Calcium-mobilizing Agents Research Articles

High concentrations of cholecystokinin octapeptide suppress protein kinase C activity in guinea pig pancreatic acini

In pancreatic acini, calcium-mobilizing agents increase intracellular calcium and stimulate the production of diacylglycerol, and then activate protein kinase C (PKC). However, there are few studies which have examined the activation of PKC in intact acini. To examine the activation of PKC in intact acini by calcium-mobilizing agents, we measured the binding of [3H]phorbol-12,13-dibutyrate (PDBu) to intact acini. Acini were incubated with 10 nM [3H]PDBu at 25 degrees C with or without agents. The binding reactions were terminated by filtration. The filters were counted by a scintillation counter after washing. Acini possessed a single class of binding sites to PDBu, with Kd = 70 nM. CCK-8 and carbachol upregulated the binding affinity of PKC to PDBu in the acini. The ability of calcium-mobilizing agents to increase binding of [3H]PDBu to the acini had a close correlation to their ability to stimulate the amylase secretion from the acini, and higher concentrations of CCK-8 for amylase secretion suppressed binding of [3H]PDBu to the acini. 8Br-cAMP, 8Br-cGMP, and calcium ionophore did not inhibit the maximal activation of PKC induced by CCK-8. The calmodulin inhibitor W7 did not reverse the inhibitory effect of higher concentrations of CCK-8 on PKC activation. These results indicate that calcium-mobilizing agents upregulate the binding affinity of PKC to PDBu in intact acini, and that higher concentrations of CCK-8 for amylase secretion may activate the intracellular mechanism that inhibits PKC activity in acini. This inhibitory mechanism was mediated by some other mechanism other than cAMP-, cGMP-, calcium- and calmodulin-dependent mechanisms.

Modulation of hepatocellular bile acid uptake by calcium-mobilizing agents and phorbol ester in isolated hamster hepatocytes: [formula omitted], S. Reza, L.A. Dougherty, H. Fromm, and R. Nussbaum. Division of Gastroenterology & Nutrition, Department of Medicine, The George Washington University Medical Center, Washington, DC

Modulation of hepatocellular bile acid uptake by calcium-mobilizing agents and phorbol ester in isolated hamster hepatocytes: [formula omitted], S. Reza, L.A. Dougherty, H. Fromm, and R. Nussbaum. Division of Gastroenterology & Nutrition, Department of Medicine, The George Washington University Medical Center, Washington, DC

Effects of thapsigargin, an intracellular calcium‐mobilizing agent, on synthesis and secretion of cartilage collagen and proteoglycan

The calcium-mobilizing agents thapsigargin and 2,5-di-(tert-butyl)-1,4- benzohydroquinone were shown to markedly elevate the intracellular calcium concentration of chick embryo chondrocytes in a dose-dependent manner. Under these conditions, the metabolism of macromolecules was variably affected. The synthesis and secretion of protein in general, and of collagen in particular, were significantly inhibited; in contrast, proteoglycan synthesis (but not glycosaminoglycan synthesis) was inhibited, whereas secretion was unaffected. Flunarizine, which prevented the thapsigargin-induced intracellular calcium elevation, and EGTA, which caused only a transient thapsigargin-induced intracellular calcium elevation, did not reverse these alterations. It was concluded, therefore, that the observed effects of thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone on chondrocyte macromolecule metabolism were not related to the ability of these drugs to increase the cytosolic free calcium concentration but may have been due to the specific depletion of the calcium sequestered in the endoplasmic reticulum. The differential effect of these drugs on protein and proteoglycan secretion suggests that the intracellular trafficking of these two classes of macromolecules may be controlled independently.

Methylxanthines and Calcium-Mobilizing Agents Inhibit the Expression of Cytokine-Inducible Nitric Oxide Synthase and Vascular Cell Adhesion Molecule-1 in Murine Microvascular Endothelial Cells

