Yasuhiro Tsunoda

Src kinase and PI 3-kinase as a transduction pathway in ceramide-induced contraction of colonic smooth muscle

Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60(src) tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60(src) and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.Ceramide mediates sustained contraction of smooth muscle cells. C2 ceramide induced a rapid increase in Src kinase activity within 15 s, peaked at 1 min, and was sustained up to 8 min. Contraction and Src kinase activity were inhibited in cells incubated in Ca2+-free medium containing 2 mM EGTA and in cells preincubated with herbimycin A, a Src kinase inhibitor. Immunoblotting using a phosphospecific anti-Src (416Y) antibody showed a ceramide-induced increase in pp60 src tyrosine phosphorylation. Immunoprecipitation using an anti-phosphotyrosine antibody followed by Western immunoblotting using a monoclonal IgG anti-phosphoinositide 3-kinase NH2 terminal-SH2 domain antibody showed a ceramide-induced increase in phosphoinositide 3-kinase (PI 3-kinase) tyrosine phosphorylation at a protein mass corresponding to 85 kDa, the regulatory subunit of PI 3-kinase, which contains the Src kinase binding site. PI 3-kinase phosphorylation was inhibited by herbimycin A and by the PI 3-kinase inhibitors wortmannin and LY-294002. Preincubation of cells with herbimycin A or PI 3-kinase inhibitors also resulted in an inhibition of mitogen-activated protein (MAP) kinase p42 and p44 activities as seen on Western blots. In summary, we found that 1) the maintenance of sustained contraction is dependent on extracellular Ca2+; 2) ceramide activates a nonreceptor tyrosine kinase pathway through activation of pp60 src and PI 3-kinase; and 3) the converging signals are probably through activation of MAP kinase.

Involvement of phosphoinositide 3-kinase and its association with pp60src in cholecystokinin-stimulated pancreatic acinar cells.

Phosphoinositide 3-kinase (PI3K) is a lipid kinase which phosphorylates the D3 position of the phosphoinositide derivatives and is known to be activated by a host of protein tyrosine kinases. PI3K has been demonstrated to play an important role in mitogenesis and cell transformation in several cell systems. However, the functional roles of PI3K in pancreatic acinar cells remain to be determined. The objective of this study was to identify and characterize the PI3K pathway and its relation to other non-receptor protein tyrosine kinases in mediating signal transduction of pancreatic acinar cells. Intact acini isolated from the rat pancreas were incubated with or without cholecystokinin octapeptide (CCK-8). A Triton X-100-soluble and 10000 rpm supernatant of the cell sonicates was used for immunoprecipitation and Western immunoblotting. When a monoclonal anti-phosphotyrosine antibody (clone 4G10) was used, two major tyrosine-phosphorylated bands were observed at the location of p85 and p60. CCK (10 pM and 10 nM) significantly enhanced the tyrosine phosphorylation of these two bands. Furthermore, when a monoclonal anti-PI3K antibody (clone UB93-3) which recognizes the N-terminal SH2 domain of the p85 regulatory subunit of PI3K was used, CCK (10 pM-10 nM) dose-dependently increased the amount of the immunodetectable PI3K band with a peak occurring at 5 min. The increase in the immunodetectable PI3K band elicited by CCK did not require the presence of extracellular Ca2+. The pp60src inhibitor, herbimycin A (6 microM), and the PI3K inhibitor, wortmannin (6 microM), both decreased intensities of the PI3K band elicited by CCK. Herbimycin A abolished phosphotransferase activities of the Src kinase following stimulation with CCK, whereas wortmannin had no effect, suggesting that Src is an upstream regulator of PI3K. Wortmannin (3-6 microM) abolished CCK-stimulated pancreatic amylase secretion. Immunoprecipitation studies using an anti-Src antibody (clone CD11) or PI3K antibody in conjunction with the anti-phosphotyrosine antibody showed that, in response to CCK, tyrosine phosphorylations of Src and PI3K were enhanced at the location of p60 and p85, respectively. Src was co-immunoprecipitated with PI3K following stimulation with CCK, suggesting that pp60src forms an immunocomplex with PI3K via the N-SH2 domain of the p85 regulatory subunit. Thus PI3K and its association with Src appear to be involved in mediating CCK-stimulated pancreatic exocytosis.

Cytosolic free calcium spiking affected by intracellular pH change.

