Immunoendocrine modulation and stimulation of hematopoiesis with the ionophore copolymer T150R1

T150R1, an 8000-dalton copolymer with sodium ionophore activity, has been shown to modulate cellular responses in multiple systems. In this article, we studied its effects on lymphoid and hematopoietic organs in the context of the adrenal-pituitary axis. When injected in mice as an oil in water emulsion, T150R1 caused a rapid, profound, and dose-dependent thymic involution accompanied by splenic hyperplasia. Time course experiments with a 2.5-mg dose revealed that the thymus size was minimal at Day 2, rose to normal by Day 14, then enlarged and gradually returned to normal by Week 6 postinjection. Thymic involution was due to cellular depletion of the cortical area, whereas thymic enlargement was due to cortical hyperplasia. Splenomegaly was seen as early as Day 4, peaked by Day 14, and gradually returned to normal by Week 6. The splenic enlargement was due to hyperplasia of the red pulp, with evidence of proliferating erythropoietic, myelopoietic, and megakaryopoietic precursors. In addition, the bone marrow was stimulated and extramedullary hematopoiesis was present in the liver. The effects of T150R1 on the thymus appeared to be mediated by corticosteroids while the effects on hematopoiesis were not. Corticosterone and ACTH levels were increased in treated animals. Adrenalectomy diminished the T150R1-induced thymic involution but enhanced the splenic hyperplasia. Hypophysectomy did not prevent thymic involution, suggesting that T150R1 has endocrine stimulatory effects. These data suggest that T150R1 represents a new class of ionophores which may act on excitable cells within the endocrine, immune, and hematopoietic systems.     

Interactions Among Lithium, Calcium, Diacylglycerides, and Phorbol Esters in the Regulation of Adrenocorticotropin Hormone Release from AtT-20 Cells

Interactions among lithium, calcium, and phorbol esters in the regulation of adrenocorticotropin hormone (ACTH) release were examined in a tumor cell line (AtT-20) of the anterior pituitary. Lithium, which blocks the phosphatase that converts inositol phosphates (IPs) to inositol, increases the levels of IPs in these cells and stimulates ACTH release. This ion potentiates the ability of calcium, an activator of phospholipase C, to raise levels of IPs in these cells and to stimulate ACTH secretion. Pretreatment of AtT-20 cells with calcium specifically abolishes the ACTH release response to lithium or calcium, a result suggesting that these secretagogues may act through a common mechanism to induce hormone secretion. Prior exposure of AtT-20 cells to either lithium or calcium also attenuates the ACTH release induced by phorbol ester, an activator of protein kinase C. To examine the link among lithium, calcium, phosphatidylinositol (PI) turnover, and phorbol ester-evoked ACTH secretion, AtT-20 cells were treated with 1-oleoyl-2-acetoyl-sn-3-glycerol (OAG), an analogue of the diacylgylcerols that are formed by phospholipase C during PI metabolism and that also activate protein kinase C. OAG itself does not alter ACTH release or the levels of IPs in AtT-20 cells. Pretreatment of AtT-20 cells with OAG, however, selectively blocks the ACTH release response to lithium, calcium, or phorbol ester. Furthermore, such pretreatment reduced the ability of lithium to increase levels of IPs. The results suggest that one mechanism of action of lithium is to potentiate selectively an action of calcium, possibly the stimulation of phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-dependent activation of human neutrophils by a synthetic ionophore

Synthetic block copolymers composed of polyoxyethylene and poly-oxypropylene have been demonstrated to possess ionophore activity selective for monovalent cations and to cause histamine release from mouse mast cells and human basophils. We now report calcium-dependent release of granule contents from human neutrophils by the most active of these agents, TI30R2. At a concentration of 100 micrograms/ml (12.5 microM), net lysozyme release ranged from 17-40% after 30 minutes incubation at 37 degrees. Lysozyme release was dose-dependent over stimulus concentrations of 5-50 micrograms/ml (0.625-6.25 microM). Release was dependent upon the presence of extracellular calcium. T130R2 did not induce the release of superoxide anions over 30 minutes of incubation. As T130R2 induces sodium influx into cells, it is likely that a depolarizing influx of sodium ions in the presence of extracellular calcium constitutes a sufficient signal for granule release but not superoxide production by human neutrophils.     

Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis

Secretin, glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this review we survey findings that also other members of the VIP-secretin-glucagon family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated.     

