The actions of Ca2+ ionophores on rat basophilic (2H3) cells are dependent on cellular ATP and hydrolysis of inositol phospholipids. A comparison with antigen stimulation

Calcium-specific ionophores are used widely to stimulate Ca2+-dependent secretion from cells on the assumption that permeabilization of the cell membranes to Ca2+ ions leads to a rise in concentration of cytosolic Ca2+ ([Ca2+]i), which in turn serves as a signal for secretion. In this way, events that precede mobilization of Ca2+ ions via receptor stimulation are bypassed. One such event is thought to be the rapid hydrolysis of membrane inositol phospholipids to form inositol phosphates and diacylglycerol. Accordingly, rat leukemic basophil (2H3) cells can be stimulated to secrete histamine either with the ionophores or by aggregation of receptors for IgE in the plasma membrane. We find, however, that ionophore A23187 stimulates secretion of histamine only at concentrations (200-1000 nM) that stimulate hydrolysis of membrane inositol phospholipids. The extent of hydrolysis of inositol phospholipids was dependent on the concentration of ionophore and the presence of external Ca2+ ions and correlated with the magnitude of the secretory response. A similar correlation between secretion and hydrolysis of inositol phospholipids was observed in response to the Ca2+-specific ionophore, ionomycin. Although this hydrolysis (possibly a consequence of elevated [Ca2+]i) was less extensive than that induced by aggregation of receptors, it may govern the secretory response to A23187. The studies revealed one paradox. The rise in [Ca2+]i depended on intracellular ATP levels, when either an ionophore or antigen was used as a stimulant irrespective of whether hydrolysis of inositol phospholipids was stimulated or not. The concept of how the ionophores act, therefore, requires critical reevaluation.     

Quantitative relationships between aggregation of IgE receptors, generation of intracellular signals, and histamine secretion in rat basophilic leukemia (2H3) cells. Enhanced responses with heavy water

RBL-2H3 cells (a tumor analog of rat mast cells) have plasma-membrane receptors that bind immunoglobulin E, which when aggregated, initiate degranulation. As in other systems, secretion is preceeded by enhanced hydrolysis of inositol phospholipids and by a rise in intracellular Ca2+. Unlike the responses of many other cells, however, both of these earlier events require extracellular Ca2+. The relationship of these events to each other and to the subsequent secretory process is thus unclear. By exposing cells to covalent oligomers of IgE one can demonstrate substantial increases in secretion of histamine by increasing the concentration and size of the oligomers or by using heavy water (D2O) in the medium. We have used such maneuvers to examine the quantitative relationships between aggregation of the receptors and the breakdown of inositol phospholipids, the increase in cytosolic Ca2+ and secretion. Our principal findings were: all treatments that increased secretion, correspondingly increased the changes that precede degranulation. These early events correlated with the degree of aggregation of the receptors even when the stimulatory conditions resulted in maximal secretion. Although the results were insufficient to prove that the hydrolysis of inositol phospholipids is required for the rise in cytosolic Ca2+, the studies with D2O and other observations supported this view. Since a plasma-membrane ion channel for Ca2+ has been implicated in the IgE-mediated rise in cytosolic Ca2+ in RBL 2H3 cells, this in turn suggests a heretofore undescribed role for hydrolysis of inositol phospholipids.     

Identification of variants of the basophilic leukemia (RBL-2H3) cells that have defective phosphoinositide responses to antigen and stimulants of guanosine 5'-triphosphate-regulatory proteins

Antigen immunoglobulin E-mediated secretion of histamine from RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and a rise in the concentration of cytosol Ca2+ (calcium signal). Such responses differed among cloned variant lines of the RBL-2H3 cell line from undetectable (1A3 bromodeoxyuridine-resistant (BUDRR), 2B1 BUDRR, and 1B3 BUDRR lines) to about 80% of those in the parent RBL-2H3 cells. In all but one line (1B3 thioguanine-resistant (TgR)), the intensities of the phosphoinositide response and of the calcium signal were correlated with the secretory response. The 1B3 TgR line had no detectable calcium signal (as measured by quin 2 fluorescence or uptake of 45Ca2+) but paradoxically showed modest rates of hydrolysis of inositol phospholipids and of secretion. The responses of the 1B3 TgR line were, however, dependent on the presence of external Ca2+ ions. The induction of secretion with antigen, therefore, was invariably associated with the hydrolysis of inositol phospholipids, but it was not necessarily associated with a change in concentration of cytosol Ca2+. All antigen unresponsive clones could secrete when synergistic signals were induced by exposure to the Ca2+ -ionophore, A23187 and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. These lines, otherwise, had immunoglobulin E receptors and had no obvious defect in their capacity to synthesize the inositol phospholipids or in their phenotypic expression of phospholipase C as measured in cell extracts. One finding of possible relevance to the role of guanosine 5'-triphosphate-regulatory proteins in the activation of phospholipase C was the inability of one antigen-nonresponsive line to respond to NaF (in intact cells) or to guanosine 5'-(3-O-thio)triphosphate (in electrically permeabilized cells).     

