Pertussis Toxin-sensitive Research Articles

Chemokine receptors are required for effector T-cell trafficking to GVHD tissues but not to bone marrow

AbstractIn allogeneic hematopoietic stem cell transplantation (allo-SCT), alloreactive donor T cells mediate the graft-versus-leukemia effect but also attack nonhematopoietic tissues, causing graft-versus-host disease (GVHD). Reducing alloreactive T-cell trafficking to GVHD target tissues while allowing their access to bone marrow (BM) and spleen, major sites of malignant hematopoiesis, is a rational strategy for reducing the GVHD risk when using alloreactive T cells as a therapeutic. Here, we show that effector T-cell (Teff) entry into BM and spleen in unmanipulated mice and in mice that received transplantation without alloreactive T cells is augmented by pertussis toxin (PTX)-sensitive chemokine receptor signaling. However, unexpectedly, in the presence of a GVH response, chemokines no longer draw T cells into BM and spleen but remain critical for their recruitment to GVHD target tissues. Consistent with this, PTX-treated Teff cells were as efficacious as untreated T cells in killing leukemia cells in BM and spleen in mice with a concurrent GVHD response. These results suggest a strategy to improve the safety of alloreactive T-cell therapeutics in treating leukemias in the context of an allo-SCT.

Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis.

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca(2+) influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca(2+) influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

The chromogranin A-derived peptides vasostatin-I and catestatin as regulatory peptides for cardiovascular functions

A range of inflammatory conditions is associated with pathologically high levels of circulating chromogranin A (CgA). This prohormone belongs to the family of uniquely acidic proteins co-stored and co-secreted with other hormones and peptides from the diffuse neuroendocrine system. Two highly conserved, CgA-derived peptides, vasostatin-I and catestatin, have been implicated as modulators of a wide range of cells and tissues, including those of the cardiovascular system. This review focuses on links between elevated circulating CgA and cardiovascular dysfunctions in inflammatory conditions in relation to potential beneficial effects of vasostatin-I and catestatin. Characteristic membrane-penetrating properties have been assigned to both peptides, and pertussis toxin sensitivity is shared by a number of their responses, notably in the vascular and cardiac endothelium. Pertussis toxin-sensitive, receptor-independent activation via heterotrimeric G proteins and Galphai/o subunits will be discussed as possible mechanisms for inhibitory effects of vasostatin-I and catestatin on vascular and cardiac responses. The accumulated evidence provides convincing support for vasostatin-I and catestatin as regulatory peptides for the cardiovascular system, converging on alleviation of significant dysfunctions as part of several inflammatory conditions.

Functional Domains of the Mouse β3-Adrenoceptor Associated with Differential G Protein Coupling

Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.

Platelet-derived growth factor mediated altered expression and regulation of ornithine decarboxylase in H-ras-transformed cell lines

This study demonstrates a novel link between alterations in platelet-derived growth factor (PDGF) regulation of ornithine decarboxylase (ODC) expression during malignant conversion. H-ras-transformed cell lines exhibited PDGF-mediated alterations in ODC gene expression. These alterations involved transcriptional, posttranscriptional, and cycloheximide-mediated events. PDGF-mediated alterations in ODC gene expression in NR3 cells (capable of only benign tumour formation) were ras-dependent, involved a tyrosine kinase activity and mitogen-activated protein (MAP) kinase-mediated signalling events, and were independent of both protein kinase C (PKC) events and pertussis toxin-sensitive (PTS) G-protein-mediated signalling. PDGF-mediated alterations in ODC gene expression in C2 cells [capable of malignant progression (metastasis formation)] were ras-dependent, required a tyrosine kinase activity, involved both MAP kinase-mediated events and phosphatidylinositol-3-kinase (PI-3-kinase)-mediated events, and were dependent upon PTS G-protein-mediated signalling but independent of PKC-mediated events. PDGF-mediated regulation of ODC gene expression changes in response to H-ras-mediated cellular transformation and malignant progression.

