S.E. van Emst-de Vries

Dose-dependent recruitment of pancreatic acinar cells during receptor-mediated calcium mobilization.

Digital-imaging microscopy of Fura-2-loaded rabbit pancreatic acinar cells was used to simultaneously monitor the cholecystokinin-octapeptide (CCK8)-induced changes in free cytosolic Ca2+ concentration, [Ca2+]i, in large numbers of individual acinar cells. CCK8 typically evoked a switchlike increase in [Ca2+]i which was preceded by a concentration-dependent latency. The threshold concentration for the CCK8-induced rise in [Ca2+]i differed greatly among individual acinar cells, resulting in the dose-dependent recruitment of acinar cells in terms of CCK8-induced Ca2+ mobilization. The EC50 value for CCK8-induced cell-recruitment was estimated to be 15 pM. The hormone was equally potent in stimulating amylase secretion from acinar cells in suspension. At a CCK8 concentration of 100 pM, virtually all cells responded to the hormone with an increase in [Ca2+]i and the number of responding cells remained unchanged upon further increase of the CCK8 concentration. The dose-response curve for cell-recruitment coincides with that of the apparent [Ca2+]i increase in a suspension of acinar cells. This suggests that the most likely interpretation of the latter dose-response curve is not a generalized increase in [Ca2+]i but an increase in the number of responding cells. The initial rise in [Ca2+]i, which was transient by nature, was followed by repetitive [Ca2+]i transients of long duration. The dose-response curve for the effect of CCK8 on the percentage of acinar cells displaying these distinct [Ca2+]i oscillations was biphasic. A maximum of 99% of the cells showing oscillatory behaviour was reached at 100 pM CCK8, beyond which concentration the number of oscillating cells dose-dependently decreased again. The latter decrease was paralleled by a dose-dependent increase of the percentage responding but non-oscillating cells, indicating that beyond 100 pM CCK8 an increasing number of acinar cells became desensitized towards hormonal induction of oscillatory changes in [Ca2+]i. CCK8 was approximately 100-fold more potent in reducing the percentage of oscillating cells than in inhibiting amylase secretion. Oscillating acinar cells responded to a stepwise increase of the medium CCK8 concentration with a rapid change in amplitude and frequency of the oscillations. Thus, with increasing CCK8 concentration the frequency gradually increased, whereas the amplitude only slightly increased at first, reached a maximum, and decreased thereafter. In some cells full extinction was reached. Again, large differences in dose-dependency were observed among individual acinar cells. The observations presented demonstrate the existence of a marked functional heterogeneity among pancreatic acinar cells in terms of CCK8-induced Ca2+ mobilization.

Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca2+ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK8) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca2+ concentration ([Ca2+]i,av) in a suspension of acinar cells. However, in the presence of 1.0 μM Staurosporine the stimulatory effect of submaximal concentrations of CCK8 was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca2+]i,av in cells prestimulated with a submaximal concentration of CCK8. The data obtained with staurosporine indicate that CCK8-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca2+ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK8-induced Ca2+ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK8-evoked increase in [Ca2+]i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK8 at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca2+]i,av evoked by the CCK8 analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca2+]i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK8 concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca2+]i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca2+ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca2+ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.

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.

A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome.

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.

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.

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme.

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.

Amino group modification of (Na+ + K+)-ATPase.

The effects of three amino group reagents on the activity of (Na++K+)-ATPase3 and its component K+-stimulatedp-nitrophenylphosphatase activity from rabbit kidney outer medulla have been studied. All three reagents cause inactivation of the enzyme. Modification of amino groups with trinitrobenzene sulfonic acid yields kinetics of inactivation of both activities, which depend on the type and concentration of the ligands present. In the absence of added ligands, or with either Na+ of Mg2+ present, the enzyme inactivation process follows complicated kinetics. In the presence of K+, Rb+, or Tl+, protection occurs due to a change of the kinetics of inactivation toward a first-order process. ATP protects against inactivation at a much lower concentration in the absence than in the presence of Mg2+ (P50 6 µM vs. 1.2 mM). Under certain conditions (100 µM reagent, 0.2 M triethanolamine buffer, pH 8.5) modification of only 2% of the amino groups is sufficient to obtain 50% inhibition of the ATPase activity. Modification of amino groups with ethylacetimidate causes a nonspecific type of inactivation of (Na++K+)-ATPase. Mg2+ and K+ have no effects, and ATP only a minor effect, on the degree of modification. The K+-stimulatedp-nitrophenylphosphatase activity is less inhibited than the (Na++K+)-ATPase activity. Half-inhibition of the (Na++K+)-ATPase is obtained only after 25% modification of the amino groups. Modification of amino groups with acetic anhydride also causes nonspecific inactivation of (Na++K+)-ATPase. Mg2+ has no effect, and ATP has only a slight protecting effect. The K+-stimulatedp-nitrophenylphosphatase activity is inhibited in parallel with the (Na++K+)-ATPase activity. Half-inactivation of the (Na++K+)-ATPase activity is obtained after 20% modification of the amino groups.