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Aggregation of the high affinity IgE receptor (FceRI) in a mast cell line resulted in activation of the p42 and the stress-activated p38 mitogen-activated protein (MAP) kinases. Selective inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to the production of the cytokine, tumor necrosis factor-α, and the release of arachidonic acid in these cells. Neither kinase, however, was essential for FceRI-mediated degranulation or constitutive production of tumor growth factor-β. Studies with SB 203580 and the p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase.

Antigen receptors on T- and B-cells activate Ras through a signaling pathway that results in the tyrosine phosphorylation of Shc and the formation of a complex of Shc with the Grb2 adaptor protein. The high affinity receptor for immunoglobulin E (FcepsilonRI) in cultured mast (RBL-2H3) cells has been reported to function differently. Here we show to the contrary that engagement of FcepsilonRI with antigen leads to increased tyrosine phosphorylation of Shc and the association of Shc with Grb2 and other proteins (p120 and p140). Like the FcepsilonRI-mediated activation of the mitogen-activated protein kinase cascade, these responses are dependent on the tyrosine kinase Syk; they are enhanced by overexpression of Syk and are blocked by expression of dominant-negative Syk. Sos is constitutively associated with Grb2 in these cells but dissociates from Shc on stimulation with antigen. These reactions are rapid, reversible, and associated with the activation of Ras. Therefore, the Syk-dependent tyrosine phosphorylation of Shc and its association with Grb2 may provide a pathway through Sos for activation of Ras by FcepsilonRI.

Stimulation of the mast cell line, RBL-2H3, with antigen via the tetrameric (alpha beta gamma 2) immunoglobulin E receptor (Fc epsilon R1) leads to the activation of cytosolic phospholipase A2 and the release of arachidonic acid. This pathway is dependent on the activation of the mitogen-activated protein (MAP) kinase. In this paper, we show that the MAP kinase/cytosolic phospholipase A2 pathway is linked to Fc epsilon R1 via the cytosolic tyrosine kinase, Syk, and that the GDP/GTP exchange factor, Vav, might be one candidate for accomplishing this link. Cross-linking of transmembrane chimeras containing the Fc epsilon R1 gamma motif, which is known to activate Syk, results in the tyrosine phosphorylation of Vav, activation of MAP kinase, and release of arachidonic acid. Cross-linking of chimeras containing the Fc epsilon R1 beta motif does not cause these events. Furthermore, stimulation of these events by antigen is enhanced by transient overexpression of a wild-type form of Syk and blocked by overexpression of a dominant negative form of Syk. By contrast, stimulation via the transfected, G protein-coupled, muscarinic m1 receptor is not influenced by either form of Syk and does not result in tyrosine phosphorylation of Vav. These data establish unequivocally that the two types of receptor are independently linked to the two types of receptor are independently linked to the MAP kinase/cytosolic phospholipase A2 pathway and demonstrate the existence of the Fc epsilon R1-Syk-MAP kinase pathway.

The phosphorylation of myosin light chains and heavy chains by protein kinase C is known to be temporally correlated with Ca(2+)-dependent secretion of granules from RBL-2H3 cells (Ludowyke, R. I., Peleg, I., Beaven, M. A., and Adelstein, R. S. (1989) J. Biol. Chem. 264, 12492-12501). We now report that whereas myosin light chains are predominantly monophosphorylated by the Ca2+/calmodulin-dependent myosin light chain kinase at serine 19 in unstimulated cells, stimulation of RBL-2H3 cells with antigen or other stimulants causes additional phosphorylation of myosin light chains by myosin light chain kinase at threonine 18, as well as by protein kinase C at serine 1 or serine 2. This diphosphorylation at serine 19 and threonine 18 by myosin light chain kinase and the monophosphorylation by protein kinase C is correlated with the rate and extent of degranulation. Secretion occurs whenever phosphorylation by both enzymes is stimulated by antigen or by the combination of low concentrations of A23187 (50 nM) and phorbol 12-myristate 13-acetate (20 nM). These phosphorylations appear to be closely associated with exocytosis in RBL-2H3 cells. Thus, phosphorylation, as well as secretion, can be blocked by the kinase inhibitors KT5926 and ML-7. More specifically, phorbol ester alone induces phosphorylation of myosin light chains by protein kinase C exclusively, but fails to induce secretion until accompanied by low concentrations of A23187, which activates myosin light chain kinase. Conversely, selective suppression of phosphorylation by protein kinase C (with Ro31-7549 in antigen-stimulated cells) suppresses degranulation, thereby indicating a requirement for protein kinase C.