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

Molecular Cloning and Characterization of Another Leukotriene B4 Receptor

Subtypes of the Muscarinic Receptor in Smooth Muscle

Muscarinic receptors are expressed in smooth muscle throughout the body. In most instances, the muscarinic receptor population in smooth muscle is composed of mainly the M2 and M3 subtypes in an 80% to 20% mixture. The M3 subtype mediates phosphoinositide hydrolysis and calcium mobilization, whereas the M2 subtype mediates an inhibition of cAMP accumulation. In addition, a variety of ionic conductances are elicited by muscarinic receptors. Muscarinic agonists stimulate a nonselective cation conductance that is pertussis toxin-sensitive and dependent on calcium. The pertussis toxin-sensitivity of this response suggests that it is mediated by M2 receptors. Following agonist induced depolarization of smooth muscle, voltage dependent calcium channels are activated to enable an influx of calcium. In some instances, muscarinic agonists enhance this conductance through a mechanism involving protein kinase C, whereas in other instances, muscarinic agonists suppress this calcium conductance. Smooth muscle often contains calcium activated potassium channels that tend to repolarize the membrane following calcium influx. Activation of muscarinic receptors suppresses this potassium conductance in some smooth muscles. Under standard conditions, muscarinic agonists elicit pertussis toxin-insensitive contractions through activation of the M3 receptor. When most of the M3 receptors are inactivated, it is possible to measure a pertussis toxin-sensitive contractile response to muscarinic agonists that is most likely mediated through M2 receptors. M2 receptors also cause an indirect contraction by inhibiting the relaxant effects of agents that increase cAMP (e.g., forskolin and isoproterenol).

Somatostatin blocks the potentiation of TRH-induced TSH secretion from perifused pituitary fragments and the change in intracellular calcium concentrations from dispersed pituitary cells elicited by prepro-TRH (PS4) or by tri-iodothyronine.

TRH and somatostatin (SRIH) are well known to stimulate and to inhibit TSH secretion respectively. However, the mechanisms underlying the effect of SRIH on thyrotrophs are still not understood. We have previously shown in vitro that the TSH response to TRH is potentiated in a Ca(2+)-dependent fashion through the activation of dihydropyridine (DHP)-sensitive Ca2+ channels by the prepro-TRH (160-169) cryptic peptide (PS4) and tri-iodo-L-thyronine (T3), when the hormone was added shortly before a TRH pulse in order to avoid its genomic effect. Using perifused rat pituitary fragments, the present study has shown that SRIH inhibits, in a dose-dependent manner, the TSH response to physiological concentration of TRH (10 nM) and reverses the Ca(2+)-dependent potentiation of that response induced either by PS4 or by T3. We have also demonstrated that the inhibition by SRIH of the T3 potentiation of TRH-induced TSH secretion is pertussis toxin-sensitive. Our data suggest that SRIH inhibits the PS4 and T3 potentiation of TRH-induced TSH secretion through the inactivation of DHP-sensitive Ca2+ channels. Using primary cultures of rat anterior pituitary cells and videomicroscopy, we have already demonstrated that TRH, as well as PS4 and T3, are able to increase intracellular Ca2+ concentration ([Ca2+]i) rapidly, in 15 s. Our study has shown that SRIH is able to abolish the acute rise in [Ca2+]i induced either by PS4 or by T3. Since [Ca2+]i responses to PS4 and T3 are also abolished by the DHP nifedipine, our results suggest that [Ca2+]i changes in PS4- or T3-sensitive pituitary cells depend directly or indirectly on the activation of DHP-sensitive Ca2+ channels and that the inhibitory effect of SRIH may be mediated by inactivation of this type of channel.

