Insulin receptors in human mononuclear leucocytes. I. Binding and degradation of insulin in normal subjects.

The binding and degradation of 125I-Insulin were investigated in mononuclear leukocytes of normal subjects. The binding data analysis show that the insulin degradation is strictly correlated with the binding of the hormone to its receptors: these data suggest that the binding of insulin to specific receptor is the possible first step for its degradation.     

An affinity label for alpha 2-adrenergic receptors in rat brain

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.     

Insulin receptors in human mononuclear leucocytes. II. Binding and degradation of insulin in obese subjects.

The binding of insulin to the receptors on circulating mononuclear cells of obese subjects is significantly decreased when compared to the binding in normal subjects. This fenomenon is due to the reduction of the number of insulin receptors rather than reduction in affinity. The insulin degradation is also reduced, but a very strong correlation, similar to that demonstrated in normal subjects exist between insulin binding to its receptors and insulin degradation.     

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.     

Different behavior of phagocytes in young and old subjects]

Ageing is a dynamic phenomenon in which there is a physiological decay in all the functions of the individual. The consequence is an increased susceptibility to infections, autoimmune diseases and cancer. Phagocytic cells as polymorphonuclear leukocytes (PMNL) and monocytes (Mo) are of prime importance in the defence against invasive agents, PMNL and Mo seek out and destroy invading micro-organisms. Chemotaxis and phagocytosis are two mechanisms that are activated by these cells for this purpose. In this study, using "in vitro" techniques, we have verified if, at the level of such functions of cell defense, there could be variations in elderly subjects with respect to younger subjects. Our results show a chemotactic activity of PMNL in the elderly that is higher and a phagocytic activity that is lower. As regards Mo, there is a lower chemotactic activity in the elderly and only a slight difference in phagocytic activity with respect to the younger subjects. These results are in agreement with those found at the clinical level showing the elderly less protected from infection with respect to younger subjects.     

Peripheral effects of endurance training in young and old subjects

The effects of 12 wk of endurance training at 70% peak O2 consumption (VO2) were studied in 10 elderly (65.1 +/- 2.9 yr) and 10 young (23.6 +/- 1.8 yr) healthy men and women. Training had no effect on weight or body composition in either group. The elderly had more adipose tissue and less muscle mass than the young. Initial peak VO2 was lower in the elderly, but the absolute increase of 5.5-6.0 ml.kg-1.min-1 after training was similar for both groups. Muscle biopsies taken at rest showed that, before training, muscle glycogen stores were 61% higher in the young. Before training, glycogen utilization per joule during submaximal exercise was higher in the elderly. Glycogen stores and muscle O2 consumption increased significantly in response to training in the elderly only. After training, the proportion of energy derived from whole body carbohydrate oxidation during submaximal exercise declined in the young only. The absolute changes that training produced in peak VO2 were similar in both age groups, but the 128% increase in muscle oxidative capacity was greater in the elderly, suggesting that peripheral factors play an important role in the response of the elderly to endurance exercise.     

Activation of alpha(2)-adrenergic receptors impairs exercise-induced lipolysis in SCAT of obese subjects

With the use of the microdialysis method, exercise-induced lipolysis was investigated in subcutaneous adipose tissue (SCAT) in obese subjects and compared with lean ones, and the effect of blockade of alpha(2)-adrenergic receptors (ARs) on lipolysis during exercise was explored. Changes in extracellular glycerol concentrations and blood flow were measured in SCAT in a control microdialysis probe at rest and during 60-min exercise bouts (50% of heart rate reserve) and in a probe supplemented with the alpha(2)-AR antagonist phentolamine. At rest and during exercise, plasma norepinephrine and epinephrine concentrations were not different in obese compared with lean men. In the basal state, plasma and extracellular glycerol concentrations were higher, whereas blood flow was lower in SCAT of obese subjects. During exercise, the increase of plasma glycerol was higher in obese subjects (115 +/- 35 vs. 65 +/- 21 micromol/l). Oppositely, the exercise-induced increase in extracellular glycerol concentrations in SCAT was five- to sixfold lower in obese than in lean subjects (50 +/- 14 vs. 318 +/- 53 micromol/l). The exercise-induced increase in extracellular glycerol concentration was not significantly modified by phentolamine infusion in lean subjects but was strongly enhanced in the obese subjects and reached the concentrations found in lean sujects (297 +/- 46 micromol/l). These findings demonstrate that the physiological stimulation of SCAT adipocyte alpha(2)-ARs during exercice-induced sympathetic nervous system activation contributes to the blunted lipolysis noted in obese men.     

