Is extracellular calcium required for insulin action?

Isolated hepatocytes and isolated adipocytes incubated in the absence of added calcium ions respond to insulin with a decrease in tissue cyclic AMP levels, and an increase in low Km phosphodiesterase activity. Isolated hepatocytes also showed a diminution of glucagon stimulated glucose output in response to insulin, while adipocytes responded with increased rates of glucose oxidation, lipid synthesis and decreased glycerol output. These responses to insulin are the same as those seen when the cells are incubated in buffers containing physiological concentrations of calcium ions. When extracellular concentrations of calcium ions were made extremely low by using either gelatine or albumin which had been pretreated to remove calcium, and/or the incubation buffers contained EGTA, both the hepatocytes and adipocytes continued to respond to insulin. These results suggest that extracellular calcium ions are not required for insulin action.     

The role of calcium ions in the mechanism of ACTH stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.     

Reevaluation of the Effect of Calcium Ions on Auxin-induced Elongation

The mechanism by which calcium ions inhibit cell elongation has been reinvestigated. Growth-inhibiting levels of calcium, when applied to isolated walls (in vitro treatment) do not decrease cell wall extensibility as measured by the Instron technique. Thus, the hypothesis that calcium inhibits growth by forming wall-stiffening calcium bridges must be abandoned. Treatment of living auxin-treated sections with calcium (in vivo treatment) does cause a decrease in the subsequently measured wall extensibility, but this decline appears to be simply a consequence of the growth inhibition rather than its cause. Growth-inhibiting levels of calcium do not appreciably reduce the rate of auxin-enhanced H(+) excretion. Pretreatment with calcium does not reduce the capacity of walls to undergo acid-activated wall loosening in the absence of calcium. High concentrations of CaCl(2) (0.02 m) cause an initial elastic shrinkage of Avena sections comparable to that caused by the same osmolarity of mannitol, but the subsequent growth inhibition is too great to be explained by an osmotic inhibition. Calcium ions do inhibit H(+)-induced extension of frozen-thawed sections under tension. The growth-inhibitory effects of calcium, then, may be ascribed to a direct inhibition exerted by calcium ions on the H(+)-induced wall-loosening process.     

Role of calcium in the insulin-dependent stimulation of DNA synthesis in mouse mammary gland in vitro

The role of extracellular Ca2+ in the control of DNA synthesis in mouse mammary tissue was studied using mammary gland explants maintained under chemically defined conditions in vitro. Chelation of calcium with ethyleneglycol-bis-(beta-aminoethyl ether) or omission of Ca2+ from the incubation media substantially reduced both basal and insulin-stimulated incorporation of [3H]thymidine into DNA. Addition of calcium to the Ca2+-deficient media restored DNA synthesis; other divalent cations could not be substituted for calcium. Insulin reduced by 5-fold the calcium concentration required to achieve half-maximal stimulation of DNA synthesis in explants, thus indicating that the Ca2+-related process may be involved in the mechanism by which insulin exerts its effect on cell multiplication. Evidence is presented that in mammary gland explants, calcium does not stimulate DNA synthesis by action on the thymidine pool size. Neither calcium nor insulin showed any effect on the activity of thymidine kinase in the mammary gland explants. On the other hand, calcium ions were shown to be necessary to maintain the activity of DNA polymerase-alpha, the enzyme involved in nuclear DNA replication.     

Acetylcholine and Calcium on Membrane Permeability and Contraction of Intestinal Smooth Muscle

Acetylcholine elicited a sustained contraction and an increase in potassium efflux in longitudinal muscle isolated from the guinea pig ileum. Stepwise increases in the calcium concentration of the bathing medium, from 0.06 to 36 mM generally reduced the increase in potassium efflux, but had a complex effect on the mechanical response. Contractions produced by high levels of acetylcholine became progressively larger or remained at a high magnitude as the calcium concentration was increased. Contractions produced by low levels of acetylcholine also improved initially, but were depressed again by the highest concentration of calcium introduced. Ethanol, in the appropriate concentration, inhibited completely the acetylcholine-induced contraction without reducing the increase in potassium efflux. Calcium reversed this effect. Both extracellular calcium and ethanol depressed the large, transient increase in muscle tone developed by fibers that were preincubated in a high calcium medium and then exposed to a calcium-free medium. These findings suggested that extracellular calcium ions react with two different sites in the membrane, a stabilizing site and a storage site. A muscle contraction is activated by calcium ions which diffuse from the storage site to the myoplasm. Calcium ions reacting with the stabilizing site impede this diffusion process. Part of the stimulatory effect of acetylcholine is derived from its capacity to counteract the action of calcium at the stabilizing site.     