The characteristics underlying cytosolic free calcium oscillation were evaluated by superfused dual wave-length microspectrofluorometry of fura-2-loaded single acinar cells from rat pancreas. Application of a physiological concentration of cholecystokinin octapeptide (CCK) (20 pM) induced a small basal increase in cytosolic free calcium concentration ([Ca2+]i) averaging 34 nM above the prestimulation level (69 nM) with superimposed repetitive Ca2+ spike oscillation. The oscillation amplitude averaged 121 nM above the basal increase in [Ca2+]i and occurred at a frequency of one pulse every 49 s. Although extracellular Ca2+ was required for maintenance of high frequency and amplitude of the spikes with increase in basal [Ca2+]i, the primary source utilized for oscillation was intracellular. The threshold of the peak [Ca2+]i amplitude for causing synchronized and same-sized oscillations was less than 300 nM. The [Ca2+]i oscillation was sensitive to intracellular pH (pHi) change. This is shown by the fact that the large pHi shift toward acidification (delta pHi decrease, 0.95) led to a basal increase in [Ca2+]i to the spike peak level with inhibiting Ca2+ oscillation. The pHi shift toward alkalinization (delta pHi increase, 0.33) led to a basal decrease in [Ca2+]i to the prestimulation level, possibly due to reuptake of Ca2+ into the Ca2+ stores, with inhibiting Ca2+ oscillation. Whereas extracellular pH (pHo) change had only minimal effects on Ca2+ oscillation (and/or Ca2+ release from intracellular stores), the extra-Ca2+ entry process, which was induced by higher concentrations of CCK, was totally inhibited by decreasing pHo from 7.4 to 6.5. Thus the major regulatory sites by which H+ affects Ca2+ oscillation are accessible from the intracellular space.

Effect of uncoupling NO/cGMP pathways on carbachol- and CCK-stimulated Ca2+ entry and amylase secretion from the rat pancreas

Abstract Nitric oxide (NO) production reportedly regulates guanosine 3′,5′-cyclic monophosphate (cGMP) formation and Ca2+ influx in pancreatic acini. We have investigated the functional roles of the NO/cGMP messenger system in rat pancreatic acini. In dispersed acini, the levels of amylase secretion, cytosolic [Ca2+]([Ca2+]i), NO synthase, and cGMP were measured. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 0.01–100 μM) had no effect on amylase secretion induced by various concentrations of carbachol, cholecystokinin octapeptide (CCK-8) or the high affinity CCK agonist, JMV-180. Similarly, L-NAME up to 100 μM did not affect the changes in Ca2+ spiking evoked by these secretagogues; nor was Ca2+ entry, refilling or oscillation altered by L-NAME. Sub- and supramaximal concentrations of these secretagogues did not change NO synthase activities compared with basal levels. While sodium nitroprusside (SNP), a NO donor, caused a 9.4-fold increase in cGMP levels compared with basal levels, carbachol, CCK-8 and JMV-180 had no effect. In addition, the guanylate cyclase inhibitor LY 83583 (10 nM to 10 μM) altered neither amylase secretion nor Ca2+ signaling induced by these secretagogues. These findings indicate that the stimulatory action of carbachol or CCK-8 is not mediated by NO or cGMP. To investigate whether cGMP stimulates pancreatic secretion we showed that both SNP and a cell-permeant cGMP analog at 0.1–1 mM stimulated amylase secretion and Ca2+ transients to a level equal to 10–15% and 13–24%, respectively, of those observed with maximal concentrations of secretagogues. The guanylate cyclase activator guanylin (1–10 μM), which increased cGMP levels 2.4-fold compared with basal levels, elicited a small amount of amylase secretion and a small Ca2+ transient. In conclusion, exogenous NO is capable of increasing endogenous cGMP, which results in a modest increase in the [Ca2+]i transient and pancreatic amylase secretion. However, the NO/cGMP system does not appear to be involved significantly in the mediation of Ca2+ signaling and amylase secretion stimulated by carbachol and CCK-8.