Histamine release from human basophils by synthetic block co-polymers composed of polyoxyethylene and polyoxypropylene and synergy with immunologic and non-immunologic stimuli

Co-polymers composed of polyoxyethylene and polyoxypropylene have been shown previously to trigger histamine release from mouse peritoneal mast cells; this property quantitatively is directly related to the ionophorous ability of these compounds to cause a functional exchange of intracellular K+ for extracellular Na+ across the cell membrane. We investigated the effect of an inflammatory copolymer, T130R2, on human basophils. The data demonstrate that T130R2 can cause calcium-dependent histamine release from human basophils in vitro. Further, at concentrations that do not cause histamine release, this co-polymer markedly augments release by suboptimal concentrations of the lectin Con A or anti-IgE antibody and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate but not the calcium ionophore A23187. Thus, these co-polymers induce mediator release from cells of both rodents and humans. In both instances it is likely that calcium-dependent cell triggering is the result of an influx of sodium ions with concomitant depolarization of the transmembrane potential. In common with the calcium ionophore A23187, the co-polymer T130R2 has the ability to synergize with stimuli which trigger the IgE receptor as well as those which directly activate the cellular calcium- and phospholipid-dependent protein kinase.     

In vitro release of histamine from murine mast cells by block co-polymers composed of polyoxyethylene and polyoxypropylene

A series of block co-polymers composed of polyoxyethylene and polyoxypropylene were investigated for their ability to induce in vitro activation of mouse mast cells. We found that six of these co-polymers could cause histamine release from mouse mast cells in vitro. At low concentrations, the most efficacious co-polymer, T130R2, caused rapid and extensive concentration-dependent release of histamine from mouse mast cells. The release process was not cytotoxic; it required metabolic energy and was not accompanied by release of lactate dehydrogenase. Optimal release of histamine was dependent on both calcium and sodium ions in the extracellular medium. The degree of in vitro histamine release correlated with in vivo inflammation and in vitro ionophore activity. We believe that this represents the first report of the activation of mediator-containing cells by an ionophore selective for monovalent cations. These copolymers may therefore represent new reagents for investigations of cellular excitation.     

R,R,R-alpha-tocopherol potentiates prostacyclin release in human endothelial cells. Evidence for structural specificity of the tocopherol molecule

Human umbilical vein endothelial cells (HUVEC) in culture synthesize prostacyclin (PGI2) as the predominant metabolite of arachidonic acid which is derived from the deacylation of phospholipids. Under basal-unstimulated condition, PGI2 release from HUVEC is extremely low; however, when endothelial monolayers were preincubated with the natural vitamin E (R,R,R-alpha-tocopherol), we found a dose-dependent potentiation of basal PGI2 release. When HUVEC were stimulated with arachidonate or ionophore A23187, there was a dose-dependent increase of PGI2 release in response to tocopherol enrichment. When HUVEC were labelled with [Me-3H]choline followed by A23187 stimulation, a significantly higher lysophosphatidylcholine was found in the tocopherol-enriched cells, suggesting a change in enzymes involved in phosphatidylcholine metabolism. Analysis of these enzymes revealed that phospholipase A2 activity was enhanced by tocopherol enrichment, whereas lysophospholipase and acyl-CoA acyltransferase were unaffected. To determine the specificity of the tocopherol molecule, different analogues were tested for their PGI2 potentiating activity. Results showed that the free hydroxyl group on the chromanol ring as well as the phytyl side-chain are absolutely required to stimulate PGI2 release, whereas, different methyl locations and substituents on the chromanol ring had no effect. These studies demonstrated that tocopherol potentiates basal PGI2 release in HUVEC and in contrast to its reported inhibitory role in rat platelets, myocardium and neutrophils, tocopherol stimulates phospholipase activity in HUVEC.     