Muscarinic receptors of rat lymphocytes--differences in young and old animals

Muscarinic receptors were studied on lymphocytes from young and old Wistar rats. Binding studies were performed by the use of [3H]-QNB, a specific muscarinic antagonist. Some differences between these two groups were observed. Maximal binding of [3H]-QNB and half time of the maximal binding is lower for lymphocytes of old rats [3H]-QNB receptor complexes could not be found in the supernatants derived from lymphocytes of old animals. Higher ability to loose or hide the muscarinic receptors was also observed in this group of rats. All these observations could reflect a more effective degradation, as well as a lower level of muscarinic receptors exposed on lymphocytes from old animals.     

Intraindividual variation of beta 2-adrenergic receptors on mononuclear leukocytes and their relationships with endogenous plasma catecholamines in man.

The interindividual and intraindividual variations of both MNL beta 2-adrenergic receptor density and dissociation constant of binding were evaluated in 19 healthy volunteers. In addition the possible relationships between catecholamine plasma levels and MNL beta 2-adrenergic receptor density were studied in 8 of these subjects. The volunteers were studied three times with ten days' interval. There was a significant inverse relationship between receptor density and norepinephrine plasma levels, only. Neither epinephrine nor dopamine were correlated with receptor density. Interindividual coefficient of variation was 29.57%. The mean value of the intraindividual coefficients of variation was 14.1%, while the mean value of the analytical coefficients of variation was 10%. Our results are at some variance with data in the literature and may contribute to elucidate the role of MNL beta 2-adrenergic receptors as an index of sympathetic function in man.     

Recall in acoustically similar and dissimilar letters by normal and deaf subjects.

Stimulus matrices with 4 X 3 letters from an acoustically similar and dissimilar alphabet were presented tachystoscopically for 5 secs to 41 Ss divided into three groups, i.e. normal and deaf apprentices and deaf children. Each S was administered 42 stimulus matrices. While the difference in the number of errors between the acoustically similar and dissimilar alphabets proved significant in the hearing Ss, it was nonsignificant in the deaf, and in contrast to these two groups, in the children the number of errors with the acoustically similar alphabet was lower than with the dissimilar one. The error matrices indicate a systematic course of errors and their different pattern for the hearing and the deaf Ss. A visual and an articulating code which is reinforced by the length of oral training, may be presumed particularly in the deaf.     

Amniotic fluid beta-2 microglobulin in normal and complicated pregnancies

Beta-2 microglobulin concentrations were measured in amniotic fluid samples obtained from normal pregnant women at various stages of gestation and complicated pregnancies during weeks 32-42 of gestation by the ELISA method. The concentration of beta-2 microglobulin in amniotic fluid increases markedly up to the 20-24th weeks of pregnancy and reaches a peak during the second trimester, occasionally reaching an eightfold value compared to the maternal serum concentration, while at term the values are similar. The decrease of amniotic fluid beta-2 microglobulin level in the third trimester reflects the maturation of foetal renal tubular function and suggests that this test may be of significance in determining foetal age. Our results revealing elevated concentrations of beta-2 microglobulin in patients with diabetes, toxaemia and placental insufficiency may indicate slower renal maturation of the foetus.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

High affinity binding of receptor-associated protein to heparin and low density lipoprotein receptor-related protein requires similar basic amino acid sequence motifs

The 39-kDa receptor-associated protein (RAP) is a specialized chaperone for members of the low density lipoprotein receptor gene family, which also binds heparin. Previous studies have identified a triplicate repeat sequence within RAP that appears to exhibit differential functions. Here we generated a series of truncated and site-directed RAP mutants in order to define the sites within RAP that are important for interacting with heparin and low density lipoprotein receptor-related protein (LRP). We found that high affinity binding of RAP to heparin is mediated by the carboxyl-terminal repeat of RAP, whereas both the carboxyl-terminal repeat and a combination of amino and central repeats exhibit high affinity binding to LRP. Several motifs were found to mediate the binding of RAP to heparin, and each contained a cluster of basic amino acids; among them, an intact R(282)VSR(285)SR(287)EK(289) motif is required for high affinity binding of RAP to heparin, whereas two other motifs, R(203)LR(205)R(206) and R(314)ISR(317)AR(319), also contribute to this interaction. We also found that intact motifs of both R(203)LR(205)R(206) and R(282)VSR(285)SR(287)EK(289) are required for high affinity binding of RAP to LRP, with the third motif, R(314)ISR(317)AR(319), contributing little to RAP-LRP interaction. We conclude that electrostatic interactions likely contribute significantly in the binding of RAP to both heparin and LRP and that high affinity interaction with both heparin and LRP appears to require mostly overlapping sequence motifs within RAP.     

