The roles of calcium and cyclic AMP in the stimulatory action of parathyroid hormone on thymic lymphocyte proliferation

Both the parathyroid hormone (PTH) and the calcium ion increase the cellular content of cyclic adenosine 3′,5′-monophosphate (cyclic AMP), promote the initiation of deoxyribonucleic acid synthesis and stimulate the proliferation of rat thymocytes maintained in vitro. The ability of cyclic AMP to serve as the mediator of the mitogenic actions of both PTH and calcium is established by the fact that cyclic AMP itself stimulates cell proliferation in the absence of PTH and extracellular calcium. Neither PTH nor calcium appear to raise the cellular cyclic AMP level by increasing the nucleotide's synthesis by adenylate cyclase (formerly adenyl cyclase); PTH concentrations as high as 50 μg per ml of medium do not increase the enzyme's activity (in the presence or absence of calcium) and mitogenic calcium concentrations inhibit it. PTH also does not directly affect isolated thymocyte phosphodiesterase, but mitogenic calcium levels inhibit the enzyme's activity. Additional experiments show that it is calcium which raises the cyclic AMP level in cells treated with PTH, and some possible calcium-mediated mechanisms by which the hormone could elevate the cellular cyclic AMP levels are discussed. Thus, the mitogenic action of PTH is primarily mediated by calcium while cyclic AMP is the ultimate implementor of the hormonal action. However, calcium has a dual role and evidence is presented which indicates that besides raising the cellular cyclic AMP level, it also controls the operation of cyclic AMP's mitogenic end-reaction.     

Stimulation by epinephrine of adenyl cyclase activity, cyclic AMP formation, DNA synthesis and cell proliferation in populations of rat thymic lymphocytes

During the first ten minutes after the beginning of a continuous exposure of rat thymocyte populations (maintained in vitro) to epinephrine, there is an increase in the cellular concentration of cyclic AMP. The hormone also increases the activity of a crude preparation of the thymocyte's cyclic AMP-forming enzyme, adenyl cyclase. Between 30 and 45 minutes after the beginning of exposure to epinephrine, an additional part of the cell population begins to synthesize deoxyribonucleic acid (DNA). These changes are finally followed two to four hours later by an increase of the flow of cells into mitosis. Since cyclic AMP itself is known to stimulate both the initiation of DNA synthesis and thymocyte proliferation, and since the mitogenic action of epinephrine is shown to be potentiated by caffeine and inhibited by imidazole, it is concluded that the mitogenic action of this hormone is mediated by the cyclic nucleotide.     

Specificity of the effect of growth hormone on DNA concentration in the nuclei of the lymphoid cells of hypophysectomized rats]

The effect of growth hormone on the DNA content in the nuclei of the thymus, spleen and the lymph node lymphocytes was studied by means of cytophotometry. In hypophysectomized rats the growth hormone increased the DNA content in the nuclei of the middle lymphocytes of these organs without altering its amount in the small lymphocytes. Thymus lymphocytes were the most sensitive to the hormone action. The DNA content in the nuclei of these cells increased as soon as one hour after the administration of the hormone; in 4 hours it reached the maximum. Other hormones with an anabolic effect (insulin, thyroxin, testosterone), induced no elevation of DNA in the thymocyte nuclei at that period of time. A conclusion was drawn on the high tropicity of the growth hormone to the cells of the lymphoid organs and particularly to the thymocytes (middle lymphocytes of the thymus).     

Independence of the lethal actions of glucocorticoids on lymphoid cells from possible hormone effects on calcium uptake