Effect of vitamin E enrichment on arachidonic acid release and cellular phospholipids in cultured human endothelial cells

The effect of (R,R,R)-alpha-tocopherol on agonist-stimulated arachidonate release and cellular lipids was investigated in cultured human umbilical cord endothelial cells. Endothelial cells in culture incorporate added tocopherol in a dose-dependent manner at both physiological (23.2 microM) or pharmacological (92.8 microM) concentrations which were well tolerated by the cells, as judged by unaltered cell number and viability. Two experiments were conducted in which cells were either incubated with (R,R,R)-alpha-tocopherol followed by labelling with [1-14C]arachidonic acid or they were labelled with arachidonate followed by incubation with tocopherol. Irrespective of the sequence of incubation with arachidonate and tocopherol, (R,R,R)-alpha-tocopherol-enriched cells released significantly more labelled arachidonate when stimulated with thrombin (2.5 U/ml) or ionophore A23187 (1 microM) for 10 min. The magnitude of [1-14C]arachidonate release was higher from ionophore A23187 stimulation than from thrombin stimulation, but the trend of increased arachidonate release in tocopherol-enriched cells was the same. Results from these studies demonstrate that (R,R,R)-alpha-tocopherol can stimulate arachidonate release in human endothelial cells. This observation is in direct contrast to the role of tocopherol, which has been shown to inhibit platelet and cardiac phospholipase A2 activity in rats, and to reduce thrombin-stimulated thromboxane release in rat platelets.     

Interactions between CRF, epinephrine, vasopressin and glucocorticoids in the control of ACTH secretion

The secretion of ACTH by corticotrophs in the anterior lobe of the rat pituitary gland is under the stimulatory influence of at least three receptors, namely that for peptidic CRF (corticotropin-releasing factor), vasopressin and alpha 1-adrenergic agents. CRF is a potent stimulator of cyclic AMP accumulation as well as adenylate cyclase activity in the rat adenohypophysis, thus suggesting an important role of cyclic AMP as mediator of CRF action on ACTH secretion. Vasopressin causes a 2-fold increase of the stimulatory effect of CRF on ACTH release in rat anterior pituitary cells in culture. The potentiating effects of vasopressin on CRF-induced ACTH release are accompanied by parallel changes of intracellular cyclic AMP levels. Vasopressin, while having no effect on basal cyclic AMP levels, causes a 2-fold increase in CRF-induced cyclic AMP accumulation without affecting the ED50 value of CRF action. ACTH secretion is also stimulated by a typical alpha 1-adrenergic receptor. Epinephrine causes a marked stimulation of ACTH release which is additive to that of CRF. Epinephrine, in analogy with vasopressin, although having no effect alone on basal cyclic AMP levels, causes a marked potentiation of CRF-induced cyclic AMP accumulation. Glucocorticoids cause a near-complete inhibition of epinephrine-induced ACTH secretion within 4 h with the following order of ED50 values: triamcinolone acetonide (0.2 nM) greater than dexamethasone (1.0 nM) much greater than cortisol (11 nM) greater than corticosterone (22 nM). Similar effects are observed for CRF- and vasopressin-induced ACTH release. Although the activity of the pituitary-adrenocortical axis in the rat is highly dependent upon sex steroids, 17 beta-estradiol, 5 alpha-dihydrotestosterone and the pure progestin R5020 have no detectable effect on basal or epinephrine-induced ACTH release, thus illustrating the high degree of specificity of glucocorticoids in their feedback control of ACTH secretion. Moreover, glucocorticoids have no effect on CRF-induced cyclic AMP accumulation, thus indicating that their inhibitory effect is exerted at a step following cyclic AMP accumulation.     

Neural cell recognition molecule F11: membrane interaction by covalently attached phosphatidylinositol

The mode of membrane insertion of F11 130 kDa protein, a neural chick cell surface glycoprotein involved in neurite fasciculation, has been investigated. Up to 41% of total F11 130 kDa is released from adult chick brain plasma membranes by phosphatidylinositol specific phospholipase C (PI-PLC), whereas no release is mediated by lecithin/cephalin specific phospholipase C (PLC). PI-PLC dependent release of F11 is also observed from embryonal chick brain plasma membranes and from the surface of intact retinal cells. Biosynthetic labelling experiments demonstrate that F11 contains ethanolamine. Taken together, these results suggest that F11 interacts with the plasma membrane at least partially through covalently linked glycosyl-phosphatidylinositol (GPI) or a structurally similar lipid.     

