulate adenylate cyclase activity; rather, the hormone merely prevented fall-off of initial rate of unstimulated (basal) enzyme activity. Only when GTP was added together with epinephrine was an unequivocal stimulation of enzyme activity observed. GTP effect was dose-dependent with half-maximal enhancement of epinephrine stimulation at 1.0 microM GTP. The GTP effect was not hormone-receptor mediated, since no shift was seen of the epinephrine dose-response curve toward higher sensitivity. GTP enhancement of epinephrine stimulation occurred over a wide range of ATP concentrations (0.01-3.0 mM) and affected the substrate Km only minimally. GTP-enhanced activity thus occurred through increased V max of the hormone-sensitive adenylate cyclase.

Adenosine Triphosphate↗
PubMed1980

Anions and cations as stimulators of liver adenylate cyclase.

The effects of inorganic salts on adenylate cyclase activities were studied in rat liver homogenates, particulates, and purified membranes. Na+ salts of N-3, NO-2, S2O32-, SO42-, and non-F- halides stimulated homogenates by a maximum of 2---5-fold; half-maximal effects were seen at concentrations ranging from 90 mM (Na2S2O3) to 410 mM (NaNo2). NaIO3 stimulated about 2-fold at relatively low salt concentrations (5---20 mM). Na+ salts were stimulatory using both ATP and the ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-P(NH)P) as enzyme substrate. The time courses of stimulation by NaCl and NaN3 were linear to at least 10 min with any of the three tissue preparations. Although salt effects clearly varied with the different anions, the magnitude and dose-response of stimulation of homogenates by Cl- salts were also dependent on the accompanying alkali cation (Li+, Na+, K+, Rb+, Cs+). MgCl2 at relatively high concentrations (50 mM) enhanced NaCl-stimulated activity in homogenates only slightly at relatively low concentrations of NaCl and not at high concentrations, suggesting a common mechanism of activation by Mg2+ and Na+ salts. NaCl- and NaN3-stimulated activities were unstable in homogenates kept at 0 degree C for 4 h, falling to 35% and 50% of their respective baseline activities. The effects on homogenates and particulates of maximally stimulatory concentrations of NaCl and NaN3 were further enhanced by GTP and the GTP analog 5'-guanylyl-beta, gama-imidodiphosphate (GMP-P(NH)P).

Adenine Nucleotides↗
PubMed1980

Thyrotroph cell adenoma of the human pituitary gland associated with primary hypothyroidism: clinical and morphological features.

The morphological features of a pituitary adenoma resected from a 39-year-old woman with hypothyroidism have been investigated. Hypothyroidism followed radioactive iodine treatment of pre-existing hyperthyroidism, presumably Graves' disease. Blood TSH was low during a euthyroid interval and was elevated above normal with the onset of hypothyroidism. The presence of a small amount of TSH was demonstrated in the tumour by radioimmunoassay. By conventional histology the tumour was a chromophobe adenoma. Some fine secretory granules stained with aldehyde thionin but were negative with PAS and contained no immunoreactive TSH. By light and electron microscopy, the tumour cells differed from thyroid deficiency cells. They were small and angular, containing numerous microtubules and small spherical secretory granules, oriented mainly along the plasma membranes. The tumour was considered to represent a thyrotroph cell adenoma, arising from protracted overstimulation secondary to chronic thyroid hormone deficiency.

Adenoma↗