The rise in concentration of free Ca2+ and of pH provides sequential, synergistic signals for secretion in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Ag stimulation of rat basophilic leukemia (RBL-2H3) cells results in hydrolysis of inositol phospholipids, a transient increase in concentration of cytosol Ca2+ [( Ca2+]i), a gradual increase in cytosolic pH (pHi) and the activation of protein kinase C. To determine whether all these changes serve as signals for secretion, studies were conducted with cells permeabilized with streptolysin O in which pHi and [Ca2+]i could be varied independently of each other and enzyme activities could be manipulated. At resting pHi (approximately 7.0) and [Ca2+]i (0.1 microM), the permeabilized cells showed little secretory response to Ag. At resting pHi, elevated levels of Ca2+ (0.33 microM) were required for maximal secretory response to Ag. At a pHi of 7.4, however, 0.1 microM [Ca2+]i was sufficient to sustain near maximal responses to Ag. Therefore, a small increase of [Ca2+]i to 0.33 microM was required to initiate secretion, but once the pHi was elevated secretion could be sustained at near basal levels of [Ca2+]i. Since elevating the [Ca2+]i and pHi, by themselves promoted little secretion, another potentiating signal must have been generated by antigen stimulation. This signal was possibly transduced via hydrolysis of inositol phospholipids and protein kinase C. Even with an elevated [Ca2+]i (0.33 microM) the hydrolysis of the phospholipids and secretion stimulated by Ag were inhibited by guanosine 5'(2-O-thio)diphosphate and neomycin. Furthermore, both protein-kinase C and the secretory response to Ag were lost after permeabilized cells were washed but both were retained if cells were exposed to PMA before permeabilization.     

Calcium-independent phosphoinositide breakdown in rat basophilic leukemia cells. Evidence for an early rise in inositol 1,4,5-trisphosphate which precedes the rise in other inositol phosphates and in cytoplasmic calcium

Aggregation of the receptor with high affinity for immunoglobulin E (IgE) in rat basophilic leukemia cells leads to a calcium-dependent and a calcium-independent hydrolysis of phosphoinositides. The increase in the levels of inositol phosphates induced in the absence of calcium is only 25% of that observed with 1 mM Ca2+. The inositol phosphates reach a new steady state level 2 min after stimulation in EGTA, whereas with calcium they continue to increase up to 15 min. A similar response is observed when the receptors are aggregated due to the interaction of bound IgE with antigen or with anit-IgE, or by the binding of IgE cross-linked chemically. The antigen-mediated response is inhibited by hapten and disruption of such antigen-antibody aggregates late after stimulation leads to a rapid decline in the levels of the inositol phosphates to basal values. Separation of the inositol phosphates by Dowex columns shows that there is a fast rise in inositol trisphosphate which peaks at 15 s and slowly declines to a lower plateau within 2 min. Analysis by high pressure liquid chromatography reveals a 5-fold increase in the levels of inositol 1,4,5-trisphosphate in less than 10 s after stimulation, which precedes any major change in the other inositol phosphates. Aggregation of the receptor in the absence of external calcium induces a transient increase in cytoplasmic calcium which reaches a maximum of approximately 25 nM over basal levels after activation. The onset of the rise in Ca2+ lags after the initial rise in the inositol 1,4,5-trisphosphate.     

