Guanine nucleotides stimulate polyphosphoinositide phosphodiesterase and exocytotic secretion from HL60 cells permeabilized with streptolysin O.

The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.     

The dual effector system for exocytosis in mast cells: Obligatory requirement for both Ca2+ and GTP

The secretory process is a coordinated cellular response, initiated by occupation of surface receptors and comprising an ordered sequence of biochemical steps subject to multiple controls. Conceptually we can divide the sequence into two main sections comprising early, receptor-mediated events leading to generation of intracellular second messengers, and later events leading to membrane fusion and exocytosis. With the discovery that occupation of Ca²⁺ mobilising receptors leads to activation of polyphosphoinositide phosphodiesterase (PPI-pde) through the mediation of a G-protein (Gp), all the early events can be ascribed to the plasma membrane. Investigation of the exocytotic stage of secretion has been simplified by the use of permeabilised cells in which the composition of the cytosol can be precisely controlled. We have used streptolysin-O, a bacterial cytolysin which generates protein-sized pores in the plasma membrane, to investigate the exocytotic mechanism of rat mast cells. We find that in addition to the activation of PPI-dpe, GTP also acts in concert with Ca²⁺ at, or close to, the exocytotic site. Exocytosis can occur after substantial depletion of cytosol lactate dehydrogenase and 3-phosphoglycerate kinase indicating that soluble cytosol proteins are unlikely to play any role. There is no absolute requirement for ATP or phosphorylating nucleotide in exocytosis though when present the effective affinities of the two obligatory effectors (i.e. Ca²⁺ and GTP) are substantially enhanced.     

Fluoroaluminates mimic guanosine 5''-[gamma-thio]triphosphate in activating the polyphosphoinositide phosphodiesterase of hepatocyte membranes. Role for the guanine nucleotide regulatory protein Gp in signal transduction.

Fluoride and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) both activate the hepatocyte membrane polyphosphoinositide phosphodiesterase (PPI-pde) in a concentration-dependent manner. AlCl3 enhances the fluoride effect, supporting the concept that [A1F4]- is the active species. Analysis of the products of inositol lipid hydrolysis demonstrate that phosphatidylinositol bisphosphate is the major lipid to be hydrolysed. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) is an inhibitor of activation of PPI-pde by both fluoride and GTP gamma S. These observations suggest that the guanine nucleotide regulatory protein (termed Gp) bears a structural resemblance to the well-characterized G-proteins of the adenylate cyclase system and the cyclic GMP phosphodiesterase system in phototransduction.     

Guanosine 5'-(gamma-thio)triphosphate stimulates arachidonic acid liberation in permeabilized rat peritoneal mast cells

The exocytotic histamine secretion from ATP-permeabilized and Mg-resealed rat peritoneal mast cells is markedly enhanced by the addition of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) at a concentration of 100 uM. GTP gamma S also caused a great enhancement of arachidonic acid liberation from these cells. The level of released arachidonic acid in permeabilized cells enhanced by GTP gamma S in the absence of Ca2+ was nearly equal to the level of permeabilized cells incubated in the presence of Ca2+ but without GTP gamma S, suggesting the Ca2+ sparing effect of GTP gamma S. From the time sequential changes in the [3H]arachidonate radioactivities in various phospholipids, it is conceivable that nucleotide-dependent arachidonic acid release was mediated via phospholipase A2 pathway. The entrapment of a diacylglycerol (DG) lipase inhibitor, RHC 80267, caused suppression of both Ca2+- and guanine nucleotide-dependent arachidonic acid liberation in mast cells, indicating contribution of DG lipase pathway for arachidonic acid generation.     

Characterization of fMet-Leu-Phe-stimulated phospholipase C in streptolysin-O-permeabilised cells

Phospholipase C (specific for inositol lipids) is known to be present both in membranes and cytosol. Receptor-mediated activation of this enzyme occurs via a guanine nucleotide regulatory protein (G-protein), designated Gp. We have compared the stimulation of this enzyme by fMet-Leu-Phe via the G-protein in HL60 membranes and in permeabilised cells. fMet-Leu-Phe stimulated phospholipase C in membranes at 2 min and the response was dependent on exogenously added GTP. GTP alone also stimulated phospholipase C activity such that at 10 min the response to fMet-Leu-Phe was minimal. In comparison, the response to fMet-Leu-Phe in permeabilised cells was greater in extent but did not require added GTP. However, it was antagonized by GDP analogues (GDP[beta S] greater than GDP greater than dGDP) and by pertussis toxin pretreatment, indicating that fMet-Leu-Phe-stimulated phospholipase C activity was also mediated via Gp. GTP and its analogue GTP[gamma S] also stimulated phospholipase C and their effects were strictly additive to the stimulation obtained with fMet-Leu-Phe. Such additivity was also observed when two receptor-directed agonists, fMet-Leu-Phe and ATP, were used to stimulate intact cells. It is concluded that (a) the size of the response with fMet-Leu-Phe in membranes is limited by the loss of a component, possibly phospholipase C, and (b) stoichiometry and physical organisation of multiple species of G-proteins and/or phospholipases C may explain the independent nature of phospholipase C activation by fMet-Leu-Phe, ATP and guanine nucleotides.     