Several inhibitors of tyrosine phosphatases, which included vanadate/H2O2, phenylarsine oxide, and diamide, blocked exocytosis in basophilic RBL-2H3 cells that had been transfected with the gene for the muscarinic m1 receptor. Because this block was observed whether the secretagogue acted through receptors (i.e. antigen and the muscarinic agonist, carbachol) or by direct activation of intracellular mechanisms (i.e. A23187, A23187 in combination with phorbol 12-myristate 13-acetate, and thapsigargin), the inhibitors appeared to act at a step distal to the mobilization of Ca2+ and activation of protein kinase C. All secretagogues caused the tyrosine phosphorylation of a 40-kDa protein, whereas the inhibitors caused a hyperphosphorylation of this protein. Therefore, both tyrosine kinase and phosphatase activities appear to regulate this phosphorylation which may, in turn, regulate secretion. The 40-kDa protein was identified as a mitogen-activated protein kinase-like protein on the basis of its reactivity to anti-mitogen-activated protein kinase antibodies. In addition, when cells were stimulated the tyrosine phosphorylated and the immunoreactive protein comigrated as a doublet on one-dimensional and as multiple phosphorylated forms on two-dimensional gel-electrophoretic systems.

Mast cells release the mediators of the immediate hypersensitivity reaction. Adenosine is known to modulate this process, but the receptor responsible for this is not the classical A1 or A2 adenosine receptors. This study was undertaken to determine whether the unique adenosine receptor (AR) previously postulated in a cultured mast cell line (RBL-2H3 cells) is the recently cloned A3AR. The receptors were quantitated by the agonist 125I-labeled APNEA (aminophenylethyladenosine), an A3AR agonist, which yielded Bmax and Kd values of 826 fmol/mg protein and 34 nM, respectively. A variety of adenosine analogs competed for 125I-APNEA binding sites with the following potency series: (R)-phenylisopropyladenosine = 5'-N-ethylcarboxamide adenosine > (S)-phenylisopropyladenosine. 125I-APNEA binding was relatively insensitive to the xanthine amine congener (XAC, 1 microM), a selective antagonist for the A1AR. Functionally, activation of these A3AR stimulated the production of inositol 1,4,5-triphosphate, leading to an increase in the level of intracellular Ca2+. Furthermore, while activation of these receptors alone produced little secretory response in RBL-2H3 cells, it enhanced antigen-induced secretion by 2-2.5-fold. Northern blotting studies using poly(A+) RNA from RBL-2H3 cells detected two transcripts of 2.0 and 3.5 kilobases, which hybridized to an A3AR cDNA but not to the A1 or A2AR cDNA probes. These data indicate that the unique AR that potentiates the secretory response to antigen in RBL-2H3 cells is exclusively the A3AR.

Previous studies indicated that rat basophilic RBL-2H3 cells contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C (PKC); of these, PKC beta and delta were the most potent transducers of signals for exocytosis in antigen-stimulated permeabilized cells. Exocytosis, nevertheless, was still dependent on an elevated free Ca2+. (Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., and Beaven, M. A. (1993) J. Biol. Chem. 268, 1749-1756). We now demonstrate that PKC alpha and epsilon, exclusively, inhibit antigen-induced hydrolysis of inositol phospholipids in the same permeabilized RBL-2H3 cells. Unlike secretion, the inhibitory actions occurred at a basal concentration (0.1 microM) of free Ca2+. The inhibitory actions of the two isozymes were potentiated by 20 nM phorbol 12-myristate 13-acetate. As indicated by the effects of the phorbol ester, the probable mechanism was reduced tyrosine phosphorylation of phospholipase C gamma 1. The negative regulation of phospholipase C was apparent in intact cells, because the PKC inhibitor Ro31-7549 or down-regulation of PKC with phorbol ester enhanced antigen-induced hydrolysis of inositol phospholipids. The concentrations of the various isozymes of PKC in RBL-2H3 cells, as estimated by immunoblotting studies, were sufficient for promotion of exocytosis (i.e. beta and delta) and inhibition of phospholipid hydrolysis (i.e. alpha and epsilon).

