Biomedical subjects

A Piiper

Publications and source records attributed to A Piiper.

At least 19 recordsLinked to original sources

PubMed2005

Apoptotic cytokeratin 18 neoepitopes in serum of patients with chronic hepatitis C.

In patients with chronic hepatitis C, alanine aminotransferase (ALT) levels do not accurately reflect the extent of liver inflammation. The discrepancy between ALT level and liver damage could be related to the mode of cell death. In the present study, we quantified serum levels of apoptotic cytokeratin 18 (CK-18) neoepitopes that are generated by activated caspases during apoptosis. Apoptotic CK-18 neoepitopes were quantified by enzyme linked immunosorbent assay in sera from patients with chronic hepatitis C and elevated ALT levels (n = 72), patients with chronic hepatitis C and persistently normal ALT levels (n = 27) and healthy controls (n = 19). Serum CK-18 neoepitope levels were strongly correlated with ALT (r = 0.659, P < 0.0001) and the histology activity index (r = 0.374, P < 0.001). Patients with chronic hepatitis C and persistently normal ALT levels had higher apoptotic CK-18 neoepitope levels than healthy controls (P = 0.03) but lower levels than patients with chronic hepatitis C and elevated ALT levels (P < 0.001). Highest serum CK-18 neoepitope levels were observed in patients with cirrhosis (P = 0.002). Hence apoptotic CK-18 neoepitopes in serum of patients with chronic hepatitis C are associated with ALT level and histological liver damage. Serum apoptotic CK-18 neoepitope levels are elevated both in patients with chronic hepatitis C and elevated ALT levels as well as in patients with normal ALT levels indicating that also patients with chronic hepatitis C and normal ALT have an increased hepatocyte loss by apoptosis.

Adult↗

PubMed2004

Electroporation of subcutaneous mouse tumors by rectangular and trapezium high voltage pulses.

The artificial electrotransfer of bioactive agents such as drugs, peptides or therapeutical nucleic acids and oligonucleotides by membrane electroporation (MEP) into single cells and tissue cells requires knowledge of the optimum ranges of the voltage, pulse duration and frequency of the applied pulses. For clinical use, the classical electroporators appear to necessitate some tissue specific presetting of the pulse parameters at the high voltage generator, before the actual therapeutic pulsing is applied. The optimum pulse parameters may be derived from the kinetic normal mode analysis of the current relaxations due to a voltage step (rectangular pulse). Here, the novel method of trapezium test pulses is proposed to rapidly assess the current (I)/voltage (U) characteristics (IUC). The analysis yields practical values for the voltage U(app) between a given electrode distance and pulse duration t(E) of rectangular high voltage (HV) pulses, to be preset for an effective in vivo electroporation of mouse subcutaneous tumors, clamped between two planar plate electrodes of stainless steel. The IUC of the trapezium pulse compares well with the IUC of rectangular pulses of increasing amplitudes. The trapezium pulse phase (s) of constant voltage and 3 ms duration, following the rising ramp phase (r), yields a current relaxation which is similar to the current relaxation during a rectangular pulse of similar duration. The fit of the current relaxation of the trapezium phase (s) to an exponential function and the IUC can be used to estimate the maximum current at a given voltage. The IUC of the falling edge (phase f) of the trapezium pulse serves to estimate the minimum voltage for the exploration of the long-lived electroporation membrane states with consecutive low-voltage (LV) pulses of longer duration, to eventually enhance electrophoretic uptake of ionic substances, initiated by the preceding HV pulses.

Animals↗

PubMed2004

Mutations in the putative HCV-E2 CD81 binding regions and correlation with cell surface CD81 expression.

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.

Adult↗

PubMed2004

Receptor tyrosine kinases are signaling intermediates of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) can utilize receptor tyrosine kinases (RTKs) to mediate important cellular responses such as proliferation, differentiation and survival. Recent advances in the field suggest that GPCR-induced transactivation of RTKs might be important for diseases such as cancer and cardiac hypertrophy. Depending on the receptor and cell type, GPCR signaling involves activation of several different RTKs. By activating different subsets of RTKs, GPCRs can fine-tune their effects on target cells. Furthermore, RTK-independent signaling pathways also initiated by GPCRs may modify the biological read out of the transactivated RTKs. This review focuses on the mechanisms how GPCRs and intracellular messengers elicit transactivation of different RTKs and the resulting different biological responses.

Cell Communication↗

PubMed2001

Subcellular distribution and function of Rab3A-D in pancreatic acinar AR42J cells.