In response to exposure to the inflammatory cytokines tumor necrosis factor-α (TNF) and interferon-γ (IFN-γ), murine brain microvascular endothelial cells (MME) synthesize the cell surface molecule, vascular cell adhesion molecule-1 (VCAM-1), and the intracellular enzyme, inducible nitric oxide synthase (iNOS). However, iNOS synthesis requires the presence of both TNF and IFN-γ, while VCAM-1 can be induced by either cytokine alone. We examined the induction of VCAM-1 and iNOS under a variety of conditions to better define the regulation of TNF and IFN-γ signal transduction pathways in MME. We utilized the analysis of steady-state levels of iNOS mRNA as well as the measurement of MME-released NO-EDRF (nitric oxide as an endothelium-derived relaxing factor) activity and accumulation of nitrite in the culture medium to define iNOS expression and activity. VCAM-1 expression was determined by flow cytometric analysis. Our data indicate that low density lipoproteins inhibited cytokine-induced iNOS activity by affecting the steady-state levels of iNOS mRNA. Methylxanthines (caffeine and theophylline) as well as several calcium-mobilizing agents inhibited the expression/activity of both iNOS and VCAM-1 in MME. The effectiveness of these agents was dependent upon the degree of disruption in cell calcium homeostasis during cytokine treatment. Cells which had been pretreated with calcium-modulating drugs and then washed and allowed to return to normal calcium homeostasis showed little to no effect from these agents. In addition, our results suggest that NO produced by iNOS acts as a metabolic switch during in flammation by inhibiting oxidative phosphorylation and forcing vascular endothelial cells to temporarily utilize anaerobic energy metabolism.

Stimulation of human neutrophils by 5-oxo-6,8,11,14-eicosatetraenoic acid by a mechanism independent of the leukotriene B4 receptor

We recently identified a novel pathway for the metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by human neutrophils, resulting in oxidation of the 5-hydroxyl group to give 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) (Powell, W. S., Gravelle, F., and Gravel, S. (1992) J. Biol. Chem. 267, 19233-19241). This pathway is quite specific for 5-HETE and other eicosanoids containing a 5(S)-hydroxyl group followed by a 6-trans double bond. In the present study we have shown that 5-oxo-ETE is very potent in raising cytosolic calcium levels in human neutrophils. This effect was reproducibly observed at concentrations as low as 0.3 nM, and the EC50 was found to be 2 nM. The mechanism of action of 5-oxo-ETE on neutrophils appeared to be distinct from that of leukotriene B4 (LTB4), since it was not blocked by the LTB4 antagonist LY255283 at a concentration which completely prevented the response to LTB4. As would be expected for a receptor-mediated mechanism, the response to 5-oxo-ETE was subject to homologous desensitization and was completely abolished by prior treatment of neutrophils with 5-oxo-ETE (100 nM) but was not affected by pretreatment of these cells with the same concentration of LTB4. 5-Oxo-15(S)-hydroxy-6,8,11,13- eicosatetraenoic acid (5-oxo-15-hydroxy-ETE), formed from 5(S),15(S)-dihydroxy-6,8,11,13- eicosatetraenoic acid (5,15-di-HETE) by the pathway responsible for the formation of 5-oxo-ETE, also raised cytosolic calcium levels in human neutrophils, with an EC50 of about 15 nM. 5-HETE, the precursor of 5-oxo-ETE, also had this effect but was about 100 times less potent than the latter substance. Desensitization experiments indicated that both 5-oxo-15-hydroxy-ETE and 5-HETE act by a mechanism similar to that of 5-oxo-ETE, but different from that of LTB4. In addition to their effects on calcium levels, both 5-oxo-ETE and 5-oxo-15-hydroxy-ETE had chemotactic effects on human neutrophils. Related eicosanoids, including 15-oxo-5,8,11,13-eicosatetraenoic acid, 5,15-diHETE, and 5(S)-hydroxy-15-oxo-6,8,11,13-eicosatetraenoic acid were much less potent, as both chemotactic and calcium-mobilizing agents. These results suggest that neutrophils possess a specific recognition mechanism for 5-oxo-ETE, which may be an important regulator of the activity of neutrophils, especially if they become desensitized to LTB4.

Inhibition of basal and tumor necrosis factor-enhanced binding of murine tumor cells to murine endothelium by transforming growth factor-beta 1.

