Growth hormone regulates cytosolic free calcium in rat fat cells by maintaining L-type calcium channels

1996 ◽  
Vol 270 (5) ◽  
pp. C1478-C1484 ◽  
Author(s):  
S. Gaur ◽  
H. Yamaguchi ◽  
H. M. Goodman

In freshly isolated individual rat adipocytes, cytosolic free Ca2+ concentration ([Ca2+]i) as measured with fura 2 slowly declined during incubation but was sustained, or even somewhat increased, by brief treatment with growth hormone (GH) at the beginning of a 3-h incubation period. GH-treated adipocytes were more permeable to Ca2+ than GH-deprived cells as indicated, using Mn2- as a surrogate and monitoring influx by the rate of quenching of fura 2 fluorescence. Blockage of Ca2- channels with 100 nM nimodipine lowered [Ca2+]i in GH-treated cells to the level seen in GH-deprived cells. Increases in [Ca2+]i or the rate of Mn2+ entry were twofold greater in GH-treated than in GH-deprived cells when extracellular K+ was increased to 30 mM. Similarly, the Ca2+ channel agonist BAY K 5552 or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol increased [Ca2+]i more in GH-treated than in GH-deprived adipocytes. Ca(2+)-ATPase activity was two times higher in plasma membranes isolated from GH-treated than from GH-deprived cells. Continued synthesis of Ca(2+)-ATPase may depend on [Ca2+]i, since the effects of GH on [Ca2+]i and Ca(2+)-ATPase were blocked by a cycloheximide or verapamil. We suggest that voltage-sensitive L-type Ca2+ channels regulate steady-state [Ca2+]i in rat adipocytes and that GH maintains the number or functional integrity of these channels.

1976 ◽  
Vol 154 (1) ◽  
pp. 11-21 ◽  
Author(s):  
J P Luzio ◽  
A C Newby ◽  
C N Hales

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.


PEDIATRICS ◽  
1968 ◽  
Vol 41 (1) ◽  
pp. 30-46
Author(s):  
Donald B. Cheek

For many years the study of growth has rested mainly on the application of anthropometric techniques and the measurement of height and weight. A few years ago Tanner9 correctly pointed out that studies on body composition were mainly related to body weight and, therefore, added little to the thinking. A more penetrating approach to the study of growth was recommended.2 The present approach,11 documented in part here, has been to apply biochemical and physiological techniques for the measurement of body cell mass, cell size, cell number and, to some extent, cell function. Body function and heat production as well as maturational age have been of concern. These studies have been made in the same children at tile same time. It is anticipated that inspection of these three dimensions of growth, size, function, and maturational age should help to elucidate problems related to growth retardation. In the clinic it is possible to predict cell-extracellular mass of children by applying equations based on relationships between body composition and height and weight. We began by presenting information on growth of muscle and the differences between the sexes with the progress of time and with respect to size and number of cells. Increments in growth rate of the male at adolescence were found. Such differences in cell growth must be related to some extent to the restrictive action of estrogens on cell multiplication in the female and to the stimulating action of androgens in the male. Growth hormone is an important hormone for the multiplication of cells, while insulin is of importance to protein synthesis. Both hormones are needed for growth. Thyroid hormone appears to play a secondary role but is important to protein synthesis especially in early postnatal life. The energy requirement for normal growth is only slightly above the basal state and the visceral cell mass is the most direct standard of reference for heat production. Restriction of nutrition can either retard growth in the size of cells, in the number of cells, or both. Current studies58 show that ingestion of protein and calories incite the secretion of growth hormone and insulin in specific patterns and at appropriate times. Growth hormone has been labelled the "feasting" hormone and insulin tile "feasting" hormone.59 Thus, the subtle relationship between nutrition and cell growth becomes apparent. Of concern is the possibility that overnutrition early in life may program excess secretion of hormones such as insulin or growth hormone. Overnutrition is a major problem in the affluent society, while conservative nutrition is compatible with longevity.6 Hirsch, et al.60 informs us that growth of adipose tissue is mainly by cell number increase–as we have seen for muscle. Again, a steady state of cell number is reached for fat cells. But, obese subjects have an excess of fat cells which do not disappear with time and diet. Such cells become increasingly insensitive to insulin as they enlarge.61 One might view the passing parade of life and growth and observe the relation of the intracellular phase to body weight from infancy to senility (Fig. 12). Here we see the upward increase of cell mass with respect to time and body weight increase. The adult data are taken from F. D. Moore.62 Clearly, with senility we can suspect that more and more of the body weight is extracellular or connective tissue and less and less of the weight is soft tissue or oxidizing protoplasm. Data on body potassium are even more remarkable in this demonstration.11 It is difficult to say with Browning: Grow old along with me! The best is yet to be.... Nevertheless, it is possible that with increased information and research the understanding of these stages of cell growth will be achieved and, no doubt, the departure from the steady state of cell population which occurs at the autumn of our existence– when cancer, and cardiovascular disease supervene–will be understood.63 However, the problems of aging can only be exposed after the physiology of growth is understood.