Members of the Rab3 subfamily have been linked to the regulation of exocytosis in secretory cells. We have recently shown by Northern blot analysis that pancreatic acinar-like AR42J cells express all four Rab3 isoforms (Rab3A-D). In the present study, we examined the subcellular distribution of endogenously expressed Rab3 proteins and their relation to the amylase-containing secretory compartment in dexamethasone-differentiated AR42J cells. Rab3A and Rab3C were enriched in the cytosol, Rab3B and Rab3D in the membrane fraction. Accordingly, confocal immunocytochemistry revealed that Rab3B and Rab3D were located in a compartment close to the plasma membrane, whereas anti-Rab3A and Rab3C mainly stained the cytosol. Sucrose density gradient centrifugation showed overlapping, but distinct localization of each Rab3 isoform. The order of banding from lighter to more dense fractions was Rab3C < Rab3A < Rab3B < Rab3D. All Rab3 proteins at least partially colocalized with amylase immunoreactivity. Transient overexpression of Rab3 proteins showed that Rab3A inhibited cholecystokinin (CCK)-induced amylase secretion, whereas overexpression of other Rab3 isoforms had no significant effect. In conclusion, our data indicate that the different Rab3 proteins show distinct subcellular distribution, suggesting different impact on exocrine secretory response in dexamethasone-differentiated AR42J cells.

Amylases↗

PubMed2001

Coupling of endothelin receptors to the ERK/MAP kinase pathway. Roles of palmitoylation and G(alpha)q.

Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ET(A) and ET(B). To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G(alpha) protein binding capacity. Endothelin-1 stimulation of wild-type ET(A) or ET(B) induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ET(A) and ET(B) mutants failed to stimulate ERK. A truncated ET(B) lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G(alpha)q, but not of G(alpha)i or G(alpha)s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ET(A)- and ET(B)-mediated ERK activation whereas blockage of phospholipase C-beta completely abrogated endothelin-promoted ERK activation through ET(A) and ET(B) in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G(alpha)q/phospholipase C-beta/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway.

Amino Acid Sequence↗

PubMed2001

Interferon alfa down-regulates CD81 in patients with chronic hepatitis C.

CD81 protein has been shown to bind hepatitis C virus (HCV) envelope 2 (E2) glycoprotein in vitro and may act as a (co)receptor for HCV. Regulation of CD81 expression by interferon alfa (IFN-alpha) and ribavirin could thereby affect the response to antiviral therapy. In the present study, the effects of IFN-alpha and ribavirin on CD81 protein and CD81 mRNA were assessed in peripheral blood lymphocytes (PBL) and isolated human hepatocytes by fluorescence-activated cell sorter (FACS) analysis and real-time polymerase chain reaction (PCR), respectively. In addition, regulation of CD81 in PBL was investigated in 10 patients treated with combination therapy. Incubation with IFN-alpha (50 U/mL) down-regulated total CD81 in PBL (81.7 +/- 11.6% of control; P =.003) and in isolated human hepatocytes (91.6 +/- 8.1% of control; P =.034). Incubation with IFN-alpha with and without ribavirin (2.2 microg/mL) significantly reduced cell surface-associated CD81 protein (83.9 +/- 10.3% of control; P =.003). PBL of untreated patients chronically infected with HCV had significantly higher levels of total CD81 protein compared with PBL obtained from healthy donors (631.1 +/- 93.3 vs. 538.9 +/- 95.2 relative fluorescence units [RFU]; P =.030). Pretreatment cell surface-associated CD81 protein was lower in patients infected with genotype HCV-3 than those infected with HCV-1 (111.8 +/- 15.0 vs. 162.0 +/- 41.3 RFU; P =.019). Furthermore, cell surface-associated CD81 protein was lower 4 weeks after initiation of therapy in patients with an initial virologic response compared with initial virologic nonresponders (110.5 +/- 8.5 vs. 139.8 +/- 27.5 RFU; P =.057). In conclusion, IFN-alpha and ribavirin regulate CD81 expression in vitro and in vivo. CD81 expression correlates with HCV genotype and initial virologic response in patients with chronic hepatitis C.

Antigens, CD↗

PubMed2000

Involvement of the platelet-derived growth factor receptor in angiotensin II-induced activation of extracellular regulated kinases 1 and 2 in human mesangial cells.