Secretagogue induced calcium mobilization in single pancreatic acinar cells

Microspectrofluorometry of fura-2 was utilized to monitor [Ca2+]i in single acinar cells stimulated with a cholinergic agonist and cholecystokinin. A similar amplitude of agonist induced Ca mobilization between single cell and populational approaches was observed. New findings in single cells not observable in populations of cells include: 1) the maintenance of a sustained elevation in [Ca2+]i above basal levels throughout agonist application, 2) the reloading of the agonist-sensitive Ca pool only following removal of the agonist and 3) the presence of oscillations of [Ca2+]i in response to agonist application which is enhanced at lower agonist concentrations.

High-affinity cholecystokinin type A receptor/cytosolic phospholipase A2 pathways mediate Ca2+ oscillations via a positive feedback regulation by calmodulin kinase in pancreatic acini.

In rat pancreatic acini, we previously demonstrated that depending on the agonist used, activation of cholecystokinin type A (CCKA) receptor (CCK-AR) results in the differential involvement of the cytosolic phospholipase A2 (cPLA2), phospholipase Cbeta1 (PLCbeta1) and Src/protein tyrosine kinase (PTK) pathways. The high-affinity CCK-AR appears to be coupled to the Gbeta/cPLA2/arachidonic acid (AA) cascade in mediating Ca2+ oscillations. The low-affinity CCK-AR is coupled to both the Galphaq/11/PLCbeta1/inositol 1,4,5-trisphosphate (IP3) to evoke intracellular Ca2+ release and the Src/PTK pathway to mediate extracellular Ca2+ influx. The objectives of this study were to provide evidence that cPLA2 is present in pancreatic acini and to evaluate the possibility that its activation results in Ca2+ oscillations and amylase secretion. Furthermore, we investigated the mechanism of Ca2+ oscillations mediated by the high-affinity CCK-AR. In rat pancreatic acini, immunoprecipitation studies using an anti-cPLA2 monoclonal antibody, demonstrated a cPLA2 band at the location of 110 kDa. A selective inhibitor of cPLA2, AACOCF3 (100 microM), inhibited production of AA metabolites, Ca2+ oscillations and amylase secretion elicited by the high-affinity CCK-AR agonist, CCK-OPE (10-1000 nM). In addition, through the repetitive release of intracellular Ca2+, CCK-OPE enhanced phosphotransferase activities of Ca2+/calmodulin-dependent protein kinase type IV (CaMK IV), which were inhibited by AACOCF3. The CaMK inhibitor, K252-a (1-3 microM), also abolished basal and CCK-OPE-stimulated CaMK IV activities. The CaM inhibitor, W-7 (100 microM), and K252-a inhibited Ca2+ oscillations and amylase secretion evoked by CCK-OPE without affecting the AA formation. Therefore, it appears that Ca2+ oscillations elicited by the high-affinity CCK-AR/Gbeta/cPLA2/AA pathway activate CaMK IV. Activated CaMK, in turn, regulates Ca2+ oscillations through a positive feedback mechanism to mediate pancreatic exocytosis.

Mammalian bombesin receptors are coupled to multiple signal transduction pathways in pancreatic acini