Coupling of muscarinic m1, m2 and m3 acetylcholine receptors, expressed in Chinese hamster ovary cells, to pertussis toxin-sensitive/insensitive guanine nucleotide-binding proteins

Chinese hamster ovary (CHO) cells expressing recombinant human m1 (CHO-m1 cells), m2 (CHO-m2 cells), or m3 (CHO-m3 cells) muscarinic receptors were characterised pharmacologically with [ 3H] N-methylscopolamine. Agonist-stimulated coupling of these receptors with guanine nucleotide-binding proteins (G proteins) was measured by guanine nucleotide- and pertussis toxin-modification of carbachol competition-binding curves, and pertussis toxin-sensitivity of agonist-stimulated [ 35S]guanosine 5′- O-3-thiotriphosphate) ([ 35S]GTPγS) binding, in membrane preparations of the CHO cell clones. High affinity agonist binding and agonist-stimulated [ 35S]GTP-γS binding was abolished in CHO-m2 cell membranes (expressing 99 ± 25 fmol of [ 3 H]N- methylcopolamine binding sites/ mg protein) after pertussis toxin pretreatment of cells, suggesting that muscarinic m2 receptors expressed in these cell membranes couple predominantly with pertussis toxin-sensitive G proteins.CHO-m1((713 ± 102 fmol/mg protein) and CHO-m3 (1212 ± 279 fmol/mg protein) cell membranes produced maller elevations in agonist-stimulated [ 35S]GTPγS binding considering the higher receptor levels, compared with CHO-m2 cell membranes. Pertussis toxin pretreatment of these clones clso resulted in a significant attenuation of agonist-stimulated [ 35S]GTPγS binding suggesting that, under these experimental conditions, muscarinic m1 and m3 receptors can couple with both pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. Guanine nucleotide-modification of agonist binding in CHO-m1 and CHO-m3 cell membranes was comparatively smaller than in CHO-m2 cell membranes. A proportion of pertussis toxin-sensitive high affinity agonist binding sites were also detected in CHO-m3 cell membranes, but not in CHO-m1 membranes or in a CHO cell clone (CHO-vt9) expressing muscarinic m3 receptors at a lower density (430 ± 43 fmol/mg protein) than in CHO-m3 cells. These data suggest that high affinity agonist binding to phospholipase C-linked receptors may not faithfully represent the activity of these receptors coupled to G q/11, but may also represent these receptors coupling to pertussis toxin-sensitive G proteins.

Evidence for Gβγ-mediated Cross-talk in Primary Cultures of Lung Alveolar Cells: PERTUSSIS TOXIN-SENSITIVE PRODUCTION OF cAMP

In the presence of activated Gs alpha, the beta gamma complex of heterotrimeric G proteins (beta gamma) stimulates adenylyl cyclase (AC) in membranes prepared from cells expressing recombinant AC II or AC IV. Conditional stimulation of AC by beta gamma has been hypothesized to integrate cross-talk between Gs- and non-Gs-coupled regulation of cellular cAMP (Tang, W. J., and Gilman, A. G. (1991) Science 254, 1500-1503). Although observations in cells expressing recombinant receptors, G alpha s, and AC support this hypothesis (Federman, A. D., Conklin, B. R., Schrader, K. A., Reed, R. R., and Bourne, H. R. (1992) Nature 356, 159-161), this mechanism has not been investigated in differentiated cells. Expression of AC II has been reported only in lung, olfactory, and brain tissue. We found that rat lung alveolar type II cells express AC II and IV. Therefore, we hypothesized that beta gamma conditionally stimulates AC in type II cells. Consistent with this hypothesis, we found that the alpha 2-adrenergic agonist UK14304 did not affect basal cAMP in type II cells but potentiated terbutaline-stimulated cAMP accumulation. Treatment of cells with pertussis toxin partially inhibited terbutaline-stimulated cAMP accumulation and completely inhibited the effects of UK14304. In type II cell membranes, purified beta gamma tripled the terbutaline-stimulated increase in AC activity. In contrast, beta gamma inhibited AC activity in the absence of terbutaline. The addition of purified Go alpha blocked beta gamma-induced effects. In summary, type II cells expressing endogenous AC II and IV regulate cAMP accumulation and AC activity in a manner consistent with conditional stimulation by beta gamma. These observations support the overall hypothesis that conditional stimulation of AC by beta gamma integrates cross-talk between signal transduction systems in differentiated cells.