In mesangial cells angiotensin II (Ang II) has been shown to activate extracellular regulated kinases 1 and 2 (ERK1/2). Here, we studied the role of the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) in Ang II-induced ERK1/2 activation in human mesangial cells. Ang II induced activation of ERK1/2 via the AT(1) receptor, and this response was blocked by the PDGFR-selective tyrosine kinase inhibitor AG1295, but not by AG1478, an EGFR-selective tyrosine kinase inhibitor, indicating participation of the PDGFR, but not of the EGFR in Ang II-induced ERK1/2 activation. In agreement with this assumption, Ang II caused tyrosine phosphorylation of the PDGFR and the adapter protein Shc in an AG1295-sensitive fashion. In conclusion, our data show that Ang II-induced activation of mitogenic signalling cascade in human mesangial cells involves ligand-independent activation of the PDGFR, but not of the coexpressed EGFR.

Angiotensin II↗

PubMed2000

Polo-like kinase1, a new target for antisense tumor therapy.

The Polo-like kinase 1 (Plk1) is a highly conserved mitotic serine/threonine kinase which is commonly overexpressed in cancer cell lines. Plk1 positively regulates mitotic progression by activating the CDC25C-CDK1 amplification loop and by regulating late mitotic events, primarily the ubiquitin-dependent proteolysis. In the present study, an antisense strategy against Plk1 mRNA was developed to specifically inhibit cell proliferation of cancer cells in cell culture and in the nude-mouse tumor model. Among 41 phosphorothioate antisense oligodeoxynucleotides tested, the 20-mer JWG2000 strongly inhibited expression of Plk1 in cultured A549 cells, leading to loss of cell viability, and exhibited anti-tumor activity in nude mice A549 xenograft. JWG2000 did not inhibit growth and viability of primary human mesangial cells and human amnion fibroblasts.

Animals↗

PubMed2000

Pertussis toxin inhibits cholecystokinin- and epidermal growth factor-induced mitogen-activated protein kinase activation by disinhibition of the cAMP signaling pathway and inhibition of c-Raf-1.

Pertussis toxin (PTx), which inactivates G(i/o) type G proteins, is widely used to investigate the involvement of G(i/o) proteins in signal transduction. Activation of extracellular-regulated kinases 1 and 2 (ERK1/2) by G protein-coupled receptors has been described to occur either through a PTx-insensitive pathway involving activation of phospholipase C and protein kinase C (PKC), or through a PTx-sensitive pathway involving G(i)betagamma-mediated activation of Src. Cholecystokinin (CCK) activates ERK1/2 by a PKC-dependent, and thus presumably PTx-insensitive, pathway. However, CCK has recently been shown to induce activation of G(i) proteins in addition to G(q/11). In the present study, PTx partially inhibited CCK-induced ERK1/2 activation in pancreatic AR42J cells, although activation of phospholipase C was not reduced. PTx also inhibited ERK1/2 activation in response to the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) as well as activation of c-Raf-1 by EGF and CCK. In contrast, PTx, CCK, and EGF had only minor effects on A-Raf and B-Raf activity. Forskolin, a direct activator of adenylyl cyclase, inhibited CCK- and EGF-induced activation of c-Raf-1 and ERK1/2 in a manner similar to that of PTx. In PTx-treated cells, the cAMP content was increased and forskolin did not further inhibit CCK- and EGF-induced activation of c-Raf-1 or ERK1/2. In conclusion, the present study shows that PTx-sensitivity of receptor-induced ERK1/2 activation could be a consequence of disinhibition of the adenylyl cyclase signaling pathway, which in turn causes inhibition of c-Raf-1 activation rather than indicating involvement of a PTx-sensitive G protein in this signaling pathway.

Adenylate Cyclase Toxin↗

PubMed1999

Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki- ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki- ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.

Enzyme Activation↗

PubMed1999

Lipoprotein(a) stimulates growth of human mesangial cells and induces activation of phospholipase C via pertussis toxin-sensitive G proteins.

BACKGROUND: Renal disease is commonly associated with hyperlipidemia and correlates with glomerular accumulation of atherogenic lipoproteins, for example, lipoprotein(a) [Lp(a)], and mesangial hypercellularity. Specific binding of Lp(a) to mesangial cells and induction of c-myc and c-fos expression has been demonstrated. Therefore, in this study, we investigated a possible growth stimulatory effect and mode of action of Lp(a) in human mesangial cells. METHODS: Lp(a) was purified from the regenerate fluid of a dextran sulfate column-based low-density lipoprotein apheresis system. Human mesangial cells were isolated by a sequential sieving technique from patients undergoing tumor nephrectomy. DNA synthesis was measured by [3H]-thymidine incorporation. The intracellular calcium concentration ([Ca2+]i) was determined by Fura 2-fluorescence, and inositol 1,4,5-trisphosphate (1,4,5-IP3) concentration was measured by a radioreceptor assay. RESULTS: The data show that Lp(a) bound to the cells with a Kd of 17.0 micrograms/ml and increased DNA synthesis and cell proliferation. Lp(a) caused a rapid increase in 1,4,5-IP3 and [Ca2+]i via a pertussis toxin-sensitive mechanism. The phospholipase C (PLC) inhibitor U73122 abolished Lp(a)-induced cell proliferation. In contrast, vasopressin-induced increase in 1,4,5-IP3 and [Ca2+]i was pertussis toxin insensitive. CONCLUSION: This study revealed that Lp(a) stimulates growth of human mesangial cells. Lp(a)-induced signaling involves binding to a receptor and stimulation of PLC via Gi proteins. Stimulation of PLC appears to be essential for the growth stimulatory effect of Lp(a). Whether these effects of Lp(a) contribute to the pathophysiology of renal disease needs to be determined.