We investigated the structural requirements for bombesin (BB)-like peptides to stimulate amylase secretion in rat pancreatic acini and examined the responsible intracellular signal transduction pathways. The tetradecapeptide BB-(1-14) was a full agonist, whereas the heptapeptide BB-(8-14) did not evoke amylase secretion. The mammalian BB analog neuromedin C decapeptide [NMC-(5-14)] was as potent as BB-(1-14) in stimulating amylase secretion, suggesting that Gly5-Asn6-His7(or Gln7) of the COOH-terminal decapeptide are essential amino acids for full biological activity. BB and NMC equipotently stimulatedd- myo-inositol 1,4,5-trisphosphate production, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. BB and NMC also stimulated protein tyrosine kinase (PTK) activities. The half-maximal effective concentration (EC50) for NMC-activated PTK was 2 log units less than the EC50 for BB-activated PTK. NMC was 10-34 times more potent than BB in increasing leukotriene C4 (an index of arachidonic acid production). The production of leukotriene C4 was inhibited by the phospholipase A2(PLA2) inhibitor ONO-RS-082. NMC is structurally homologous to BB-(5-14) except that Gln7 in BB is replaced by His7 in NMC. Therefore, substitution of Gln7 for His7 may alter the signal transduction systems to include the PTK and PLA2 pathways. U-73122 inhibited Ca2+ spiking and amylase secretion induced by NMC and BB. However, the PTK inhibitor genistein and the PLA2 inhibitor ONO-RS-082 inhibited secretion induced by NMC but not that induced by BB. In contrast to nonmammalian BB receptors, which primarily use the PLC pathway, the rat BB receptor is linked to three different signal transduction systems: PLC, PTK, and PLA2 pathways.We investigated the structural requirements for bombesin (BB)-like peptides to stimulate amylase secretion in rat pancreatic acini and examined the responsible intracellular signal transduction pathways. The tetradecapeptide BB-(1-14) was a full agonist, whereas the heptapeptide BB-(8-14) did not evoke amylase secretion. The mammalian BB analog neuromedin C decapeptide [NMC-(5-14)] was as potent as BB-(1-14) in stimulating amylase secretion, suggesting that Gly5-Asn6-His7 (or Gln7) of the COOH-terminal decapeptide are essential amino acids for full biological activity. BB and NMC equipotently stimulated D-myo-inositol 1,4,5-trisphosphate production, which was inhibited by the phospholipase C (PLC) inhibitor U-73122. BB and NMC also stimulated protein tyrosine kinase (PTK) activities. The half-maximal effective concentration (EC50) for NMC-activated PTK was 2 log units less than the EC50 for BB-activated PTK. NMC was 10-34 times more potent than BB in increasing leukotriene C4 (an index of arachidonic acid production). The production of leukotriene C4 was inhibited by the phospholipase A2 (PLA2) inhibitor ONO-RS-082. NMC is structurally homologous to BB-(5-14) except that Gln7 in BB is replaced by His7 in NMC. Therefore, substitution of Gln7 for His7 may alter the signal transduction systems to include the PTK and PLA2 pathways. U-73122 inhibited Ca2+ spiking and amylase secretion induced by NMC and BB. However, the PTK inhibitor genistein and the PLA2 inhibitor ONO-RS-082 inhibited secretion induced by NMC but not that induced by BB. In contrast to nonmammalian BB receptors, which primarily use the PLC pathway, the rat BB receptor is linked to three different signal transduction systems: PLC, PTK, and PLA2 pathways.

Diazepam-binding inhibitor33–50 elicits Ca2+ oscillation and CCK secretion in STC-1 cells via L-type Ca2+ channels

We recently isolated and characterized 86-amino acid CCK-releasing peptide from porcine intestinal mucosa. The sequence of this peptide is identical to that of porcine diazepam-binding inhibitor (DBI). Intraduodenal administration of DBI stimulates the CCK release and elicits pancreatic secretion in rats. In this study we utilized a murine tumor cell line (STC-1 cells) that contains CCK to examine if DBI directly acts on these cells to stimulate CCK release. We investigated the cellular mechanisms responsible for this action. We showed that DBI33-50, a biologically active fragment of DBI1-86, significantly stimulated CCK secretion in STC-1 cells. This action was abolished by Ca2+-free medium. The mean basal intracellular Ca2+ concentration ([Ca2+]i) was 52 nM in fura 2-loaded STC-1 cells. DBI33-50 (1-1,000 nM) elicited Ca2+ oscillations; DBI33-50 (10 nM) increased the oscillation frequency to 5 cycles/10 min and elicited a net [Ca2+]iincrease (peak - basal) to 157 nM. In contrast, bombesin and forskolin caused an initial transient [Ca2+]ifollowed by a small sustained [Ca2+]iplateau. Withdrawal of extracellular Ca2+ abolished Ca2+ oscillations stimulated by DBI33-50. L-type Ca2+ channel blockers nifedipine and diltiazem (3-10 μM) markedly attenuated DBI-stimulated Ca2+ oscillations. In other cell types L-type Ca2+ channels are activated by cAMP-protein kinase A. DBI33-50 failed to stimulate cAMP formation in STC-1 cells. Similarly, DBI33-50 had no effect on myo-inositol 1,4,5-trisphosphate concentration ([IP3]), whereas bombesin caused an eightfold increase in [IP3] over basal. In addition, inhibitors of phospholipase C (U-73122), phospholipase A2 (ONO-RS-082), and protein tyrosine kinase (genistein) did not alter the Ca2+ oscillations elicited by DBI33-50. It appears that DBI33-50 acts directly on STC-1 cells to elicit Ca2+oscillations via the voltage-dependent L-type Ca2+ channels, resulting in the secretion of CCK. Mediation of this action is by intracellular mechanisms independent of the traditional signal transduction pathways, including phospholipase C, phospholipase A2, protein tyrosine kinase, and cAMP systems.