Studies of protein kinase C in the rat basophilic leukemia (RBL-2H3) cell reveal that antigen-induced signals are not mimicked by the actions of phorbol myristate acetate and Ca2+ ionophore

Exogenous activators of protein kinase C such as PMA in combination with a Ca2+ ionophore (A23187), cause secretion in rat basophilic (RBL-2H3) cells,but they do so through stimulatory signals that are not the same as those generated by Ag or oligomers of IgE. On the one hand, the synergy between PMA and A23187 and the suppression of Ag-mediated signals (hydrolysis of inositol phospholipids and rise in concentration of cytosolic Ca2+) by PMA were totally dependent on protein kinase C. The loss of synergistic and inhibitory actions of PMA, for example, correlated with the loss of protein kinase C (as determined by immunoblotting techniques) when cells were continuously exposed to PMA. Furthermore, the permeabilization of RBL-2H3 cells resulted in the loss of both protein kinase C and the inhibitory action of PMA, but both were retained if cells were exposed to PMA before permeabilization Ag-induced secretion, on the other hand, was not as dependent on the presence of protein kinase C. The potent inhibitor of this enzyme, staurosporine, which blocked completely the secretory response to the combination of PMA and A23187, did not inhibit Ag-induced secretion except at concentrations (greater than 10 nM) that inhibited Ag-stimulated hydrolysis of inositol phospholipids as well. Also RBL-2H3 cells still showed some secretory-response (approximately 25% of normal) to Ag when cells were depleted (greater than 98%) of protein kinase C by prolonged treatment with PMA. Previous studies have indicated that the secretory response to PMA and A23187 is much lower than that elicited by Ag when the concentrations of stimulants were matched to give the same increase in concentrations of cytosolic Ca2+.     

On the mechanism of action of dexamethasone in a rat mast cell line (RBL-2H3 cells). Evidence for altered coupling of receptors and G-proteins

Prolonged exposure of rat basophilic leukemia (RBL-2H3) cells, a cultured analog of rat mast cells, to 0.1 microM dexamethasone resulted in global suppression of various stimulatory events in response to Ag and a global enhancement of the same stimulatory events to the adenosine analog, N-(ethylcarboxamide)adenosine (NECA). We had previously shown that Ag and NECA both activate phospholipase C but by different mechanisms; cells that had been treated with cholera or pertussis toxin, for example, responded to Ag but not to NECA with the release of inositol phosphates, increase in levels of cytosolic Ca2+, and secretion. Because the toxins still inhibited the responses to NECA in dexamethasone-treated cells, the effects of dexamethasone may have been exerted at the level of receptor/G-protein coupling rather than at the level of effector systems. Additional evidence for this was the following: 1) NECA-induced hydrolysis of the inositol phospholipids was still enhanced after permeabilizing (with streptolysin O or Staphylococcus alpha-toxin) and washing the cells; 2) the response to the G-protein stimulant, guanosine 5'-(3-O-thio)triphosphate was also enhanced in permeabilized, dexamethasone-treated cells and 3) binding and kinetic studies suggested that the enhanced responsiveness to NECA was attributable in part to an increase in receptor number. The suppressive action of dexamethasone on Ag-induced hydrolysis of inositol phospholipids, however, was readily lost by permeabilizing RBL-2H3 cells. The results indicate, therefore, that treatment with dexamethasone leads to changes in receptor-coupling mechanisms that are either resistant to (i.e., NECA-mediated responses) or reversed by (i.e., Ag-mediated responses) cell permeabilization.     

Pertussis toxin does not inhibit muscarinic-receptor-mediated phosphoinositide hydrolysis or calcium mobilization.

Pertussis toxin was used to examine the role of the inhibitory guanine nucleotide regulatory protein, Ni, in muscarinic-receptor-mediated stimulation of phosphoinositide turnover and calcium mobilization. In cultured chick heart cells, pertussis-toxin treatment inhibited muscarinic-receptor-mediated attenuation of isoprenaline-stimulated cyclic AMP accumulation. This finding is consistent with the proposal that pertussis toxin blocks the capacity of Ni to couple muscarinic receptors to adenylate cyclase. In contrast, treatment of chick heart cells or 1321N1 human astrocytoma cells with pertussis toxin did not block muscarinic-receptor-mediated stimulation of phosphoinositide hydrolysis, as measured by [3H]inositol phosphate accumulation in the presence of Li+. Pertussis-toxin treatment also had little effect on basal and muscarinic-receptor-stimulated phosphatidylinositol synthesis, as measured by the incorporation of [3H]inositol into phosphatidylinositol. Activation of muscarinic receptors also enhances the rate of unidirectional 45Ca2+ efflux in 1321N1 cells; this response, like phosphoinositide hydrolysis, was not prevented by pertussis-toxin treatment. Our data suggest that muscarinic receptors are not coupled to phosphoinositide hydrolysis or calcium mobilization through Ni.     