We have examined the possibility that hormone-induced increases in calcium uptake might initiate the lethal actions of glucocorticoids in two types of lymphoid cells. Hormone-induced increases in nuclear fragility are used as the measure of hormone action, since in both rat thymus cells and in mouse P1 798 lymphosarcoma cells increased nuclear fragility (the inability of nuclei to survive lysis of the cells by hypotonic shock) precedes other indices of cellular deterioration by several hours. In the case of the tumor cells, those from corticosteroid-sensitive lines are less able to withstand incubation in vitro than resistant cells. Such differences in cell survival are predicted both by earlier changes in nuclear fragility and also by differences in calcium uptake. However, there is no detectable early glucocorticoid effect on calcium uptake that precedes or coincides with the substantial hormone-induced increases in nuclear fragility that develop in the sensitive cells by 2 h. In rat thymus cells the absence of calcium in the medium does prevent some of the increase in nuclear fragility and cell disintegration that occurs spontaneously during incubation in vitro. Nevertheless, when cells are exposed to hormones the glucocorticoid effect on nuclear fragility develops in the absence of calcium and is similar in magnitude to that seen in the presence of calcium. We conclude that calcium seems to enhance the spontaneous deterioration of lymphoid cells, and there is a large increase in calcium uptake that occurs as cells deteriorate. It nevertheless seems unlikely that hormone-induced changes in calcium uptake initiate the lethal actions of glucocorticoids. The data also support a proposal made earlier [2] that resistance to glucocorticoids in tumor cells may develop by the selection of cells with hardier membranes.     

Role of calcium in the regulation of adenohypophysial hormone release.

It is now accepted by most investigators that the initial action of most peptide hormones involves an interaction with a specific receptor on (or in) the plasma membrane of the target cell. A cascade of intracellular events results and culminates in the physiological response characteristic of the interaction of the particular hormone with its target cell. The regulation of hormone release from the adenohypophysis by the hypothalamic releasing hormones is presumed to occur via a similar process. The nature of the interaction at the cell surface as well as the details and sequence of the subsequent intracellular events are largely unknown. We do know, however, that two of the key factors regulating the intracellular secretory machinery in most cells are 1) the adenylate cyclase — cyclic AMP — protein kinase system and 2) the divalent cation, calcium. Since there have been several recent reviews (1–3) which have covered the role of the cyclic nucleotides in pituitary hormone secretion, this discussion will be restricted to a consideration of the regulatory role played by calcium.As was the case with tissues, the early work regarding calcium and the adenohypophysis followed the pattern of determining the ability of secretagogues to release pituitary hormones subsequent to various manipulations designed to remove what was often implicity considered to be extracellular calcium.     

Comparison of the effect of hormones on the hormone synthesis of Tetrahymena in medium or salt solution

Tetrahymena pyriformis was maintained in TYM (tryptone‐yeast medium) as well as in Losina salt solution. One hour treatment of 10^?15 M histamine, serotonin or insulin was given before the histamine, serotonin, triiodothyronine and adrenocorticotropin contents of the cells were measured by flow cytometry after immunocytochemical staining. Maintenance in salt solution increased the hormone level in the cells, and use of the treatment hormone treatments further increased the endogenous hormone content relative to that in medium. The cells in salt mimic better the natural conditions, which means that the effects of hormones under more natural conditions are expressed to a greater extent than the exogenously given hormones in TYM typically used under laboratory conditions. Intercellular hormonal communication between the cells of a Tetrahymena population might assist in the survival of the individual cells.     

Possible role of PEPT1 in gastrointestinal hormone secretion

Oligopeptides originating from ingested meal stimulate the secretion of various gastrointestinal hormones, but the mechanism is unknown. In this study, we show that transfection of oligopeptide transporter 1 (PEPT1) in STC-1 cells, a murine enteroendocrine cell line, evokes di-peptide-stimulated hormone secretion in a pH-dependent manner. Measurement of membrane potentials shows that PEPT1- transfected STC-1 cells are depolarized by di-peptide glycyl-glycine but not by glycine monomer. Glycyl-glycine stimulation induces a rise in the intracellular calcium concentration in PEPT1-transfected STC-1 cells. The secretion induced by glycyl-glycine in PEPT1-transfected STC-1 cells was blocked by nifedipine, a Ca(2+) channel blocker, suggesting that the secretion is triggered by Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. These data suggest that PEPT1 mediates oligopeptide-induced hormone secretion in enteroendocrine cells.     

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.     

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.     