ACTH, alpha-MSH, and control of cortisol release: cloning, sequencing, and functional expression of the melanocortin-2 and melanocortin-5 receptor in Cyprinus carpio

Cortisol release from fish head kidney during the acute phase of the stress response is controlled by the adrenocorticotropic hormone (ACTH) from the pituitary pars distalis (PD). Alpha-melanocyte-stimulating hormone (alpha-MSH) and beta-endorphin, from the pars intermedia (PI), have been implicated in cortisol release during the chronic phase. The present study addresses the regulation of cortisol release by ACTH and alpha-MSH in common carp (Cyprinus carpio) and includes characterization of their receptors, namely, the melanocortin-2 and melanocortin-5 receptors (MC2R and MC5R). We could not demonstrate corticotropic activity of alpha-MSH, beta-endorphin, and combinations of these. We do show a corticotrope in the PI, but its identity is as yet uncertain. Carp restrained for 1 and 7 days showed elevated plasma cortisol and alpha-MSH levels; cortisol is still elevated but lower at day 7 than day 1 of restraint. Interrenal response capacity is unaffected, as estimated by stimulation with a maximum dose ACTH in a superfusion setup. MC2R and MC5R appear phylogenetically well conserved. MC2R is predominantly expressed in head kidney; a low abundance was found in spleen and kidney. MC5R is expressed in brain, pituitary PD, kidney, and skin. Quantitative PCR analysis of MC2R and MC5R expression in the head kidney of restrained fish reveals MC2R mRNA downregulation after 7 days restraint, in line with lower plasma cortisol levels seen. We discuss regulation of corticosteroid production from a phylogenetic perspective. We propose that increased levels of alpha-MSH exert a positive feedback on hypothalamic corticotropin-releasing hormone release to sustain a mild stress axis activity.     

CD2 triggering stimulates a phospholipase A2 activity beside the phospholipase C pathway in human T lymphocytes

In previous studies we demonstrated the triggering of the phospholipase C (PLC) pathway during the activation of an Ag-specific human CD4+ T lymphocyte clone by a mitogenic pair of CD2 (X11,D66) mAb. Similar conditions were applied to investigate a possible involvement of a phospholipase A2 (PLA2) acting as an additional alternative pathway during human T cell activation. Our results show that arachidonic acid or its derivatives are released after CD2 triggering. This release is largely independent of PLC activation and is mediated by a PLA2 because: 1) phosphatidylcholine is the preferential source of [3H]arachidonate release; 2) [3H]arachidonic acid release and phosphatidylcholine hydrolysis are blocked by two inhibitors of solubilized PLA2, mepacrine, and 4-p-bromophenacylbromide; and 3) we evidenced a PLA2 activity in cell homogenates. Extracellular calcium appears to play a critical role because the effects of CD2 mAb were inhibited in a Ca2(+)-depleted medium. In contrast, protein kinase C is not implicated since PMA, a protein kinase C activator, neither stimulated arachidonic acid release nor modulated CD2-induced arachidonic acid release. Cyclic AMP which has been proved to regulate the activity of the PLC in T lymphocytes does not appear to play an important role in the regulation of PLA2 activity since PGE2 has only a minimal effect on [3H]-arachidonate release. Altogether, these findings suggest that CD2 triggering stimulates a PLA2 activity in T lymphocytes via an extracellular Ca2(+)-dependent PLC protein kinase C independent mechanism.     

Phospholipase A2-mediated release of arachidonic acid in stimulated guinea pig alveolar macrophages: interaction with lipid mediators and cyclic AMP

The stimulation of cultured guinea pig alveolar macrophages by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine, or by the phospholipid inflammatory mediator platelet activating factor (PAF) induced an increase in arachidonic acid release and its cyclooxygenase products. This release, which was mimicked by the association of threshold concentrations of the calcium ionophore A 23187 and of the protein kinase C activator tetradecanoyl phorbol acetate arose mainly from diacyl- and alkyl-acyl-phosphatidylcholine and phosphatidylinositol. Using [1-14C]arachidonic acid-labeled membranes as an endogenous substrate as well as dioleoyl-phosphatidyl [14C]ethanolamine as an exogenous substrate, we showed that phospholipase A2 activity of stimulated macrophages increases upon stimulation. Treatment of macrophages by prostaglandin E2 decreased the arachidonic acid release elicited by the chemotactic peptide and PAF. Furthermore, prostaglandin E2 increased and PAF decreased the cellular content in cyclic AMP. From these results we suggest that an initial stimulation of alveolar macrophages by a bacterial signal initiates the sequential activation of a phospholipase C and of phospholipase A2, leading to the release of PAF and eicosanoids. These mediators may in turn modulate the cell response by increasing or decreasing cyclic AMP, Ca2+, or diacyglycerol macrophage content.     