A possible role for Ca2+/calmodulin-dependent protein kinase IV during pancreatic acinar stimulus–secretion coupling

Ca(2+)/calmodulin-dependent protein kinases (CaMKs) are important intracellular mediators in the mediation of stimulus-secretion coupling and excitation-contraction coupling in a wide variety of cell types. We attempted to identify and characterize the functional roles of CaMK in mediating pancreatic enzyme secretion. Immunoprecipitation and immunoblotting studies using a CaMKII or CaMKIV antibody showed that rat pancreatic acini expressed both CaMKII and CaMKIV. Phosphotransferase activities of CaMKs were measured by a radioenzyme assay (REA) using autocamtide II, peptide gamma and myosin P-light chain as substrates. Although CaMKII and CaMKIV use autocamtide II as a substrate, peptide gamma is more efficiently phosphorylated by CaMKIV than by CaMKII. Intact acini were stimulated with cholecystokinin (CCK)-8, carbachol (CCh) and the high-affinity CCK-A receptor agonist, CCK-OPE, and the cell lysates were used for REA. CCK-8, CCh and CCK-OPE caused a concentration-dependent increase in CaMKs activities. When autocamtide II was used, maximal increases were 1.5-1.8-fold over basal (20.2+/-2.0 pmol/min/mg protein), with peaks occurring at 20 min after cell stimulation. In separate studies that used peptide gamma, CCK-8, CCh and CCK-OPE dose-dependently increased CaMKIV activities. Maximal increases were 1.5-2.4-fold over basal (30.7+/-3. 2 pmol/min/mg protein) with peaks occurring at 20 min after cell stimulation. Peak increases after cell stimulation induced by peptide gamma were 1.8-2.8-fold higher than those induced by autocamtide II. CCK-8, CCh and CCK-OPE also significantly increased phosphotransferase activities of myosin light chain kinase (MLCK) substrate (basal: 4.4+/-0.7 pmol/min/mg protein). However, maximal increases induced by MLCK substrate were less than 10% of those occurring in peptide gamma. Characteristics of the phosphotransferase activity were also different between autocamtide II and peptide gamma. When autocamtide II was used, elimination of medium Ca(2+) in either cell lysates or intact cells resulted in a significant decrease in the activity, whereas it had no or little effect when peptide gamma was used. This suggests that Ca(2+) influx from the extracellular space is not fully required for CaMKIV activity and Ca(2+) is not a prerequisite for phosphotransferase activity once CaMKIV is activated by either intracellular Ca(2+) release or intracellular Ca(2+) oscillations. The specific CaMKII inhibitor KN-62 (50 microM) had no effect on the CaMKIV activity and pancreatic enzyme secretion elicited by CCK-8, CCh and CCK-OPE. The specific MLCK inhibitor, ML-9 (10 microM), also did not inhibit CCK-8-stimulated pancreatic amylase secretion. In contrast, wide spectrum CaMK inhibitors, K-252a (1 microM) and KT5926 (3 microM), significantly inhibited CaMKIV activities and enzyme secretion evoked by secretagogues. Thus, CaMKIV appears to be an important intracellular mediator during stimulus-secretion coupling of rat pancreatic acinar cells.

Secretagogue-induced Ca2+ oscillations in isolated canine gastric chief cells

Agonist-induced changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) of isolated canine gastric chief cells were evaluated by microspectrofluorometry of superfused fura-2 loaded cells. Application of high concentrations of carbachol (CCh, 10(-5) M) or cholecystokinin octapeptide (10(-8) M) resulted in biphasic Ca2+ mobilization comprising an initial large transient followed by a small sustained elevation above the prestimulation level. Submaximal concentrations of CCh (10(-6) M) or cholecystokinin (10(-9) M) led to either a transient series of large amplitude Ca2+ spike(s) or a higher frequency of sustained Ca2+ oscillations of smaller amplitude. Cholecystokinin at 10(-10) M induced only sustained Ca2+ oscillations. Elimination of Ca2+ from the medium had no immediate effect on oscillations indicating an intracellular source of Ca2+. Thus the Ca2+ signalling mode in chief cells is dependent on agonist concentrations.