Treatment with dexamethasone down-regulates IgE-receptor-mediated signals and up-regulates adenosine-receptor-mediated signals in a rat mast cell (RBL-2H3) line

As reported by other workers, the treatment of rat basophilic leukemia RBL-2H3 cells with dexamethasone resulted in a marked decrease in responsiveness to Ag-stimulation. All responses measured, which included hydrolysis of inositol phospholipids, increase in concentration of cytosolic Ca2+, release of arachidonic acid and the secretion of serotonin, were suppressed, but once the cells were permeabilized the inhibitory actions of dexamethasone were no longer apparent. This suggested that all the necessary components of the stimulatory cascade were intact in the dexamethasone-treated cells. The measurement of phospholipase C activity in cell extracts and studies with phorbol ester also indicated that the cells contained a normal complement of phospholipase C and protein kinase C activity. We had previously shown that both Ag and the adenosine analog, 5'-(N-ethylcarboxamide)-adenosine, can activate phospholipase C, but they do so through different G proteins. Interestingly, the activation of phospholipase C by 5'-(N-ethylcarboxamide)-adenosine and the ensuing stimulatory events were markedly enhanced in dexamethasone-treated cells. The treatment with dexamethasone thus did not result in direct suppression of effector systems, but instead resulted in the selective modulation of the coupling between receptors and the effector systems by mechanisms that require soluble cytosolic factors.     

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.     

IgE receptor-activated calcium permeability pathway in rat basophilic leukemia cells: measurement of the unidirectional influx of calcium using quin2-buffered cells

The intracellular calcium indicator and buffer quin2 has been used to generate a large calcium buffering capacity in the cytoplasm of rat basophilic leukemia cells. Above 3 mM intracellular quin2, there is no further increase in the initial rate of antigen-induced 45Ca uptake, suggesting that 45Ca buffering by quin2 is now sufficient to prevent the immediate efflux of 45Ca from the cells. Thus, the initial rate of 45Ca uptake should reflect the true unidirectional influx of calcium that occurs when immunoglobulin E (IgE) receptors are aggregated by antigen. The antigen-induced calcium permeability pathway appears to be saturable, with a Km of about 0.7 mM and a Vmax of 0.9 nmol of calcium (10(6) cells)-1 min-1. Although net 45Ca uptake reaches a plateau a few minutes after antigen stimulation, the increase in plasma membrane permeability is maintained for at least an hour, provided that receptors for IgE remain aggregated. The initial rate of 45Ca influx correlates well with the subsequent secretion of [3H]serotonin in response to different concentrations of antigen. Both 45Ca uptake and [3H]serotonin secretion are maximal when only 10% of the receptors are occupied with antigen-specific IgE. Thus, 45Ca influx correlates more closely with secretion than with the number of IgE receptors aggregated by antigen.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.     

IgE receptor-mediated hydrolysis of phosphoinositides by cytoplasts from rat basophilic leukemia cells

Cytoplasts (plasma membrane sacs containing cytoplasm, endoplasmic reticulum, and few organelles) were prepared from rat basophilic leukemia cells by treatment with cytochalasin B and centrifugation at 33 degrees C through stepwise gradients of Ficoll. To compare the relative ability of cytoplasts and cells to generate second-messengers (inositol phosphates, Ca2+) in response to stimulation of the high affinity receptor for IgE, we normalized our results per recovered receptor by using the tightly bound IgE as a marker. This marker correlated well with other estimates of plasma membrane recovery. Furthermore, data normalized on this basis correlated well with data expressed as percentage of phosphoinositides hydrolyzed. The purest fraction of cytoplasts (containing about 6% of the receptors) was satisfactorily devoid of organelles and, at early times, generated about 50% as much inositol phosphates per receptor as did the intact, untreated cells. This response of the cytoplasts, like that of the cells, was totally dependent upon aggregation of the receptors. The response by the cytoplasts (in the 5-min time frame which we examined), unlike that of the cells, was not enhanced by the presence of extracellular Ca2+. Furthermore, unlike the cells, the cytoplasts failed to raise their intracellular free Ca2+ levels after addition of polyvalent Ag. This result suggests that aggregation of the receptors may be insufficient, by itself, to open the normal Ca2+ channels.     