1989 ◽  
Vol 256 (3) ◽  
pp. E375-E379
Author(s):  
R. W. Holl ◽  
M. O. Thorner ◽  
D. A. Leong

Digital imaging microscopy using the calcium-sensitive indicator probe fura-2 was combined with a reverse hemolytic plaque assay (RHPA) for growth hormone (GH) secretion. This technique allows dynamic measurements of the cytosolic free calcium concentration ([Ca2+]i) in individual pituitary somatotropes. Stimulation by growth hormone-releasing factor (GRF) increases, whereas somatostatin (SRIF) reduces [Ca2+]i in this cell type. [Ca2+]i increased in somatotropes when the cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) was elevated by 1) activating cellular adenylate cyclase with forskolin (5 microM) and 2) treatment with the cAMP-analogues dibutyryl-cAMP (1 mM) or 8-bromo-cAMP (5 mM). The forskolin-induced calcium rise was abolished in the absence of extracellular calcium. This indicates that cAMP increases the influx of calcium into the cytosol and thereby stimulates hormone release. When forskolin was given in combination with SRIF (10 nM), [Ca2+]i decreased to the same level reached with SRIF treatment alone, indicating a site of action distal to the generation of cAMP. Activating protein kinase C with the phorbol ester 12,13-phorbol dibutyrate (PDB; 100 nM) increased [Ca2+]i as well. Again, this effect was dependent on extracellular calcium and blocked when PDB and SRIF were applied simultaneously. Combined stimulation with GRF plus PDB did not augment the response of [Ca2+]i over GRF treatment alone.


1986 ◽  
Vol 238 (2) ◽  
pp. 345-351 ◽  
Author(s):  
M Bajaj ◽  
T L Blundell ◽  
R Horuk ◽  
J E Pitts ◽  
S P Wood ◽  
...  

Insulin from a hystricomorph rodent, coypu (Myocaster coypus), was isolated and purified to near homogeneity. Like the other insulins that have been characterized in this Suborder of Rodentia, coypu insulin also exhibits a very low (3%) biological potency, relative to pig insulin, on lipogenesis in isolated rat fat-cells. The receptor-binding affinity is significantly higher (5-8%) in rat fat-cells, in rat liver plasma membranes and in pig liver cells, indicating that the efficacy of coypu insulin on receptors is about 2-fold lower than that of pig insulin. The primary structures of the oxidized A- and B-chains were determined, and our sequence analysis confirms a previous report [Smith (1972) Diabetes 21, Suppl. 2, 457-460] that the C-terminus of the A-chain is extended by a single residue (i.e. aspartate-A22), in contrast with most other insulin sequences, which terminate at residue A21. In spite of a large number of amino acid substitutions (relative to mammalian insulins), computer-graphics model-building studies suggest a similar spatial arrangement for coypu insulin to that for pig insulin. The substitution of the zinc-co-ordinating site (B10-His----Gln) along with various substitutions on the intermolecular surfaces involved in the formation of higher aggregates are consistent with the observation that this insulin is predominantly ‘monomeric’ in nature. The c.d. spectrum of coypu insulin is relatively similar to those of casiragua insulin and of bovine insulin at low concentration.


Cell Calcium ◽  
1998 ◽  
Vol 23 (4) ◽  
pp. 207-217 ◽  
Author(s):  
J.L. Ramírez ◽  
R. Torronteras ◽  
M.M. Malagón ◽  
J.P. Castalño ◽  
S. García-Navarro ◽  
...  

Platelets ◽  
1991 ◽  
Vol 2 (1) ◽  
pp. 19-24 ◽  
Author(s):  
O.K. Bellinger ◽  
C.M. Kirchmaier ◽  
A. Schirmer ◽  
H.K. Breddin

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