Effect of hypo-osmotic environmental changes on the expression of gonadotropin-releasing hormone,its receptor,and gonadotropin hormone subunit mRNA in adult chum salmon (Oncorhynchus keta)

Migrating fish such as salmonids are affected by external environmental factors and salinity changes are particularly important, influencing spawning migration. The aim of this study was to test whether changes in salinity would affect the expression of the hypothalamic-pituitary-gonadal (HPG) axis hormones (gonadotropin-releasing hormones (GnRHs) [salmon GnRH and chicken GnRH-II], GnRH receptors [GnRHR1 and GnRHR5], and mRNA of the gonadotropin hormone [GTH] subunits [GTHα, follicle stimulating hormone β, and luteinizing hormone β]) in chum salmon (Oncorhynchus keta). Fish were progressively transferred from seawater (SW) through 50% SW to freshwater (FW), and the relationship between the osmoregulatory hormone prolactin (PRL) and sexual maturation was determined. The expression and activity of HPG hormones and their receptors, and levels of estradiol-17β and PRL increased after fish were transferred to FW, demonstrating that changes in salinity stimulate the HPG axis and PRL production in migrating chum salmon. These findings reveal details about the role of the endocrine system in maintaining homeostasis and stimulating sexual maturation and reproduction in response to salinity changes in this species.     

Role of calcium in plant hormone action

Abstract

The role of calcium as a second messenger in plant cells has been recognized in a number of physiological processes. As described for animal systems, plant cells contain all the elements necessary for coupling the external signals to a specific response by regulation of calcium levels. However, the evidence that Ca₂₊ can be considered a second messenger for hormone response in plants is still circumstantial, besides several reports on the subject have been produced. All the hormone effects may in some tissues be regulated by calcium metabolism, but only for few of them a precise role of this cation has been established. The studies on the different hormones will be reviewed and discussed.     

RESPONSIVENESS OF YEAST CELLS TO AUXIN, ANIMAL SEX HORMONES AND YEAST SEXUAL HORMONES IN RELATION TO SEX CONTROLLING GENES

Genetic control of the ability of yeast cells to respond to cell-expanding action of animal, plant and yeast hormones was studied. Mating type specificity in the ability to respond to yeast sexual hormones was changed by introduction of homothallism-controlling genes, D and HM. Auxin, a plant hormone can expand cells of most homothallic strains, but not those of heterothallic strains as far as tested. Animal sex hormones showed the action similar to that of yeast sexual hormones.     

Hypoplasia of the bone marrow in rats following removal of the parathyroid glands

Removal of the parathyroid glands caused a marked and permanent lowering of the mitotic activity in rat bone marrow, which was followed by a pronounced decline (ca. 40%) in the size of the marrow's nucleated cell population. These changes occurred only when the hypocalcemia induced by the lack of parathyroid hormone (PTH) was maintained by feeding the animals a calcium deficient diet. Since the overall marrow hypoplasia was almost entirely due to strikingly large reductions (ca. 70%) in the size of the erythroid and lymphoid subpopulations, it is concluded that PTH and calcium are major physiological regulators of the proliferation of erythroid and lymphoid cells.     

Release of insulin-like growth factor carrier proteins by osteoblasts: stimulation by estradiol and growth hormone

Osteoblast-like rat calvaria cells release specific insulin-like growth factor (IGF) carrier proteins (CPs). As analyzed by SDS-PAGE under nonreducing conditions, Western blotting and detection by 125I-IGFs, CPs migrating with the IGF-binding subunits of the major CP species of rat serum (42/45/49 kDa) accumulate in cell culture medium. Treatment of the cells with growth hormone and estradiol increases the abundance of this glycosylated CP species. Since the two hormones were previously found to stimulate osteoblast replication via an IGF I-dependent mechanism, the data indicate that hormones may control local IGF action not only by regulating synthesis of IGFs and their receptors but also their presentation by CPs.     

N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) protects thymocytes from programmed cell death.