Modulation of Phosphoinositide Hydrolysis by NaF and Aluminum in Rat Cortical Slices

NaF stimulated phosphoinositide hydrolysis in rat cortical slices. The production of [3H]inositol monophosphate was rapid for the first 15 min of incubation with NaF, followed by a plateau. The major product detected was [3H]inositol monophosphate, although significant amounts of [3H]inositol bisphosphate and [3H]inositol trisphosphate were also produced. The stimulation of [3H]inositol monophosphate production by NaF was concentration dependent between 2 and 20 mM NaF. Addition of 10 or 100 microM AlCl3 or aluminum maltol did not alter the effect of NaF, whereas at 500 microM, these aluminum preparations resulted in significant inhibition. Increasing the concentration of K+ from 5 to 20 mM potentiated [3H]inositol monophosphate production induced by carbachol but not by NaF. Incubation with 1 microM phorbol 12-myristate 13-acetate, a phorbol ester, inhibited carbachol-induced, but not NaF-induced, [3H]inositol monophosphate production. These results further support the hypothesis that a guanine nucleotide binding protein that can be activated by NaF is involved in phosphoinositide hydrolysis in brain. The use of NaF provides a means to bypass receptors to study intracellular regulatory sites of phosphoinositide metabolism without disrupting cells.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

Astrocytes possess prostaglandin F2 alpha receptors coupled to phospholipase C.

We examined the effect of prostaglandin (PG) F2 alpha on phosphoinositide (PI) hydrolysis in rat cultured astrocytes. PGF2 alpha stimulated the formation of [3H]inositol phosphates in [3H]inositol-labeled astrocytes with the ED50 value of 23 nM, whereas PGD2 and PGE2 were much less effective than PGF2 alpha. Transformation of astrocytes was accompanied by an increase in the stimulatory response of PGF2 alpha. Pretreatment of the astrocytes with pertussis toxin and cholera toxin did not affect the PGF2 alpha-evoked PI hydrolysis. In the digitonin-permeabilized astrocytes, PGF2 alpha significantly enhanced the GTP gamma S-evoked PI hydrolysis in the presence of Ca2+. These results indicate that rat cultured astrocytes possess PGF2 alpha receptors coupled to phospholipase C.     

Hydrolysis of inositol phospholipids induced by stimulation of the T cell antigen receptor complex in antigen-specific, murine helper T cell clones. Requirement for exogenous calcium

Two murine, keyhole limpet hemocyanin-specific, Th cell clones were studied for their ability to respond to antibody-mediated stimulation of the TCR complex or to Ag-pulsed accessory cells by hydrolyzing inositol phospholipids. Both clones were positive for the determinant expressed on the epsilon chain of CD3 that is recognized by the mAb, 145-2C11 (2C11 mAb); one clone also expressed the V beta 8 epitope of the alpha/beta chains of the TCR recognized by the F23.1 mAb. Treatment of these cells with 2C11 or F23.1 mAb adsorbed onto polystyrene beads induced a time-dependent accumulation of inositol phosphates (IP). Keyhole limpet hemocyanin-pulsed accessory cells which expressed the appropriate MHC phenotype also induced IP accumulation, whereas no response was induced by medium-treated or MHC congenic accessory cells. The hydrolysis of inositol phospholipids induced by TCR perturbation depended upon the presence of exogenous Ca2+; Mg2+ did not substitute for Ca2+. Treatment of cells with ionomycin at concentrations up to 30 microM was unable to induce hydrolysis of inositol phospholipids, indicating that entrance of Ca2+ was itself insufficient to generate IP. Stimulated IP generation was rapidly blocked upon addition of EGTA to the incubation medium. Reducing the level of exogenous Ca2+ decreased the production of inositol mono-, bis-, and trisphosphate isomers similarly, suggesting that extracellular Ca2+ was required for the initiation of the hydrolysis rather than affecting phospholipase C affinity for its substrates. We concluded that activation of inositol phospholipid hydrolysis by perturbation of the TCR complex in the Th cell clones under investigation displays a Ca2+-dependent component which is likely to be proximal to IP generation.