Rat basophilic RBL-2H3 cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen, contained the Ca(2+)-dependent alpha and beta and the Ca(2+)-independent delta, epsilon, and zeta isoforms of protein kinase C. These isoforms associated, to variable extents (i.e. delta the most and zeta the least), with the membrane fraction upon antigen stimulation but without external Ca2+; only the Ca(2+)-independent isoforms did so. Both types of isozymes were probably necessary for optimal responses to antigen as indicated by the following observations. All Ca(2+)-dependent isozymes were degraded in cells treated with 20 nM phorbol 12-myristate 13-acetate for 6 h, whereas the Ca(2+)-independent isozymes were not degraded and were retained when the cells were subsequently permeabilized and washed. Cells so treated still exhibited antigen-induced secretion (25-33% of normal) which was suppressed by selective inhibitors of protein kinase C (Ro31-7549 and calphostin C) thereby indicating a possible contribution of the Ca(2+)-independent isozymes in secretion. Normally, washed permeabilized cells lost all isozymes of protein kinase C and failed to secrete in response to antigen. A full secretory response to antigen could be reconstituted by the subsequent addition of nanomolar concentrations of either beta or delta isozymes of protein kinase C (other isozymes were much less effective) but only in the presence of 1 microM free Ca2+ to indicate distinct roles for Ca2+ and protein kinase C in exocytosis.

An increase in concentration of cytosolic Ca2+ ([Ca2+]i) is associated with an accelerated influx of 45Ca2+ when cultured RBL-2H3 cells are stimulated with either antigen or analogs of adenosine although these agents act via different receptors and coupling proteins (Ali, H., Cunha-Melo, J.R., Saul, W.F., and Beaven, M.A. (1990) J. Biol. Chem. 265, 745-753). The same mechanism probably operates for basal Ca2+ influx in unstimulated cells and for the accelerated influx in stimulated cells. This influx had the following characteristics. 1) It was decreased when cells were depolarized with high external K+; 2) it was blocked by other cations (La3+ greater than Zn2+ greater than Cd2+ greater than Mn2 = Co2+ greater than Ba2+ greater than Ni2+ greater than Sr2+) either by competing with Ca2+ at external sites (e.g. La3+ or Zn2+) or by co-passage into the cell (e.g. Mn2+ or Sr2+); and 3) the inhibition of influx by K+ and the metal ions had exactly the same characteristics whether cells were stimulated or unstimulated even though influx rates were different. The dependence of various cellular responses on influx of Ca2+ was demonstrated as follows. The stimulated influx of Ca2+, rise in [Ca2+]i, and secretion, could be blocked in a concentration-dependent manner by increasing the concentration of La3+, but concentrations of La3+ (greater than 20 microM) that suppressed influx to below basal rates of influx markedly suppressed the hydrolysis of inositol phospholipids (levels of inositol 1,4,5-trisphosphate were unaffected). Some metal ions, e.g. Mn2+ and Sr2+, however, supported the stimulated hydrolysis of inositol phospholipid and some secretion in the absence of Ca2+. Thus a basal rate of influx of Ca2+ was required for the full activation of inositol phospholipid hydrolysis, but in addition an accelerated influx was necessary for exocytosis.

5'-(N-Ethyl)carboxamidoadenosine (NECA), an analog of adenosine, transiently stimulated a rat tumor mast cell (RBL-2H3 cells) to cause a release of inositol phosphates and an increase in levels of Ca2+ in the cytosol. It failed, however, to stimulate a sustained uptake of 45Ca2+ or secretion. The effects of other agents that act on P1- or P2-purinergic receptors suggested that NECA and other adenosine agonists acted via a novel subtype of adenosine membrane receptor. Although the order of potency of agonists was characteristic of A2-adenosine receptors, there was no indication of the involvement of adenylate cyclase, and antagonists such as isobutylmethylxanthine, 8-phenyltheophylline, and 8-p-sulfophenyltheophylline inhibited the responses to either NECA or antigen. The fact that stimulation of inositol phospholipid hydrolysis by NECA in washed, permeabilized RBL-2H3 cells was blocked by pertussis toxin as well as by cholera toxin suggested instead that the NECA-sensitive receptor activated phospholipase C via a G-protein. In contrast to NECA, antigen stimulation resulted in a pertussis toxin-resistant, sustained hydrolysis of inositol phospholipids, increases in free intracellular Ca2+, accelerated influx of 45Ca2+, and secretion from RBL-2H3 cells. In combination with NECA, all responses to antigen were markedly enhanced, and the enhancement was selectively blocked by pertussis toxin. The ability of antigen, but not NECA, to provoke secretion may be dependent primarily on the sustained activation of a cholera toxin-sensitive Ca2+ influx pathway that serves to amplify stimulatory signals for secretion. These studies also suggested that phospholipase C could be activated through different G-proteins via different receptors within the same cell.