The adherence of cells to microvascular endothelium is important in a number of processes, including inflammatory responses and metastasis. It has been demonstrated that in human models, cytokines such as TNF, IL-1, IFN-gamma increase the adhesiveness of endothelium for cells of the immune and inflammatory system by stimulating the expression of cell adhesion molecules on endothelial cell surfaces. We and others have shown similar cytokine-induced endothelial adhesiveness for tumor cells in murine and human models. In contrast to the effect of those modulators, transforming growth factor-beta (TGF-beta) has been shown to inhibit the binding of human neutrophils and T lymphocytes to human endothelium, although the mechanism of TGF-beta action remains unknown. Little is known about the effect of TGF-beta on tumor cell-endothelial interaction. In the present study, we demonstrate that TGF-beta inhibits basal and TNF-enhanced binding of murine P815 mastocytoma cells to murine microvascular endothelium (MME). The alterations in MME mediated by TGF-beta, also lead to the inhibition of adherence of murine splenocytes, thymocytes, and human lymphoblastoid cells but do not inhibit adherence of murine B16 melanoma cells. The effect of TGF-beta is transient and inhibition of the endothelial adhesive phenotype is strongest 12 to 24 h after addition of the factor to MME. The TGF-beta-mediated inhibition of P815 basal binding to endothelium is dependent on protein synthesis because cycloheximide reverses the TGF-beta effect. TGF-beta does not appear to activate classical signal transduction pathways. Inhibitors of G proteins do not abolish TGF-beta action, protein kinase C and protein kinase A activators elicit an effect opposite to that of the factor, TGF-beta does not increase intracellular cAMP levels, and finally calcium-mobilizing agents do not mimic, but rather inhibit the effect of TGF-beta. However, TGF-beta-mediated inhibition of both basal binding and TNF-enhanced P815 binding to MME is completely abolished in the presence of the protein phosphatase inhibitor okadaic acid which suggests that TGF-beta may elicit its effect by stimulating protein phosphatase activity.

Maitotoxin increases inositol phosphates in rat anterior pituitary cells

Maitotoxin is a potent marine poison that mobilizes calcium in most vertebrate cell types and accelerates secretion from anterior pituitary cells. It is not known whether voltage-sensitive calcium channels or other mechanisms initiate the effects of maitotoxin on anterior pituitary cells. Changes in intracellular Ca2+ levels may also be achieved by releasing internal calcium stores via inositol trisphosphate (InsP3). Indeed, maitotoxin rapidly increased inositol phosphate accumulation in a concentration-dependent manner. Calcium channel antagonists such as nifedipine and verapamil did not block this response nor did calcium-mobilizing agents (BAYk8644, A23187) mimic this effect. These data suggest that the mechanism by which maitotoxin acts at the pituitary may include the activation of an enzyme that produces the calcium-mobilizing signal InsP3.

Direct and cytokine-mediated activation of protein kinase C induces human immunodeficiency virus expression in chronically infected promonocytic cells

The chronically infected promonocytic clone U1 expresses low-to-undetectable constitutive levels of human immunodeficiency virus (HIV). Virus replication in these cells can be increased up to 25-fold by phorbol esters (phorbol-12-myristate-13-acetate), recombinant cytokines such as tumor necrosis factor-alpha, and cytokine-enriched mononuclear cell supernatants. We have tested specific activators of protein kinases (PK) and PK inhibitors (isoquinolinesulfonamide derivatives), as well as calcium-mobilizing agents, for their effect on constitutive and induced virus expression in U1 cells. Virus expression was measured by reverse transcriptase, Western blot, and nuclear run-on analysis. Activation of PKC by 1-oleyl,2-acetylglycerol, a synthetic analog of the natural ligand 1,2-diacylglycerol, and bryostatin 1 (a recently described specific PKC activator) resulted in a two- to eightfold increase in virus production. In contrast, activators of cyclic-nucleotide-dependent PKs were not effective in inducing virus expression. PK inhibitors were tested for their effect on HIV upregulation by cytokines and other inducing agents. The isoquinolinesulfonamide derivative H7, a potent inhibitor of PKC activation, effectively blocked (70 to 90%) HIV induction by cytokines and phorbol-12-myristate-13-acetate. The derivative HA1004, which is more selective for cyclic-nucleotide-dependent kinases, did not suppress viral induction. In addition, increases in intracellular calcium levels dramatically enhanced HIV production induced by both specific PKC activators and cytokines. These results indicate that activation of PKC is a common pathway involved in the upregulation of HIV expression in chronically infected cells stimulated by cytokines and other inducing agents.