Inhibition by glucocorticoids of PGE2 and ACTH secretion induced by phorbol esters and EGF in rat pituitary cells

In rat pituitary cells in primary culture glucocorticoids specifically inhibit PGE2 and ACTH secretions induced by TPA, a potent phorbol ester derivative (triamcinolone acetonide greater than dexamethasone greater than cortisol greater than or equal to corticosterone). However, while PGE2 secretion can be inhibited up to 80%, ACTH secretion can only be inhibited up to 40%. Similar inhibitory effects are observed with mepacrine, an inhibitor of phospholipase A2 (PLA2). Glucocorticoids having also been described as PLA2-inhibitors, their inhibitory effect on TPA-induced secretions could thus be related to their anti-PLA2 activity. Their inhibitory effect on PLA2 has been attributed to their ability to induce the synthesis of lipocortin, the activity of which could be regulated by activation of kinase C or EGF-receptor kinase. Since in our model, EGF-induced PGE2 secretion is also inhibited by dexamethasone, these results suggest that a lipocortin-like protein could be present in pituitary cells and involved in the effect of TPA and EGF on PGE2, and, at least partly, on ACTH release.     

The biosynthesis and secretion of adrenocorticotropin by the ovine anterior pituitary is predominantly regulated by arginine vasopressin (AVP). Evidence that protein kinase C mediates the action of AVP

This study was undertaken to define the roles of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of adrenocorticotropin (ACTH) release and biosynthesis in cultured ovine anterior pituitary cells and to define the intracellular mechanisms responsible for their action. At 4 h, CRF and AVP increased both ACTH release and total ACTH content, with AVP clearly the more potent agonist (maximal ACTH release: AVP, 22.8-fold; CRF, 7.6-fold; maximal increment in total ACTH content: AVP, 1.9-fold; CRF, 1.1-fold; EC50 for ACTH release: AVP, 2.3 +/- 0.5 nM; CRF, 9.2 +/- 5.0 nM). The increase in total ACTH content was interpreted to reflect an augmentation of ACTH biosynthesis since it was abolished by 10 microM cycloheximide. Exposure of the anterior pituitary cells to increasing concentrations of forskolin or 8-bromo-cAMP elicited increases in ACTH release and total ACTH content that were similar to those caused by CRF. A 30-min incubation with phorbol 12-myristate 13-acetate (PMA) caused a dose-related translocation of protein kinase C from the cytosol to the cell membrane; after 4 h, the increases in ACTH release and total ACTH content in response to increasing concentrations of PMA were similar to those caused by AVP. Chronic (24 h) exposure to 150 nM PMA caused an almost total depletion of both cytosolic and membrane-bound protein kinase C activities. When protein kinase C-depleted cells were subsequently exposed to AVP, the increases in ACTH release and total ACTH content were markedly attenuated, but the responses to CRF were preserved. Finally, the combination of CRF and AVP, CRF and PMA, or AVP and 8-bromo-cAMP increased ACTH release and total ACTH content in a synergistic manner. We conclude that: 1) in ovine anterior pituitary cells, AVP is the predominant regulator of ACTH secretion and biosynthesis; 2) the action of AVP is predominantly mediated by activation of protein kinase C, whereas the action of CRF is likely to be mediated by activation of the cAMP-dependent protein kinase (protein kinase A); and 3) the ability of CRF and AVP to increase total ACTH content and secretion in a synergistic manner provides a demonstration in normal pituitary cells that protein kinases C and A may interact in a unidirectional manner to regulate ACTH biosynthesis in addition to ACTH release. This interaction may take place within, or between, individual corticotropes.     

Defining the role of protein kinase c in calcium-ionophore-(A23187)-mediated activation of phospholipase A2 in pulmonary endothelium.