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.

Following the addition of indomethacin to exponentially growing rat hepatoma cell (HTC) cultures, cells accumulated in the G1 phase. Over the course of several hours, specific changes in membrane transport accompanied this inhibition. Indomethacin stimulated the uptake of 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), inhibited the Na+-dependent active transport of alpha-aminoisobutyric acid (AIB), and methylaminoisobutyric acid (MeAIB), but had no effect on the uptake of thymidine, uridine, or 2-deoxyglucose. The stimulation of BCH transport was immediate and reversible. The uptake of BCH was Na+-independent and only slightly depressed by metabolic inhibitors (10 to 20%). Inhibition of AIB and MeAIB uptake, which was of gradual onset, and was observed with several anti-inflammatory drugs and was dependent on the concentration of drugs. Upon removal of drug, AIB and MeAIB transport gradually increased and reached a maximum prior to resumption of DNA synthesis and cell division. Thee effects involved changes in Vmax only. Neither the apparent affinity (Km) for amino acids nor rate of exodus (k, 0.7h-1 for BCH, 1.2h-1 for AIB) were altered. The uptake of MeAIB (and AIB) occurred by a low and a high affinity (Km = 0.27 mM) component. Indomethacin inhibited specifically the high affinity component which was shown to be Na+- and energy-dependent. Although this component was inhibited by treatment with agents that lowered ATP levels or blocked protein synthesis, the anti-inflammatory drugs appeared not to act through these mechanisms.

Rat basophil leukemic (2H3) cells ( Siraganian , R.P., McGivney , A., Barsumian , E. L., Crews, F. T., Hirata , F., and Axelrod , J. (1982) Fed. Proc. 41, 30-34) loaded with fluorescent Ca2+ indicator quin 2 ( Tsien , R. Y. (1980) Biochemistry 19, 2396-2404) showed a rapid increase in free cytosol calcium concentration [( Ca]i) when histamine release was induced. Intracellular quin 2 concentrations up to 7 mM did not affect release of histamine in response to antigen (aggregated ovalbumin) or concanavalin A with cells primed with antigen-specific monoclonal IgE, or in response to Ca2+ ionophores. The [Ca]i increased from approximately 105 nM to a maximum of approximately 1200 nM within 2 to 3 min after antigenic stimulation and then declined slowly over 30 min toward the level in unstimulated cells. Histamine release was most rapid as [Ca]i reached the maximum value and then decreased continuously with [Ca]i over the subsequent 30 min. Neither the Ca signal nor histamine release was observed when the Ca2+ concentration in the medium [( Ca]o) was less than 50 microM, but both responses were restored on readdition of Ca2+ to 1 mM. The maximal Ca signal was obtained when [Ca]o was approximately greater than 1 mM and was half-maximal at [Ca]o congruent to 0.4 mM. In marked contrast [Ca]i in unstimulated cells varied very little with [Ca]o from 0.1 to 1 mM. Maintenance of the Ca signal required the continuous presence of stimulating ligand, external Ca2+, and the maintenance of cellular ATP; metabolic inhibitors blocked or reversed the Ca signal. La+ ions also caused a rapid and reversible block of the Ca signal and histamine release. The data are interpreted in a model in which the Ca signal is generated by a La3+-sensitive signal influx pathway that is functionally independent of the normal Ca2+ influx pathway in unstimulated cells, and that allows a 10-fold or greater increase in rate of Ca2+ entry. The Ca signal is maintained dynamically by the balance between the increased Ca2+ influx and active Ca2+ efflux across the plasma membrane.