Norepinephrine spillover at rest and during submaximal exercise in young and old subjects

Mazzeo, Robert S., Chakravarthi Rajkumar, Garry Jennings,and Murray Esler. Norepinephrine spillover at rest and during submaximal exercise in young and old subjects. J. Appl. Physiol. 82(6): 1869-1874, 1997.Aging isassociated with elevations in plasma norepinephrineconcentrations. The purpose of this investigation was toexamine total body and regional norepinephrine spillover as anindicator of sympathetic nerve activity. Eight young (26 ± 3 yr)and seven old (69 ± 5 yr) male subjects were studied at rest andduring 20 min of submaximal cycling exercise at 50% of peak workcapacity. Norepinephrine spillover was determined by continuousintravenous infusion of[³H]norepinephrine.Arterial norepinephrine concentrations were significantly greater atrest for old vs. young subjects (280 ± 36 vs. 196 ± 27 ng/ml,respectively). Whereas total norepinephrine spillover did not differbetween groups at rest, hepatomesenteric norepinephrine spillover was50% greater in old subjects compared with their young counterparts (51 ± 7 vs. 34 ± 5 ng/min, respectively). Additionally,norepinephrine clearance rates at rest were significantly lower for theold subjects (23%). During exercise, plasmanorepinephrine concentrations increased compared with rest, with oldsubjects again demonstrating greater values than the young group.Hepatomesenteric norepinephrine spillover was significantly greater(+36%) during exercise for old subjects compared with young; however,no difference was found for whole body spillover rates between agegroups. Norepinephrine clearance rates remained depressed(30%) in the old subjects during exercise. Clearance ofepinephrine mirrored that for norepinephrine both at rest and duringexercise across age groups. It was concluded that in old subjects, areduction in norepinephrine clearance and an increase in regionalnorepinephrine spillover can account for the higher plasmanorepinephrine concentrations observed at rest. This relationship isnot exacerbated by the stress imposed during an acute bout of exercise.

     

Ventilatory and arousal patterns during sleep in normal young and elderly subjects

Since elderly subjects have lower chemosensitivity, we postulated that ventilation might be more state dependent in the elderly. To address this we investigated the changes in ventilation, measured by respiratory inductive plethysmography, with sleep in 12 healthy young (19-29 yr) and 13 elderly (greater than 65 yr) subjects. Ventilation was measured in representative periods in each sleep state. These data showed that there is no difference between the elderly and the young either in mean ventilation or in the variability of ventilation awake or in the different states of sleep. In both groups ventilation was variable in stage 1-2 sleep and least variable in stage 3-4 sleep. The variability in stage 1-2 sleep was due to periodic breathing (cycle time approximately 45 s) in both age groups. Although within a sleep state no differences were observed, over the night of study the elderly behaved differently from the young. Apneas occurred more frequently in the elderly, and 5 of 13 elderly met the criteria for sleep apnea syndrome compared with 1 of 12 young subjects. Apneas tended to occur predominantly in stage 1-2 sleep and seem to be an exaggeration of the periodicity that is typical of this state. Four of the elderly with apnea remained in this stage of sleep throughout the night of study. The apneic episodes usually terminated with an electroencephalogram arousal that occurred prior to or simultaneously with the onset of ventilation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central alpha 2-adrenergic receptors in the rat: effect of sodium in vitro

Characteristics of the (3H) Rauwolscine binding were studied in homogenates of rat cerebral cortex, particularly in presence of NaCl. We had shown that (3H) Rauwolscine specifically bind the alpha 2-adrenoceptors. The Scatchard plot analysis had demonstrated 2 sites: a high affinity site (B 41,06 +/- 5,21 KD = 1,31 +/- 0,16) and a low affinity site (B = 203,41 +/- 13,37 KD = 11,62 +/- 0,55 nM). Incubation of cerebral cortex homogenates with NaCl 150 mM induced an increase (53,97 p less than 0.001) of the high affinity site density. These results indicate that (3H) Rauwolscine is a useful alpha 2-adrenoceptor ligand to study in animal models various central nervous system abnormalities or diseases induced by ion disorders.     

Overexpression of alpha 2-adrenergic receptors in fetal rat heart: receptors in search of a function.

alpha 2-Adrenergic receptors are transiently overexpressed by many types of developing cells. In the current study, the developmental profile and cellular function of these receptors were examined in the fetal and neonatal rat heart. alpha 2-Receptors, assessed with [3H]rauwolscine binding, were extremely high in fetal hearts on gestational day 19, 30-fold higher than values seen in adults. Receptor binding decreased by two-thirds by gestational day 21 and dropped by half again by postnatal day 3. To assess potential cellular functions controlled by the alpha 2-receptors, the capability of an alpha 2-agonist (clonidine) to inhibit membrane-associated adenylate cyclase activity was measured in three different settings: basal enzyme activity, the enzymatic response to isoproterenol (dependent upon beta-receptor linkages to the regulatory protein, Gs), and the response to forskolin (independent of receptor-Gs interactions). Despite the high number of alpha 2-receptors in fetal hearts, clonidine failed to alter any of the adenylate cyclase activity measures. In light of the postulated role of alpha 2-receptors in the maintenance of fetal/neonatal atrioventricular conduction, the excess alpha 2-receptors are probably linked to other cellular events, such as movement of calcium into the cell.