Blood Proteins↗

PubMed1998

Lipoprotein (a) stimulates mitogen activated protein kinase in human mesangial cells.

Evidence suggests an important role of elevated serum lipoproteins in the progression of renal glomerulosclerosis. We report here that lipoprotein (a) (Lp(a)) increased phosphorylation and activity of mitogen activated protein kinase (MAPK) in human mesangial cells. When protein kinase C (PKC) was depleted by long-term incubation with the phorbol 12-O-myristate 13-acetate the effect of Lp(a) on MAPK activation was completely inhibited. Forskolin, a stimulator of the adenylyl cyclase, and dibutyryl-cAMP reduced the effect of Lp(a) on MAPK phosphorylation and activation. We conclude that Lp(a) stimulates the MAPK cascade via activation of PKC and that activation of protein kinase A counteracts Lp(a) induced MAPK activation in human mesangial cells.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1997

Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-beta1.

We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-beta1, -beta3, -gamma1 and -delta1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-beta1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-beta1 but not PLC-beta3, -gamma1 or -delta1. P1 also activated recombinant PLC-beta1, indicating direct activation of PLC-beta1 by Rab3 effector-domain peptides. To investigate further the structure-function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue)[KVKTVYRH (EC50 of 1 nM to stimulate amylase release) approximately TVGIDFKVKTVYRH > TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-beta1 isoenzyme.

Amino Acid Sequence↗

PubMed1997

Differential expression of Rab3 isoforms during differentiation of pancreatic acinar cell line AR42J.

Evidence is accumulating that low molecular weight GTPases of the Rab3 subfamily regulate exocytosis in secretory cells. The different isoforms of Rab3 (Rab3AD) probably have opposite roles in secretory processes. In the present study we have investigated differentiation-dependent expression of Rab3 isoforms in the pancreatic acinar carcinoma cell line AR42J. The data show that AR42J cells express all four different isoforms of Rab3. Dexamethasone treatment, which increases the number of secretory granules and secretagogue-induced exocytosis several-fold, was accompanied by down-regulation of Rab3A, Rab3C, and Rab3D mRNA, whereas Rab3B mRNA and Rab3 protein were found up-regulated at the mRNA and protein level, suggesting that Rab3B might be involved in the secretory function of AR42J cells.

Amino Acid Sequence↗

PubMed1997

Epidermal growth factor inhibits bombesin-induced activation of phospholipase C-beta1 in rat pancreatic acinar cells.

BACKGROUND & AIMS: Epidermal growth factor (EGF) inhibits bombesin-induced activation of phosphoinositide-specific phospholipase C (PLC) in pancreatic acini. The aim of this study was to investigate the mechanism by which EGF inhibits bombesin-induced activation of PLC. METHODS: Intact pancreatic acini were pretreated with pertussis toxin to study the role of Gi/o-type heterotrimeric guanosine triphosphate-binding regulatory proteins (G proteins) in EGF-induced modulation of PLC activity. To identify the PLC isoenzyme(s) and Gi/o protein subtype(s) involved in EGF-induced signaling, PLC activity was measured in isolated pancreatic acinar membranes that had been preincubated with immunoneutralizing antibodies raised against various PLC-beta isoenzymes or G protein alpha-subunits. The association of PLC-beta1 and Gi/o-type G proteins was studied by pertussis toxin-catalyzed [32P]adenosine diphosphate ribosylation of PLC-beta1 immunoprecipitates. RESULTS: Pertussis toxin pretreatment of pancreatic acini abolished the inhibitory effect of EGF on bombesin-induced PLC activation and amylase release. Anti-PLC-beta1, -beta3, and Gq/11alpha antibodies inhibited bombesin-induced PLC activity by 50%, 35%, and 65%, respectively. Anti-Gi1-2alpha, but not a Gi3alpha-specific antibody, abolished the inhibitory effect of EGF on bombesin-induced PLC activity. Pertussis toxin-sensitive G proteins coimmunoprecipitated with PLC-beta1 in an EGF-dependent fashion. CONCLUSIONS: EGF inhibits bombesin-induced activation of PLC-beta1 by a mechanism involving activation of Gi1-2 proteins in pancreatic acinar membranes.