Factors affecting dense and alpha-granule secretion from electropermeabilized human platelets: Ca(2+)-independent actions of phorbol ester and GTP gamma S.

Electropermeabilized human platelets containing 5-hydroxy[14C]tryptamine ([14C]5-HT) were suspended in a glutamate medium containing ATP and incubated for 10 min with (in various combinations) Ca2+ buffers, phorbol 12-myristate 13-acetate (PMA), guanine nucleotides, and thrombin. Release of [14C]5-HT and beta-thromboglobulin (beta TG) were used to measure secretion from dense and alpha-granules, respectively. Ca2+ alone induced secretion from both granule types; half-maximal effects were seen at a -log [Ca2+ free] (pCa) of 5.5 and maximal secretion at a pCa of 4.5, when approximately 80% of 5-HT and approximately 50% of beta TG were released. Addition of PMA, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), GTP, or thrombin shifted the Ca2+ dose-response curves for secretion of both 5-HT and beta TG to the left and caused small increases in the maximum secretion observed. These results suggested that secretion from alpha-granules, like that from dense granules, is a Ca(2+)-dependent process stimulated by the sequential activation of a G-protein, phospholipase C, and protein kinase C (PKC). However, high concentrations of PMA and GTP gamma S had distinct effects in the absence of Ca2+ (pCa greater than 9); 100 nM PMA released approximately 20% of platelet 5-HT but little beta TG, whereas 100 microM GTP gamma S stimulated secretion of approximately 25% of each. Simultaneous addition of PMA greatly enhanced these effects of GTP gamma S. Phosphorylation of pleckstrin in permeabilized platelets incubated with [gamma-32P]ATP was used as an index of the activation of PKC during secretion. In the absence of Ca2+, 100 nM PMA caused maximal phosphorylation of pleckstrin and 100 microM GTP gamma S was approximately 50% as effective as PMA; neither GTP gamma S nor Ca2+ enhanced the phosphorylation of pleckstrin caused by 100 nM PMA. These results indicate that, although activation of PKC promoted secretion, GTP gamma S exerted additional stimulatory effects on secretion from both dense and alpha-granules that were not mediated by PKC. Measurement of [3H]inositol phosphate formation in permeabilized platelets containing [3H]phosphoinositides showed that GTP gamma S did not stimulate phosphoinositide-specific phospholipase C in the absence of Ca2+. It follows that in permeabilized platelets, GTP gamma S can both stimulate PKC and enhance secretion via G-protein-linked effectors other than this phospholipase.     

Epidermal growth factor stimulates diacylglycerol kinase in isolated plasma membrane vesicles from A431 cells

We have examined the effect of epidermal growth factor(EGF) on three kinds of kinases activities, phosphatidylinositol(PI) kinase, phosphatidylinositol 4-phosphate[PI(4)P] kinase and diacylglycerol(DG) kinase that make important roles in the regulation of inositol phospholipids metabolism. When isolated plasma membrane vesicles from A431 cells were incubated at 30 degrees C with [gamma-32P]ATP and exogenously added DG, EGF enhanced the activity of DG kinase approximately 2-fold. This stimulation is found to be dose-dependent with a half maximal activation at 1 nM. In this case, EGF increased Vmax without changing Km Value for ATP or DG. Although this activation was observed in the absence of detergent, it was more evident when membrane vesicles were treated with 1 mM deoxycholate. Interestingly, the effect of EGF was only detected in magnesium containing medium. The use of manganese instead of magnesium diminished the stimulatory effect in either condition, presence or absence of deoxycholate. On the other hand, the stimulation of PI kinase or PI(4)P kinase activity was not caused by EGF. These results suggest that DG kinase activation by EGF makes important roles in cellular responses leading to cell growth.     

Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells

In order to clarify the mechanism(s) by which cyclic GMP inhibits the generation of inositol phosphates in rat aorta segments and cultured bovine aortic smooth muscle cells, we studied phosphoinositide hydrolysis and GTPase activity in homogenates and membrane preparations of cultured bovine aortic smooth muscle cells. Pretreatment of homogenate preparations with cyclic GMP plus ATP did not inhibit [8-arginine, 3H] vasopressin (AVP) binding, but resulted in a total suppression of the AVP-induced GTPase activation. The pretreatment with cyclic GMP and ATP also inhibited the formation of inositol phosphates induced by AVP in the presence of low concentrations of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), or by high concentrations of GTP gamma S alone. However, the formation of inositol phosphates by high concentrations of Ca2+ alone was not blocked. These results suggest that the ability of cyclic GMP to inhibit phosphoinositide hydrolysis results from an inhibition of a guanine nucleotide regulatory protein activation, and the interaction between guanine nucleotide regulatory protein and phospholipase C. While the precise site of this inhibition is not presently known, the inhibition by cyclic GMP is dependent upon the addition of ATP and probably entails a phosphorylation event since adenylylimidodiphosphate can not substitute for the ATP requirement.     

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Protein phosphorylation and the dependence on Ca2+ and GTP-gamma-S for exocytosis from permeabilised mast cells

Exocytosis in permeabilised mast cells requires only that the concentrations of Ca2+ and GTP-gamma-S (the essential effectors) are elevated into the micromolar range of concentrations. These act through an unidentified Ca2(+)-binding protein and an uncharacterized G-protein (GE). There is no requirement for ATP in the final stages of the secretory pathway. However, mast cells permeabilised in the absence of ATP rapidly become refractory to stimulation due to a reduction in the affinity for the essential effectors. Here, we show that responsiveness may be restored by the addition of ATP. The characteristics of such ATP-dependent secretion have been examined. Preincubation (prior to permeabilization) of the cells with phorbol ester enhances affinity to Ca2+, and introduction of neomycin reduces Ca2+ affinity. AMG.C16, an ether-linked analogue of diglyceride, inhibits secretion in a manner which can be partially reversed by elevating the concentration of ATP. These observations indicate that while protein phosphorylation does not comprise a step in the triggering of exocytosis, a primed condition most likely involving a state of protein phosphorylation, and maintained by reactions catalysed by protein kinase C, is essential.     

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils.

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.     

Phorbol ester inhibits polyphosphoinositide phosphodiesterase activity stimulated by either Ca2+, fluoride or GTP analogue in HL60 membranes and in permeabilized HL60 cells

The effect of PMA (phorbol 12-myristate, 13-acetate) on PPI-pde (polyphosphoinositide phosphodiesterase) activity in the promyelocytic cell-line HL60 was examined. HL60 cells were pretreated with PMA in a time- and concentration-dependent manner and PPI-pde activity was monitored both in streptolysin O-permeabilized cells and in membranes. PPI-pde activity was stimulated by either GTP gamma S (guanosine 5'-[gamma-thio]triphosphate), fluoride or Ca2+. Both the Ca2(+)-stimulated and the G protein-mediated PPI-pde activity in permeabilized HL60 cells is maximally inhibited (70-90%) after 60 min pretreatment of intact cells with 10nM PMA. PPI-pde activity can also be observed in membranes prepared from HL60 cells although this activity represents only 10% of the total activity seen in permeabilized cells. In membranes, where PPI-pde activity can also be stimulated by either via the G-protein or directly by Ca2+, PMA pretreatment was also inhibitory regardless of the mode of activation. We suggest that both the membrane-bound PPI-pde activity and that present in the permeabilized cells are targets for protein phosphorylation by protein kinase C leading to inhibition of the catalytic function.     

Copper and signal transduction: platelets as a model to determine the role of copper in stimulus-response coupling.

Platelets from copper-deficient rats have been used as a model to investigate the role of copper in receptor-mediated cellular responses. Copper deficiency doubles the rate of dense granule secretion and increases myosin association with the platelet cytoskeleton following thrombin stimulation. Mechanisms underlying the effects of copper deficiency on thrombin-induced signals that elicit dense granule secretion involve suppression of protein kinase C activity and impairment of Ca2+ release from intracellular stores. Copper deficiency also reduces the cellular GTP content of platelets. This may limit receptor effector coupling through GTP-dependent regulatory proteins leading to protein kinase C activation and the release of Ca2+ from intracellular stores. The reduction in GTP content during copper deficiency results from its utilization to maintain cellular ATP levels in response to severely inhibited cytochrome c oxidase activity in platelet mitochondria. Thus, the role of copper in maintaining normal signal transduction may be indirectly related to its biological function in mitochondria.     

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.     

