R.L.L. Smeets

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

The aminosteroid 1-(6-¿[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-amino¿hexyl)- 1H-pyrrole-2,5-dione (U73122) and its inactive analogue 1-(6-¿[17beta-3-methoxyestra-1,3,5(10)-trien- 17-yl]-amino¿hexyl-2,5-pyrrolidine-dione (U73343) are widely used to study the involvement of G protein-coupled 1-phosphatidylinositol-phosphodiesterase, or phospholipase C, in receptor-mediated cell activation. The present work shows that both aminosteroids inhibit cholecystokinin-(26-33)-peptide amide (CCK-8)-induced phospholipase D activation equipotently in Chinese hamster ovary cells expressing the cholecystokinin-A receptor (CHO-CCK(A) cells). In addition, the two aminosteroids virtually completely inhibited thapsigargin- and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced phospholipase D activation. Since the latter two drugs mimic inositol 1,4,5-trisphosphate-mediated Ca2+ mobilisation and 1,2-diacylglycerol-mediated protein kinase C activation. respectively, this suggests that both U73122 and U73343 act downstream of phospholipase C to inhibit receptor-mediated phospholipase D activation. U73122, but not U73343. effectively inhibited both TPA/Ca2+-stimulated phospholipase D activation and TPA/phosphatidylserine-stimulated protein kinase C activation in a homogenate of CHO-CCK(A) cells. The data presented suggest that U73122 may act at the level of protein kinase C to inhibit activation of phospholipase D. The exact site of action of U73343 is presently unknown.

Recovery from TPA inhibition of receptor-mediated Ca2+ mobilization is paralleled by down-regulation of protein kinase C-alpha in CHO cells expressing the CCK-A receptor

Digital-imaging microscopy of Fura-2-loaded Chinese hamster ovary cells, stably expressing the cholecystokinin-A receptor, revealed that both the C-terminal octapeptide of cholecystokinin (CCKB) and its analogue JMV-180, which acts as an agonist at the high-affinity CCK-A receptor, recruited CHO-CCK-A cells dose-dependently in terms of receptor-mediated Ca2+ mobilization. Agonist-evoked cell recruitment was inhibited by short-term (10 min) pretreatment with 0.1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA). In the case of CCKB, inhibition was overcome with increasing of the hormone concentration. In contrast, increasing of the JMV-180 concentration did not reverse the inhibitory action of TPA. CHO-CCK-A cells gradually regained their responsiveness to JMV-180 during prolonged TPA pretreatment. Complete recovery was observed within 1 h following addition of TPA. Western blot analysis using antibodies directed against the various PKC isotypes revealed that recovery was paralleled by the disappearance of PKC-alpha. Surprisingly, short-term (10 min) TPA pretreatment virtually completely inhibited the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to CCKB concentrations at which the effect on cell recruitment was not affected by short term phorbol ester pretreatment. Together with the finding that JMV-180 does not detectably increase the cellular Ins(1,4,5)P3 content, this suggests a large overproduction of this second messenger by CCKB concentrations supramaximal in terms of cell recruitment. Again, full responsiveness was observed after long term TPA pretreatment. The present observations are in agreement with the idea that in CHO-CCK-A cells activation of PKC-alpha leads to inhibition of agonist-evoked Ca2+ mobilization through inhibition of receptor-stimulated Ins(1,4,5)P3 formation.

Protein-Kinase-C Activation Inhibits Receptor-Evoked Inositol Trisphosphate Formation and Induction of Cytosolic Calcium Oscillations by Decreasing the Affinity-State of the Cholecystokinin Receptor in Pancreatic Acinar-Cells