Human plasmin induces a receptor-mediated arachidonate release coupled with G proteins in endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 10(-7) M plasmin increased arachidonate release coupled with the increase in prostacyclin production. The stimulatory effect of plasmin on arachidonate release could be divided into the early and late phases according to its calcium dependency and pertussis toxin sensitivity. The early phase of plasmin-induced arachidonate release was a calcium-dependent and pertussis toxin-sensitive response, which was observed within 20 min after plasmin treatment. The late phase was a calcium-independent and pertussis toxin-insensitive response, which was induced gradually from 20 to 60 min. Induction of the early phase of plasmin's effect required both the lysine binding and catalytic sites in plasmin molecule because it was inhibited either by the binding antagonist tranexamic acid or by the serine protease inhibitor aprotinin. Guanosine 5'-O-(2-thiotriphosphate) potentiated the effect of plasmin in permeabilized or nonpermeabilized cells, indicating that the early phase effect was mediated by a pertussis toxin-sensitive guanosine 5'-triphosphate (GTP)-binding protein. The late phase of plasmin's effect was due to the catalytic activity because it was inhibited by aprotinin but not by tranexamic acid. Microplasmin structurally having the catalytic sites induced a similar late phase effect. Plasmin did not elicit the metabolism of phosphatidyl polyphosphoinositides. These studies demonstrate that the activation of phospholipase A2, which results in arachidonate release, in the early phase of plasmin's effect is a receptor-mediation via GTP-binding protein that is not coupled through phospholipase C activation.

Pertussis toxin-mediated ribosylation of G proteins blocks the hypnotic response to an α2-agonist in the locus coeruleus of the rat

Biologic responses mediated by adrenoceptors are transduced by a receptor-effector mechanism that involves a guanine nucleotide binding protein (G protein). Recently, we determined that the transduction mechanism for the hypnotic response to dexmedetomidine, a highly selective α 2-agonist, is located in the locus coeruleus (LC) of the rat. In this study, we examined the role of pertussis toxin-sensitive (PTX) G proteins in the LC for the hypnotic response to dexmedetomidine. The LC of rats were stereotactically cannulated and treated with PTX, 0.34 μg, or vehicle. Five days later, the hypnotic response to dexmedetomidine, 7 μg into the LC or 50 μg · kg −1 IP, was tested. On the following day, the LC was harvested and assayed to determine whether the G proteins had been ribosylated by pretreatment with PTX in vivo. Quantitative immunoblotting of G 0α, G iα1,2, and G iα3, the α-subunit of three PTX-sensitive proteins, was also performed. In vivo treatment with PTX into the LC blocked the hypnotic response to LC-administered desmedetomidine and, to a lesser extent, IP-administered dexmedetomidine. The in vivo PTX treatment effectively ribosylated the G proteins. No alteration in the amount of the different species of PTX-sensitive α-subunit was produced by in vivo PTX treatment. These data suggest a pivotal role for PTX-sensitive G proteins in the LC in the hypnotic response to α 2-agonists in the rat.

Activation of phosphoinositide turnover and protein kinase C by neurotransmitters that modulate calcium channels in embryonic chick sensory neurons

Gamma aminobutyric acid (GABA) and norepinephrine modulate the excitability of primary chick sensory neurons by decreasing the voltage dependent Ca current. Although previous electro-physiological studies indicate that neurotransmitter modulation of the Ca current in these neurons involves protein kinase C, the biochemical aspects of this mechanism have not been examined directly. We find that both norepinephrine (via a unique a receptor subtype) and GABA (via GABAb receptors) linked to pertussis toxin sensitive pathways, stimulate the metabolism of membrane phosphatidylinositol phospholipids in primary chick sensory neurons. In addition, norepinephrine causes the rapid translocation of C kinase activity from cytosolic to membrane associated distribution, consistent with its rapid activation in response to applied neurotransmitter. The pharmacology, pertussis toxin sensitivity and time course of the biochemical changes due to neurotransmitter treatment parallel the effects of these transmitters on calcium current modulation. These biochemical studies confirm the hypothesis that activation of protein kinase C is critically involved in calcium channel modulation in embryonic chick sensory neurons.