We sought to investigate the mechanisms by which the calcium ionophore A23187 triggers arachidonic acid release in bovine pulmonary endothelial cells and to test the hypothesis that protein kinase C is involved in this process. Our results indicate that the mechanism by which A23187 increases phospholipase A2 activity and arachidonic acid release in bovine pulmonary arterial endothelial cells depends upon the concentration studied. At concentrations of 1 microM and 2.5 microM, A23187 increases phospholipase A2 activity and arachidonic acid release without stimulating protein kinase C. At concentrations of 5-12.5 microM, A23187 increases arachidonic acid release and phospholipase A2 activity in conjunction with a dose-dependent activation of membrane-bound protein kinase C. To test the hypothesis that these doses of A23187 increase phospholipase A2 activity by stimulating protein kinase C, we studied the effect of prior treatment with the protein kinase C inhibitor sphingosine. Sphingosine inhibits the increase in phospholipase A2 activity and arachidonic acid release caused by A23187 over the range 5-12.5 microM. To investigate further the potential role of protein kinase C, we studied the effects of the inactive phorbol ester 4 alpha-phorbol 12 beta-myristate 13 alpha-acetate (4 alpha-PMA) and an active phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (4 beta PMA). Neither 4 alpha-PMA nor 4 beta-PMA affected basal arachidonic acid release. 4 alpha-PMA also did not augment the effects of A23187. In contrast, 4 beta-PMA significantly augments the increase in phospholipase A2 activity and arachidonic acid release caused by lower doses of A23187. Under these conditions, sphingosine completely inhibits the stimulatory effects of 4 beta-PMA on protein kinase C translocation, phospholipase A2 and arachidonic acid release. Thus, at low doses (1 microM and 2.5 microM) A23187 increases phospholipase A2 activity and arachidonic acid release by a mechanism that does not involve protein kinase C. At these A23187 doses, activating membrane-bound protein kinase C with 4 beta-PMA causes a synergistic increase in phospholipase A2 activity and arachidonic acid release. At higher doses (5-12.5 microM), A23187 acts in large part by stimulating protein kinase C translocation. Overall, our results indicate that activating membrane-bound protein kinase C by itself is an insufficient stimulus to increase phospholipase A2 activity and arachidonic acid release in pulmonary endothelial cells, but activating protein kinase C can substantially augment the increase in phospholipase A2 activity and arachidonic acid caused by a small increase in intracellular calcium.     

Stimulation of monovalent ion fluxes and DNA synthesis in 3T3 cells by melittin and vasopressin is not mediated by phospholipid deacylation

Melittin at subtoxic concentrations stimulates monovalent ion fluxes and, together with insulin, synergistically increases DNA synthesis, but has little effect on phospholipase activity. The Na⁺ ionophore monensin increases Na-K pump activity without affecting phospholipase activity; whereas, the divalent cation ionophore A23187 has reciprocal effects. Finally, vasopressin and insulin potently stimulate ion fluxes and DNA synthesis but have no effect on phospholipase activity. Thus, the polypeptide mitogens melittin, vasopressin, and insulin alter membrane function and subsequently stimulate DNA synthesis in quiescent 3T3 cells by mechanisms independent of phospholipid deacylation.     

Exocytosis of CTLA-4 is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated during activation of regulatory T cells

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.     

Temperature effects in the stimulus-secretion process from isolated chromaffin cells

Temperature effects on the stimulus-secretion coupling process was studied by inducing release of catecholamines (CA) from isolated chromaffin cells of the bovine adrenal medulla. Use was made of three different secretagogues: acetylcholine (ACH), high potassium concentration, and the calcium ionophore A23187, at various incubation temperatures. The latter two agents induced a monotonic increase in secretion with rise in temperature, suggesting different regions of the dependence of total release on temperature. The ACH-induced secretion was, however, markedly different and exhibited a maximal release at 30 degrees C. Kinetic experiments using ACH stimulus revealed that this maximum is produced by different temperature dependence in the stages of activation and desensitization. A proposed model for the total release process yields temperature-dependent parameters that can be divided into three regions of initial rates of secretory activity corresponding to the above independent findings using high K+ concentration and the calcium ionophore. The transitions between the various regions indicate possible transitions in the physical properties of the plasma and secretory granule membranes. Elucidation of the interaction between the membranes is of primary importance in the determining mechanism of CA secretion from the isolated adrenal medulla cell.     

A small phospholipase inhibitory factor released by cultured cell lines

Cells of the mouse macrophage-like cell line RAW264 release a dialysable inhibitor of phospholipase activity into their culture medium. This inhibitor can be detected in saline solution, Hanks solution and a variety of tissue culture media in the presence or absence of serum. The inhibitor is stable at 4 degrees C, unaffected by trypsin, nucleases, or boiling, and partially extractable with chloroform/methanol. The release of both arachidonic acid and prostaglandins from mouse macrophages or human monocytes is inhibited by this material. A variety of other cell types release the inhibitor, which is effective against stimulation of arachidonic acid release from cultured macrophages by zymosan, serum, immune complexes and the calcium ionophore A23187.