Digital-imaging microscopy of Fura-2-loaded pancreatic acinar cells revealed that the C-terminal octapeptide of cholecystokinin (CCK8) dose-dependently recruited 94% of freshly isolated acinar cells in terms of receptor-evoked Ca2+ mobilization. Maximal and half-maximal cell-recruitment were reached with 0.1 nM and 16.8 pM CCK8, respectively. The upstroke of the dose-recruitment curve consisted of cells displaying oscillatory changes in free cytosolic Ca2+ concentration ([Ca2+]i). After having reached its maximum, the percentage oscillating cells dose-dependently decreased upon further increasing of the CCK8 concentration. Pretreatment of the acinar cells with 0.1 microM TPA caused a rightward shift of the dose-recruitment curve but did not change the maximal effect of CCK8 on the recruitment of oscillating cells. Half-maximal recruitment was obtained with 287 pM CCK8. This observation demonstrates that high levels of protein kinase C activation do not inhibit Ca2+ oscillations at a level downstream to receptor activation. Moreover, this observation demonstrates that protein kinase C-mediated inhibition of Ca2+ oscillations evoked by submaximal CCK8 concentrations occurs at the receptor level, converting it from a high-affinity state into a low-affinity state. This conclusion is supported by the observation that TPA completely inhibited the recruitment of acinar cells in response to the high-affinity receptor agonist JMV-180. The inhibitory action of TPA on CCK8-evoked cell-recruitment was paralleled by an inhibitory effect of the phorbol ester on the CCK8-evoked peak increase in average inositol trisphosphate concentration in a population of acinar cells. This observation indicates that low concentrations of CCK8 interact with the high-affinity CCK receptor to increase [Ca2+]i through the intermediation of inositol trisphosphate.

Protein kinase C-mediated inhibition of transmembrane signalling through CCKA and CCKB receptors

1 The rat CCKA and CCKB receptors were stably expressed in Chinese hamster ovary (CHO‐09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2 Spectrofluorophotometry of Fura‐2‐loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin‐(26‐33)‐peptide amide (CCK‐8‐S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCKA cells, measured as an increase in Fura‐2 fluorescence emission ratio, 1000 fold more potently than its non‐sulphated form (CCK‐8‐NS) (EC50 values of 0.19 nM and 0.18 μM, respectively). By contrast, CCK‐8‐S and CCK‐8‐NS were equally potent in CCKB cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCKA receptor agonist JMV‐180 increased [Ca2+]i only in CCKA cells. Likewise, pentagastrin increased [Ca2+]i only in CCKB cells. Finally, CCK‐8‐S‐induced Ca2+ signalling through the CCKA receptor was most potently inhibited by the CCKA receptor antagonist L364,718, whereas the CCKB receptor antagonist L365,260 was more potent in CCKB cells. 3 Receptor‐mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3‐isobutyl‐1‐methylxanthine. CCK‐8‐S and, to a lesser extent, CCK‐8‐NS, but not JMV‐180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCKA cells. By contrast, none of these agonists increased cyclicAMP in CCKB cells. 4 Short‐term (3 min) pretreatment with the PKC activator 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) evoked a rightward shift of the dose‐response curve for the Ca2+ mobilizing effect of CCK‐8‐S in both cell lines. In addition, short‐term TPA pretreatment markedly reduced CCK‐8‐S‐induced cyclicAMP accumulation in CCKA cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF‐109203X and staurosporine, whereas no inhibition was observed with the inactive phorbol ester, 4‐α‐phorbol 12‐myristate 13‐acetate. 5 During prolonged TPA treatment, the cells gradually recovered from phorbol ester inhibition and in the case of CCK‐8‐S‐induced Ca2+ mobilization complete recovery was achieved after 24 h of TPA treatment. Western blot analysis revealed that this recovery was paralleled by down‐regulation of PKC‐α, suggesting the involvement of this PKC isotype in the inhibitory action of TPA. 6 This study demonstrates that following expression in CHO cells (i) both CCKA and CCKB receptors are coupled to Ca2+ mobilization, (ii) only CCKA receptors are coupled to cyclicAMP formation and (iii) with both receptors signalling is inhibited by PKC.

Mutational analysis of the putative devazepide binding site of the CCKA receptor

Recently a molecular model was proposed for the binding site of the antagonist 3S(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepine-3-yl) -1H-indole-2-carboxamide (devazepide) on the cholecystokinin-A (CCK(A)) receptor (Van der Bent et al., 1994. Drug Design Discov. 12, 129-148). Fifteen amino acids were identified, including hydrophilic ones such as Ser139, Asn349 and Ser379, that might interact with the carboxamide moiety in devazepide. To provide mutational evidence for this model, wild-type and mutant receptors (S139A, N349A and S379A) were transiently expressed and compared with respect to the ability of devazepide to inhibit binding of radiolabelled cholecystokinin-(26-33)-peptide amide (CCK-8) and CCK-8-evoked Ca2+ mobilization. The data presented suggest the involvement of the three residues in antagonist binding, although to a different extent. However, it does not seem likely that hydrogen bonds are the driving force in view of the relatively minor changes in receptor affinity and activity.