Rho controls cortical F-actin disassembly in addition to, but independently of, secretion in mast cells

Localized disassembly of cortical F-actin has long been considered necessary for facilitation of exocytosis. Exposure of permeabilized mast cells to calcium/ATP induces cortical F-actin disassembly (calmodulin-dependent) and secretion (calmodulin-independent). The delay in the onset of secretion is characteristic for the calcium/ATP response and is abolished by GTP. Here we report that a constitutively active mutant of Rho (V14RhoA) enhanced both secretion and cortical F-actin disassembly. In addition, V14RhoA mimicked GTP by abolishing the delay in secretion. Inhibition of Rho by C3 transferase prevented both secretion ( approximately 80%) and F-actin disassembly (approximately 20%). Thus, both Rho GTPase and calcium/calmodulin contribute to the control of cortical F-actin disassembly. Stabilization of actin filaments by high concentrations of phalloidin or by a calmodulin-inhibitory peptide (based on the calmodulin-binding domain of myosin light chain kinase) did not affect the extent of secretion or the secretion-enhancing effects of V14RhoA. These results further support the existence of divergent, Rho-dependent, pathways regulating actin and exocytosis. Furthermore, compound Y-27632, a specific inhibitor of Rho-associated protein kinase (p160(ROCK)), attenuated the Rho-induced loss of cortical F-actin without affecting secretion. A model is presented in which Rho regulates secretion and cortical F-actin in a manner dependent on and/or synergistic with calcium.     

Calcium- and guanine-nucleotide-dependent exocytosis in permeabilized rat mast cells. Modulation by protein kinase C.

We have used a digitonin-permeabilized cell system to study the signal transduction pathways responsible for stimulus-secretion coupling in the rat peritoneal mast cell. Conditions were established for permeabilizing the mast cell plasma membrane without disrupting secretory vesicles. Exocytotic release of histamine from digitonin-permeabilized cells required a combination of micromolar concentrations of Ca2+ and the stable guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]), but was independent of exogenous ATP. In the presence of 40 microM-GTP[S], exocytosis was half-maximal at 1.3 microM-Ca2+ and maximal at 10 microM-Ca2+; GTP[S] alone (100 microM) had no effect on histamine release in the absence of added Ca2+. In the presence of 10 microM free Ca2+, 5 microM-GTP[S] was required for half-maximal exocytosis. To examine the possible role of protein kinase C (PKC) in exocytosis, we utilized 12-O-tetradecanoylphorbol 13-acetate (TPA) to activate PKC and studied its effect on histamine release from permeabilized mast cells. Cells that had been incubated with TPA (25 nM for 5 min) exhibited increased sensitivity to both GTP[S] and Ca2+. The PKC inhibitor staurosporine blocked the effect of TPA without inhibiting normal exocytosis in response to the combination of GTP[S] and Ca2+. In addition, down-regulation of mast-cell PKC by long-term TPA treatment (25 nM for 20 h) blocked the ability of the cells to respond to TPA and inhibited exocytosis in response to Ca2+ and GTP[S] by 40-50%. These results suggest that the sensitivity of the exocytotic machinery of the mast cell can be altered by PKC-catalysed phosphorylation events, but that activation of PKC is not required for exocytosis to occur.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.     

Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes.

We studied the regulation of arachidonic acid (AA) release by guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) and Ca2+ in electropermeabilized HL60 granulocytes. Stimulation of AA release by GTP gamma S and Ca2+ was mediated by phospholipase A2 (PLA2) and required the presence of MgATP (EC50: 100-250 microM). The nucleotide effects were Ca(2+)-dependent (maximal effects detected at 1 microM free cation). UTP and ATP gamma S, which stimulate AA release in intact HL60 granulocytes with potencies and efficacies similar to those of ATP, were ineffective in supporting the effects of GTP gamma S in electropermeabilized cells. Pretreatment with pertussis toxin affected stimulation of AA release by ATP in intact cell, without altering the nucleotide effects in permeabilized cells. We observed the protein kinase C-dependent phosphorylation of PLA2 in permeabilized HL60 granulocytes, together with a correlation between the effects of phorbol esters and staurosporine on this reaction and on AA release. ATP-independent activation of PLA2 by GTP gamma S and/or Ca2+ was measured in subcellular fractions prepared from HL60 granulocytes. These data appear consistent with a model in which PLA2 activity in resting HL60 granulocytes is subjected to an inhibitory constraint that prevents its activation by Ca2+ and G-proteins. Removal of this constraint, either by the protein kinase C-dependent phosphorylation of the enzyme in vivo or physical disruption of the regulatory assembly (e.g. by N2 cavitation), allows its activation by Ca2+ and G-proteins.