Effects of angiotensin II and [Arg]vasopressin on cytosolic free Ca2+ concentration ([Ca2+]i) and phosphoinositide metabolism were studied in cultured aortic smooth muscle cells obtained from Wistar-Kyoto rats and their spontaneously hypertensive substrain. [Ca2+]i was measured using the fluorescent Ca2+ indicator quin2. No clear differences in basal [Ca2+]i were detected between cells derived from the two strains. High concentrations of angiotensin II (greater than or equal to 10 nM) and [Arg]vasopressin (greater than or equal to 100 nM) elicited large and rapid increases in [Ca2+]i, followed by a rapid return to control values. Low concentrations of these peptides (less than or equal to 1.0 nM) elicited small and slow increases in [Ca2+]i that persisted for minutes. These responses were blocked by specific antagonists for each of these peptides. Only high concentrations of angiotensin II caused [Ca2+]i increases in "Ca2+-free" medium, which suggested that high concentrations of angiotensin II could release Ca2+ from intracellular pools. A high concentration of angiotensin II and [Arg]vasopressin elicited progressive accumulations of inositol phosphates. Only high concentrations of angiotensin II caused inositol phosphate accumulation in Ca2+-free medium. Maximal accumulation of inositol phosphate elicited by angiotensin II and [Arg]vasopressin was found to be additive. A desensitization to the effects of both peptides on Ca2+ mobilization occurred despite the continued accumulation of inositol phosphates. These observations indicated that angiotensin II and [Arg]vasopressin interacted with independent receptors, both of which are linked to phosphoinositide breakdown and Ca2+ mobilization.

Phosphatidylinositol (PI) and its phosphorylated derivatives are rapidly broken down in 2H3 cells stimulated with antigen, with a time course which coincides with the generation of the Ca signal. Stimulated PI breakdown is absolutely dependent on Ca2+ in the medium with a concentration dependence similar to that of the Ca signal and histamine release described in the preceding paper. However, PI breakdown does not depend on the rise in free cytoplasmic Ca2+ concentration in stimulated cells over the range 100 nM to 1 microM. Thus, stimulation by the ionophore A23187 causes only a small increase in PI breakdown and the Ca signal stimulated by antigen can be selectively blocked with appropriate concentrations of Zn2+ (100 microM) or La3+ (10-100 microM) which have small or negligible effects on stimulated PI breakdown. Both PI breakdown and the Ca signal appear to depend on a common external Ca2+ site (or sites) with Km approximately equal to 0.4 mM, and the data are consistent with either independent activation of PI phosphodiesterase and the Ca signal after antigenic stimulation, or with PI breakdown as a component of the mechanism by which the Ca signal is generated.

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.

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.

We have re-examined, by high pressure liquid chromatographic (HPLC) procedures, the hydrolysis of 3H-labeled inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells. Previous studies showed no clear correlation between the release of any particular inositol metabolite and the calcium signal in these cells. Paradoxically no responses were observed when the cells were stimulated with the antigen, aggregated ovalbumin, in the absence of external Ca2+. We report here that in the absence of external Ca2+ aggregation of the IgE receptor by agents other than aggregated ovalbumin causes the release of small amounts of [3H]inositol phosphates and a small increase in levels of cytosol Ca2+ (approximately 25 nM). The response, however, varied with the type of stimulant used. Within seconds after addition of 24 mol of dinitrophenol conjugated with 1 mol of bovine serum albumin to cells primed with dinitrophenol-specific IgE there was a small burst in release of [3H]inositol 1,4,5-triphosphate, [3H]inositol 1,3,4,5-tetrakisphosphate, and [3H]inositol 1,3,4-trisphosphate which was followed by a gradual rise in inositol 1,3,4-trisphosphate, inositol bisphosphate, and inositol monophosphate. Eventually, all inositol phosphates reached different steady state levels which were maintained for at least 40 min. In contrast, the initial response to oligomeric IgE, which aggregates receptors at a relatively slow rate, was muted although the subsequent development of the response was the same. The levels of inositol pentakisphosphate and hexakisphosphate remained unchanged. These and other studies with cell extracts support the conclusion that inositol 1,4,5-trisphosphate, a putative messenger for release of intracellular Ca2+, was converted to inositol 1,3,4,5-tetrakisphosphate and thence to inositol 1,3,4-trisphosphate. Both trisphosphate metabolites were dephosphorylated in sequential fashion by phosphatase enzymes in the cytosolic and membrane fractions. However, the appearance of several isomers of inositol monophosphates and bisphosphates suggested that degradation proceeded through multiple pathways in the intact cell.