Effect of Inhibin on Activators of Protein Kinase-C and Calcium-Mobilizing Agents which Stimulate Secretion of Gonadotropins in Vitro: Implication of a Postgonadotropin-Releasing Hormone Receptor Effect of Inhibin on Gonadotropin Release*

To further characterize the subcellular mechanisms by which inhibin suppresses GnRH-stimulated gonadotropin release, anterior pituitary cells from adult male Sprague-Dawley rats were treated on day 2 of culture with or without purified 31-kDa bovine inhibin (1-300 pM) for a further 3 days. On day 5, the pretreated cells were washed and incubated in the absence or presence of various secretagogues for 4 h. At the end of the stimulation, the media were saved, and cells were lysed for measurement of both extracellular and intracellular FSH and LH by specific RIAs. Released hormone was expressed as the proportion of total (released plus intracellular) hormone that was available for release in each case. This manipulation of the data corrects for the differential effect of the inhibin pretreatments to suppress intracellular FSH before the stimulation period. Pretreatment for 3 days with inhibin suppressed the proportions of FSH and LH released during 4 h in response to 1) phorbol 12-myristate 13-acetate (100 nM), an activator of protein kinase-C, by maxima of 48% and 53% with inhibin median inhibitory concentrations (IC50) of 17 and 18 pM, respectively; 2) mezerein (100 nM), another type of activator of protein kinase-C, by maxima of 49% and 50% with inhibin IC50 of 19 and 20 pM, respectively; 3) high extracellular K+ (60 mM) by 42% (P less than 0.01) and 38% (P less than 0.01), respectively, with 130 pM inhibin; 4) the calcium ionophore, A23187 (100 microM) by maxima of 54% and 56% with IC50 of 18 and 17 pM, respectively; and 5) GnRH (10 nM) by maxima of 52% and 53% with IC50 of 18 and 19 pM, respectively. However, inhibin had no effect on the proportional release of gonadotropin induced by melittin, an activator of phospholipase-A2. Finally, inhibin had no effect on ACTH release either under basal conditions or in response to CRF (10 nM), phorbol 12-myristate 13-acetate (100 nM), or A23187 (100 microM). We conclude that inhibin suppresses the stimulated release of hormones from gonadotrophs in part by a mechanism common to both gonadotropins that is independent of the previously described inhibitory effect of inhibin on the GnRH receptor. The results are consistent with an action at a site(s) beyond the GnRH receptor, such as protein kinase-C and calmodulin.

Synergistic effects of calcium‐mobilizing agents and adenosine on histamine release from rat peritoneal mast cells

1. Adenosine and its metabolically stable analogue N-ethyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when these are stimulated by calcium-mobilizing agents. NECA and adenosine shift the concentration-response curve of the calcium ionophore A23187 to lower concentrations. 2. The potencies of NECA or adenosine in enhancing A23187-induced histamine release are dependent on the level of stimulated release in the absence of adenosine analogues. At high levels of release their potencies are up to 20 times higher than at low levels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing histamine release are shallow. 3. The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low levels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high levels of release, however, NBTI alone enhances the release of histamine. 4. It is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The levels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role.

Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH, and TSH secretion from anterior pituitary cells.

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.

Binding of somatostatin to pancreatic acinar cells.

The binding of 125I-[Tyr11]somatostatin to guinea pig pancreatic acini was saturable and temperature, protein, and radioligand concentration dependent. Dissociation rate was very slow (t1/2 = 193 +/- 24 min). Scatchard analysis revealed a single class of binding sites with a Kd of 0.28 +/- 0.02 nM and a maximal binding capacity of 72 +/- 10.6 fmol/mg prot. There was a strong correlation between binding capacity and extracellular calcium concentration. Incubating acini in EGTA-containing medium with no added Ca2+ caused a 50% decrease in maximal binding capacity with a decrease in receptor affinity. Furthermore, in the absence of calcium, bound somatostatin was rapidly released (t1/2 = 14 +/- 1 min). Subcellular fractionation studies and acid treatment of acini incubated with the tracer showed that most of the somatostatin binding sites were located on the cell surface. Agents that altered cellular calcium in pancreatic acini, such as analogues of cholecystokinin and cholinergic agents, also inhibited the binding of 125I-[Tyr11]somatostatin by a calcium-dependent process. We conclude that somatostatin binds to specific plasma membrane receptors to form a slowly reversible complex that is highly reactive with calcium. Cell calcium-mobilizing agents decrease the affinity of acinar somatostatin receptors for somatostatin.