Some effects of varied calcium nutrition on the growth and composition of tomato plants

Summary Tomato plants were grown in nutrient solutions containing 0.05, 0.2, 1.0, 5.0, 15.0 and 30.0 meq Ca/l. Plants grown at the lowest level of calcium suffered from calcium deficiency and produced the least amount of dry matter. Dry matter yield was optimal from plants grown in the solution containing only 0.2 meq Ca/l and decreased at higher levels of substrate calcium. Despite large differences in the concentrations of the individual ions in the dried material, highly significant (P < 0.001) relationships were found between total cations and total anions and between insoluble calcium and oxalate.An explanation for the observed response to calcium is suggested based on the stimulation of the synthesis of oxalate by tissue calcium at the expense of carbohydrates. re]19760812     

Cellular localization of the Ca2+ binding TCH3 protein of Arabidopsis

TCH3 is an Arabidopsis t ou ch ( TCH ) gene isolated as a result of its strong and rapid upregulation in response to mechanical stimuli, such as touch and wind. TCH3 encodes an unusual calcium ion-binding protein that is closely related to calmodulin but has the potential to bind six calcium ions. Here it is shown that TCH3 shows a restricted pattern of accumulation during Arabidopsis vegetative development. These data provide insight into the endogenous signals that may regulate TCH3 expression and the sites of TCH3 action. TCH3 is abundant in the shoot apical meristem, vascular tissue, the root columella and pericycle cells that give rise to lateral roots. In addition, TCH3 accumulation in cells of developing shoots and roots closely correlates with the process of cellular expansion. Following wind stimulation, TCH3 becomes more abundant in specific regions including the branchpoints of leaf primordia and stipules, pith parenchyma, and the vascular tissue. The consequences of TCH3 upregulation by wind are therefore spatially restricted and TCH3 may function at these sites to modify cell or tissue characteristics following mechanical stimulation. Because TCH3 accumulates specifically in cells and tissues that are thought to be under the influence of auxin, auxin levels may regulate TCH3 expression during development. TCH3 is upregulated in response to low levels of exogenous indole-3-acetic acid (IAA), but not by inactive auxin-related compounds. These results suggest that TCH3 protein may play roles in mediating physiological responses to auxin and mechanical environmental stimuli.     

Calcium as a Crucial Cofactor for Low Density Lipoprotein Receptor Folding in the Endoplasmic Reticulum

The family of low density lipoprotein (LDL) receptors mediate uptake of a plethora of ligands from the circulation and couple this to signaling, thereby performing a crucial role in physiological processes including embryonic development, cancer development, homeostasis of lipoproteins, viral infection, and neuronal plasticity. Structural integrity of individual ectodomain modules in these receptors depends on calcium, and we showed before that the LDL receptor folds its modules late after synthesis via intermediates with abundant non-native disulfide bonds and structure. Using a radioactive pulse-chase approach, we here show that for proper LDL receptor folding, calcium had to be present from the very early start of folding, which suggests at least some native, essential coordination of calcium ions at the still largely non-native folding phase. As long as the protein was in the endoplasmic reticulum (ER), its folding was reversible, which changed only upon both proper incorporation of calcium and exit from the ER. Coevolution of protein folding with the high calcium concentration in the ER may be the basis for the need for this cation throughout the folding process even though calcium is only stably integrated in native repeats at a later stage.     

A diacylglycerol kinase is involved in the regulation of interleukin-2 synthesis in Jurkat T cells.

Diacylglycerol (DAG) plays a prominent role in several activation processes because of its ability, in the presence of calcium ions and phosphatidylserine, to activate C kinase. In T lymphocytes, however, DAG, produced in response to activating mAb or lectins, is rapidly metabolized into phosphatidic acid (PA) which may participate in further steps of activation. We thus investigated the involvement of a DAG kinase in several events subsequent to the activation of Jurkat T cells by CD3 mAb or phytohemagglutinin (PHA). We showed that three inhibitors of DAG kinase abrogated PA production and impaired calcium release from intracellular compartment, restored phosphatidylserine synthesis, and finally blocked IL-2 production in CD3- and PHA-stimulated cells. Postactivation DAG levels were not modified and DAG kinase inhibitors lowered IL-2 secretion even in the presence of phorbol ester that directly activates the C kinase. These results clearly demonstrate that, beside the effect of DAG on C kinase, DAG kinase dependent production of PA is crucial for further steps of T cell activation.     

Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.     

Platelet aggregation. II. Adenyl cyclase, prostaglandin E1, and calcium

In exploration of the proposal that prostaglandin E1 (PGE1) inhibits platelet aggregation via stimulation of adenyl cyclase, the temporal relationship of adenosine cyclic 3',5' monophosphate (cyclic AMP) synthesis and inhibition of ADP-induced aggregation in response to PGE1 was studied. The requirement for calcium in aggregation led to the investigation of the effects of calcium ions on platelet adenyl cyclase activity. PGE1 stimulated the synthesis of cyclic AMP from adenosine-5'-triphosphate-8-14-C by platelet membrane fractions and also increased cyclic AMP synthesis in intact platelets previously incubated for 2 hours with adenosine-14-C. The accumulation of cyclic AMP increased signficiantly at low concentrations of PGE1 and reached a maximum at about 1 mug. Regardless of the inducing agent, calcium ions are an absolute requirement for the aggregation of platelets.     