Previous studies with antigen-stimulated rat basophilic leukemia (RBL-2H3) cells indicated the formation of multiple isomers of each of the various categories of inositol phosphates. The identities of the different isomers have been elucidated by selective labeling of [3H]inositol 1,3,4,5-tetrakisphosphate with [32P]phosphate in the 3'-or 4',5'-positions and by following the metabolism of different radiolabeled inositol phosphates in extracts of RBL-2H3 cells. We report here that inositol 1,3,4,5-tetrakisphosphate, when incubated with the membrane fraction of extracts of RBL-2H3 cells, was converted to inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate. Further dephosphorylation of the inositol polyphosphates proceeded rapidly in whole extracts of cells, although the process was significantly retarded when ATP (2 mM) levels were maintained by an ATP-regenerating system. The degradation of inositol 1,4,5-trisphosphate proceeded with the sequential formation of inositol 1,4-bisphosphate, the inositol 4-monophosphate (with smaller amounts of the 1-monophosphate), and finally inositol. Inositol 1,3,4-trisphosphate, on the other hand, was converted to inositol 1,3-bisphosphate and inositol 3,4-bisphosphate and subsequently to inositol 4-monophosphate and inositol 1-monophosphate (stereoisomeric forms were undetermined). The possible implications of the apparent interconversion between inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in regulating histamine secretion in the RBL-2H3 cells are discussed.

Types I, II, and III protein kinase C have been shown to be products of, respectively, gamma, beta, and alpha genes of this enzyme family (Huang, F. L., Yoshida, Y., Nakabayashi, H., Knopf, J. L., Young, W. S., III, and Huang, K.-P. (1987) Biochem. Biophys. Res. Commun. 149, 946-952). Incubation of the highly purified rat brain protein kinase C isozymes with trypsin (kinase/trypsin (w/w) = 100) under identical conditions results in a preferential degradation of types I and II enzymes, whereas the type III enzyme was relatively resistant to tryptic proteolysis. Degradation of the type III enzyme by trypsin could be facilitated with the addition of Ca2+, phosphatidylserine, and dioleoylglycerol; none of these components alone was effective. Limited proteolysis of the three protein kinase C isozymes generated distinctive fragments for each isozyme, indicating that each isozyme has different trypsin-sensitive sites. Tryptic digestion of the type III protein kinase C was used as a model to determine the effects of various modulators on protein kinase C degradation. While Ca2+ and phosphatidylserine together were sufficient to convert the type III protein kinase C from a trypsin-insensitive to a -sensitive form, addition of dioleoylglycerol greatly reduced the Ca2+ requirement for such a conversion. Among the various phospholipids tested, in the presence of either dioleoylglycerol or phorbol ester, phosphatidylserine, cardiolipin, and phosphatidic acid were the most effective, and phosphatidylcholine and phosphatidylethanolamine were the least effective in supporting the digestion of type III protein kinase. Other acidic phospholipids, such as lysophosphatidylserine and phosphatidylinositol, were also effective in supporting the degradation in the presence of phorbol ester but not in the presence of dioleoylglycerol. The relevance of these proteolytic reactions to physiological responses was assessed with phorbol ester on rat basophilic leukemia RBL-2H3 cells, which contained both types II and III protein kinase C. Immunoblot analysis with the isozyme-specific antibodies revealed that phorbol ester induced a faster degradation of type II than that of type III isozyme in these cells. The results demonstrate that the various protein kinase C isozymes have different susceptibilities to proteolysis in vitro, when tested with trypsin, as well as to endogenous proteases in intact cells.

The concentration-dependent equilibrium of glucagon in mildly alkaline solution involves the formation of associated forms of higher helicity. The equilibrium may be followed by the change of optical rotation with concentration. In acid solution, the effect is still present but much diminished. The association is accompanied by the generation of a new Cotton effect at 298 mµ, which is attributed to the single tryptophan residue. A further consequence of the association is a shift in the spectrophotometric titration curve of the tyrosine residues. Both at high and at low glucagon concentration, the ionization curves are fitted by two pKa values, which are 9.9 and 10.7 for the disaggregated material and 10.1 and 10.7 for the associated form. From the sequence, it is possible to deduce that it is Tyr-13 which undergoes a pKa shift on association, whereas the remaining tyrosine, Tyr-10, seems to be unaffected. It is suggested that the association should be viewed in terms of two linked equilibria, the associated state acting as a thermodynamic trap for the structured conformation. It is shown that the true association constants must differ from the apparent ones by a factor which may correspond to several kilocalories per mole in free energy. It is suggested that such a mechanism forms a general basis for interrelations between aggregation state and conformation and is relevant to such phenomena as refolding pathways of denatured proteins.