Amylases↗

PubMed1997

CCK, carbachol, and bombesin activate distinct PLC-beta isoenzymes via Gq/11 in rat pancreatic acinar membranes.

Four different isoforms of phospholipase C-beta (PLC-beta 1-4) have been discovered, raising the important question of whether a distinct receptor activates a single PLC-beta isoenzyme or a subset of PLC-beta isoenzymes. The present study was designed to investigate activation of PLC-beta isoenzymes by three different PLC-activating agonists that bind to different receptor entities, i.e., cholecystokinin octapeptide (CCK-8), bombesin, and carbachol in rat pancreatic acinar membranes. PLC activity was measured using exogenous [3H]phosphatidylinositol 4,5-bisphosphate as substrate. Western blot analysis of pancreatic acinar membranes revealed the presence of PLC-beta 1, -beta 3, -gamma 1, and -delta 1, but not of PLC-beta 2, -beta 4, -gamma 2, and -delta 2. Preincubation of the membranes with anti-PLC-beta 1 or -beta 3 antibody reduced agonist-induced activation of PLC. The order of sensitivity toward inhibition by anti-PLC-beta 1 antibody was CCK-8 > bombesin > carbachol. An opposite order of sensitivity was found for inhibition of PLC activity by anti-PLC-beta 3 antibody (carbachol > bombesin > CCK-8). Anti-PLC-beta 2, -beta 4, -gamma 1, -gamma 2, -delta 1, and -delta 2 antibodies had no effect. Preincubation of the membranes with an antibody raised against the COOH terminus of the alpha-subunit of Gq/11 proteins inhibited PLC activity in response to all three different receptor agonists to a similar extent, whereas anti-Gi alpha 1-2 and anti-Gi alpha 3 antibodies had no effect. In conclusion, the data of the present study indicate that CCK-8 and carbachol activate PLC-beta 1 and PLC-beta 3, respectively, whereas bombesin activates both PLC-beta 1 and PLC-beta 3. Activation of PLC-beta by these receptor agonists is mediated by Gq/11.

Animals↗

PubMed1997

Epidermal growth factor activates phospholipase C-gamma 1 via G(i)1-2 proteins in isolated pancreatic acinar membranes.

In the present study, isolated pancreatic acinar membranes were used to investigate the mechanism of epidermal growth factor (EGF)-induced activation of phospholipase C (PLC). The data show that EGF caused a rapid and strong increase in tyrosine phosphorylation of the EGF receptor, with a maximum 5-15 s after the beginning of the incubation followed by a decline. With use of [3H]phosphatidylinositol 4,5-bisphosphate as an exogenous substrate, PLC activity increased fourfold on exposure of the membranes to EGF (85 nM). In contrast, EGF-induced tyrosine phosphorylation of PLC-gamma 1 was rather small, indicating that tyrosine phosphorylation of PLC-gamma 1 is not proportional to changes in PLC activity. EGF-induced activation of PLC was strongly inhibited by pretreatment of the membranes with pertussis toxin, by an antibody raised against a COOH-terminal sequence shared by alpha-subunits of the inhibitory G proteins G(i)1 and G(i)2, and by an anti-PLC-gamma 1 antibody, whereas anti-G(i) alpha 3, anti-Gq/11 alpha, and anti-PLC-beta 1 antibodies had no effect. In contrast, pertussis toxin or the anti-G(i) alpha 1-2 antibody had no effect on EGF-induced tyrosine phosphorylation of PLC-gamma 1. EGF promoted association of G(i) proteins with both the EGF receptor and PLC-gamma 1 with similar kinetics as EGF-receptor autophosphorylation. All EGF-induced responses were abolished by the specific tyrosine kinase inhibitor pp60v-arc (137-157), suggesting that EGF-receptor tyrosine kinase activity is essential for G(i)1-2-mediated activation of PLC-gamma 1. However, there was no evidence of tyrosine phosphorylation of G(i) alpha 1-2. Taken together, these data show that EGF causes activation of PLC-gamma 1 by a mechanism requiring activation of G(i)1-2 and only a small increase in tyrosine phosphorylation of PLC-gamma 1.

Animals↗