The in vivo modulation of dopamine synthesis by calcium ions: influences on the calcium independent release

To investigate the contribution of the dopamine (DA) synthesis to both the calcium-dependent and the carrier-mediated, mechanisms of DA release in the striatum, anaesthetized rats were locally superfused in the striatum with a push–pull cannula supplied with an artificial CSF containing tritiated tyrosine. DA, dihydroxyphenylacetic acid (DOPAC) and their respective specific activity were measured in effluent and used to evaluate changes in the DA synthesizing rate. Excluding calcium ions from the CSF only partially reduced spontaneous DA release (70%) still leaving a possible carrier-mediated DA release. This effect was not additive with a local superfusion with 0.1 mM a-methyl-p-tyrosine, a blocker of DA synthesis, suggesting that synthesis could already be reduced by calcium-free superfusion. Local superfusion with 100 μM cadmium in the presence or not of calcium ions, increased the DA release (220 and 350%, respectively), simultaneously reducing DA synthesis. Local application of 1 μM calcium ionophore (A23187) was without effect on the basal release of DA but enhanced DA synthesis and increased the amphetamine-evoked and carrier-mediated amine release. We conclude that DA synthesis can be a modulatory process of the firing-independent and carrier-mediated amine release while it weakly affects the classical calcium-dependent release.     

A role of G-proteins and calcium in light-regulated primary leaf formation in Sorghum bicolor

Primary leaf development of Sorghum bicolor is a phytochrome-mediated response. Primary leaves are not produced in Sorghum seedlings even after 10 d of germination if grown in darkness. However, 5 min irradiation with white light or red light given to 5 d etiolated seedlings resulted in the formation of etiolated leaves. This effect of red light was reversed by far-red light. When calcium (3-5 mM) was added exogenously, complete leaf formation was obtained in darkness; however, the kinetics of the response was slower than that seen with light irradiation. This effect was also obtained with potassium ions but magnesium ions had no effect. Light- and calcium-mediated leaf development could be arrested at the stage of leaf emergence or leaf expansion by the addition of inhibitors of G-proteins or by calcium channel blockers suggesting a role of G-proteins and calcium in phytochrome signal transduction during primary leaf development.Key words: Leaf formation, G-proteins, calcium, potassium, Sorghum bicolor.      

Calcium ions are required for cell fusion mediated by the CD4-human immunodeficiency virus type 1 envelope glycoprotein interaction.

Calcium ions are required for fusion of a wide variety of artificial and biological membranes. To examine the role of calcium ions for cell fusion mediated by interactions between CD4 and the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (gp120-gp41), we used two experimental systems: (i) cells expressing gp120-gp41 and its receptor CD4, both encoded by recombinant vaccinia viruses, and (ii) chronically infected cells producing low levels of HIV-1. Fusion was measured by counting the number of syncytia and by monitoring the redistribution of fluorescence dyes by video microscopy. Syncytia did not form in solutions without calcium ions. Addition of calcium ions partially restored the formation of syncytia. EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] blocked syncytium formation in culture media containing calcium ions. Membrane fusion as monitored by fluorescence dye redistribution also required calcium ions. Cell fusion increased with an increase in calcium ion concentration from 100 microM to 10 mM but was not affected by magnesium ions in the concentration range from 0 to 30 mM. Fibrinogen and fibronectin did not promote fusion in the absence or presence of Ca2+. Binding of soluble CD4 to gp120-gp41-expressing cells was not affected by Ca2+ and Mg2+. We conclude that Ca2+ is involved in postbinding steps in cell fusion mediated by the CD4-HIV-1 envelope glycoprotein interaction.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal–conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal–conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid‐ to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal–conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen‐responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal–conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.     

Concanavalin A and the initiation of thymic lymphoblast DNA synthesis and proliferation by a calcium-dependent increase in cyclic GMP level

Exposure of a thymic lymphocyte population (suspended in serum-free synthetic medium) to the phytomitogen concanavalin A (Con A) causes brief (within the first 8 to 12 minutes) rises in the cellular contents of cyclic AMP and cyclic GMP. However, the rise in the cyclic GMP level is calcium (extracellular)-dependent, but the cyclic AMP rise is not. These changes are followed during the next hour by the initiation of DNA synthesis by a large fraction of the lymphoblast subpopulation which, like the preceding cyclic GMP rise, is calcium-dependent. The stimulated lymphoblasts eventually progress into mitosis. Additional observations indicate that Con A operates by sensitizing lymphoblasts to calcium ions which, in turn, cause the initiation of DNA synthesis by a process mediated by cyclic GMP, but not cyclic AMP.     

Rhizobium-lnduced calcium spiking in Lotus japonicus

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.