Gamma-irradiation, glucocorticoid hormones, and calcium ionophores stimulate a suicide process in thymocytes, known as apoptosis or programmed cell death, that involves internucleosomal DNA fragmentation by a Ca(2+)- and Mg(2+)-dependent nuclear endonuclease. In this study we report that N-(2-mercaptoethyl)-1,3-propanediamine (WR-1065) blocked DNA fragmentation and cell death in thymocytes exposed to gamma-radiation, dexamethasone, or calcium ionophore A23187. WR-1065 protected the thymocytes from radiation-induced apoptosis when incubated with cells after irradiation but not before and/or during irradiation. WR-1065 inhibited Ca(2+)- and Mg(2+)-dependent DNA fragmentation in isolated thymocyte nuclei. Our results suggest that WR-1065 protects thymocytes from apoptosis by inhibiting Ca(2+)- and Mg(2+)-dependent nuclear endonuclease action.     

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding,G proteins,phosphoinositide breakdown,cyclic AMP generation,and calcium influx

Summary 1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A₁-adenosine receptors.2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTPS to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins.3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin.N-Ethylmaleimide inhibits the response to both peptides.4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTPS-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects.5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered byN-ethylmaleimide.6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.     

Morphological studies on endocytosis of chondroitin sulphate proteoglycan by rat liver endothelial cells

Summary Endocytosis via the hyaluronic acid/chondroitin sulphate receptor of rat liver endothelial cells was studied ultrastructurally, by use of a probe consisting of chondroitin sulphate proteoglycan attached to 15-nm gold particles. The probe bound to the surface of the cells exclusively in coated regions of the plasma membrane. Internalization at 37° C took place in less than one minute during which time interval the bound probe was transferred to coated vesicles. Further transfer to lysosomes was delayed in association with an accumulation of probes in a prelysosomal compartment consisting of large vacuoles in which probes lined the inner aspect of the membrane. Transport to lysosomes occurred only after a lag phase of at least 40–60 min at 37° C.Abbreviations CS chondroitin sulphate - CSPG chondroitin sulphate proteoglycan - CSPG-Au CSPG-gold complex - EM electronmicroscopical or electron microscopy - HA hyaluronic acid - KC Kuppfer cells - LEC liver endothelial cells - PC parenchymal cells - RES reticuloendothelial system     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

Calcium influx: an intracellular message of the mitogenic action of insulin-like growth factor-I

When G0-arrested BALB/c 3T3 cells were treated sequentially with platelet-derived growth factor and epidermal growth factor, cells became responsive to insulin-like growth factor-I (IGF-I). In these primed competent cells, 1 nM IGF-I elicited an approximately 3-fold increase in the calcium influx rate. IGF-I-induced calcium influx was relatively slow in onset and continued for at least 2 h in the presence of IGF-I. When a single Ca2+ channel current was studied by the patch-clamp technique using the cell-attached mode, inward currents with unitary conductance of 19 pS were observed in the presence of 1 nM IGF-I in the patch pipette. IGF-I-sensitive inward current was independent of membrane potential and was activated by a high concentration of insulin. Accordingly, 1 nM IGF-I caused a gradual increase in cytoplasmic free calcium concentration measured by fura2. The action of IGF-I on calcium influx was dependent on extracellular calcium, and IGF-I did not stimulate calcium influx when extracellular calcium concentration was reduced to 10 microM. Both cobalt and tetramethrin blocked the action of IGF-I on calcium influx without affecting the binding of 125I-IGF-I. In primed competent cells, IGF-I-stimulated [3H]thymidine incorporation was dependent on extracellular calcium and was attenuated by cobalt and tetramethrin. When cell-bound 125I-IGF-I was cross-linked by use of disuccinimidyl suberate, a 130-kDa protein was radiolabeled. Affinity labeling of the 130-kDa protein, presumably the alpha-subunit of the IGF-I receptor, was blocked by excess amount of unlabeled IGF-I. These results suggest that relatively low concentrations of IGF-I stimulate calcium influx in primed competent BALB/c 3T3 cells by activating a calcium-permeable cation channel via the IGF-I receptor and that calcium influx may be a critical intracellular message of the progression activity of IGF-I.