Responses of pertussis toxin-treated microvascular endothelial cells to transforming growth factor beta 1. No evidence for pertussis-sensitive G-protein involvement in TGF-beta signal transduction.

Responses of bovine adrenal capillary endothelial cells (BACE) on treatment with transforming growth factor beta 1 (TGF-beta 1) have been characterized and tested for sensitivity to inactivation of pertussis toxin-sensitive G-proteins. TGF-beta 1 elicited growth inhibition, monolayer remodeling, elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) and alpha 2(1)) and TGF-beta 1, and inhibition of p34cdc2 histone H1 kinase activity in BACE cells. Pertussis toxin treatment enhanced both inhibition of BACE cell [3H]methylthymidine uptake and remodeling of BACE monolayers by TGF-beta 1. These findings contrast with studies of mink lung epithelial cells, in which TGF-beta 1 growth inhibition has been shown to be pertussis-sensitive. Further investigation revealed that pertussis toxin treatment of BACE cells had no effect on TGF-beta 1-stimulated elevation of steady state mRNA levels for collagen type 1 (alpha 1(1) or alpha 2(1)) or for TGF-beta 1. Analysis of p34cdc2 activity in BACE cells revealed potent inhibition of p34cdc2 histone H1 kinase activity by TGF-beta 1. Pertussis toxin treatment also abolished the increase in p34cdc2 activity, however, precluding the determination of the pertussis toxin sensitivity of this response to TGF-beta 1. Consistent with suppression of p34cdc2 activation, pertussis toxin also caused substantial inhibition of mitogen-stimulated BACE cell [3H]methylthymidine uptake. It is concluded that TGF-beta 1 signal transduction in this cell type does not involve G-proteins of the pertussis toxin-sensitive class and that, in view of its potent effects on DNA synthesis and p34cdc2 activation, the use of pertussis toxin to determine G-protein involvement in cytokine signalling pathways should be approached with caution.

Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors

Calcium release from intracellular stores is a point of convergence for a variety of receptors involved in cell signaling. Consequently, the mechanism(s) by which cells differentiate between individual receptor signals is central to transmembrane communication. There are significant differences in timing and magnitude of Ca2+ release stimulated by the m2 and m3 muscarinic acetylcholine receptors. The m2 receptors couple to a pertussis toxin-sensitive G protein to activate phosphatidyl inositol hydrolysis weakly and to stimulate small, delayed and oscillatory chloride currents. In contrast, m3 receptors potently activate phosphatidyl inositol hydrolysis and stimulate large, rapid and transient chloride currents by a pertussis toxin-insensitive G protein pathway. Using confocal microscopy, we now show that the m2- and m3-coupled Ca2+ release pathways can also be spatially distinguished. At submaximal acetylcholine concentrations, both receptors stimulated pulses of Ca2+ release from discrete foci in random, periodic and frequently bursting patterns of activity. But maximal stimulation of m2 receptors increased the number of focal release sites, whereas m3 receptors invariably evoked a Ca2+ wave propagating rapidly just beneath the plasma membrane surface. Analysis of pertussis toxin sensitivity and hybrid m2-m3 muscarinic acetylcholine receptors confirmed that these Ca2+ release patterns represent distinct cell signalling pathways.

Differences in signal transduction between Fc gamma receptors (Fc gamma RII, Fc gamma RIII) and FMLP receptors in neutrophils. Effects of colchicine on pertussis toxin sensitivity and diacylglycerol formation.

Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis.

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.