The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor–insulin receptor generates Ca2+ signals through the PLC-γ1 pathway

The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates.

Phosphorylation and desensitization of the pancreatic cholecystokinin-A receptor

The eukaryotic cell uses a variety of mechanisms to protect itself from overstimulation. Among these mechanisms are processes involving the receptor, including uncoupling from G proteins and movement into cellular compartments. Here, we focus on mechanisms by which the pancreatic acinar cell protects itself from overstimulation through the cholecystokinin receptor with special emphasis on the role of receptor phosphorylation.

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCKA receptor

Many G protein‐coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCKA) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild‐type (CCKAWT) and mutant (CCKAMT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. Binding of [3H]‐cholecystokinin‐(26‐33)‐peptide amide (CCK‐8) to membranes prepared from CHO‐CCKAWT cells and CHO‐CCKAMT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK‐8, respectively). The dose‐response curves for CCK‐8‐induced cyclic AMP accumulation and inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO‐CCKAMT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO‐CCKAWT cells. This demonstrates that attenuation of CCK‐8‐induced activation of adenylyl cyclase and phospholipase C‐β involves a staurosporine‐sensitive kinase, which acts directly at the potential sites of PKC action on the CCKA receptor in CCK‐8‐stimulated CHO‐CCKAWT cells. The potent PKC activator, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), evoked a rightward shift of the dose‐response curve for CCK‐8‐induced cyclic AMP accumulation in CHO‐CCKAWT cells but not CHO‐CCKAMT cells. This is in agreement with the idea that PKC acts directly at the CCKA receptor to attenuate adenylyl cyclase activation. In contrast, TPA evoked a rightward shift of the dose‐response curve for CCK‐8‐induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high‐level PKC activation inhibits CCK‐8‐induced Ins(1,4,5)P3 formation also at a post‐receptor site. TPA inhibition of agonist‐induced Ca2+ mobilization was only partly reversed in CHO‐CCKAMT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas‐7. These findings are in agreement with the idea that partial reversal of agonist‐induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. The data presented demonstrate that the predicted sites for PKC action on the CCKA receptor are the only sites involved in TPA‐induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post‐receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C‐β activation by receptors that are not phosphorylated by PKC.

Analysis of Agonist-Induced Cell Recruitment in Terms of Intracellular Calcium Mobilization in a Population of Enzymatically Dispersed Pancreatic Acinar Cells

The term cell recruitment is often used to describe the accumulation of motile cells such as neutrophils, lymphocytes, macrophages and mast cells at the site of release of some chemotactic factor. In the broadest sense of the word, however, this term applies to every situation in which a signal mobilizes cells to perform some kind of cellular activity. For instance, in a population of enzymatically dispersed pancreatic acinar cells the peptide hormone cholecystokinin (CCK) both time- and dose-dependently increases the number of cells displaying repetitive changes in free cytosolic calcium concentration (Fig. 29.1). This particular experiment clearly demonstrates that only part of the cells respond to stimulation with a submaximal concentration of 10 pM of the COOH-terminal octapeptide of CCK (CCK8), with a delay greatly differing between individual cells. As a result, maximal recruitment is reached only at 10 min following the onset of stimulation. By contrast, the vast majority of cells respond within the first minute of stimulation with a close to maximal hormone concentration of 1 nM. Such detection of differences in sensitivity among individual cells was enabled by the development of digital imaging techniques allowing simultaneous monitoring of agonist-induced changes in [Ca2+]i in large numbers of individual cells loaded with Ca2+-sensitive fluorescent dye. This chapter aims to give a detailed description of the method used in our laboratory to analyze agonist-induced cell recruitment in terms of intracellular calcium mobilization.