Biomedical subjects

M Dufresne

Publications and source records attributed to M Dufresne.

At least 19 recordsLinked to original sources

PubMed2006

Interactions of B16F10 melanoma cells aggregated on a cellulose substrate.

There is evidence that the shape of cells and their contact with a matrix direct the growth and the differentiation of both normal and cancer cells. Cells in 3D culture resemble the in vivo situation more closely than do those in conventional 2D cultures. We have studied the interactions and functions of B16F10 mouse melanoma cells, which spread and grow well on tissue culture polystyrene (tPS), when they were made to aggregate on cellulose-coated Petri dishes (CEL). This aggregation of melanoma cells on CEL was Ca2+ dependent and mediated by N-cadherins. The levels of N-cadherin and beta-catenin transcripts in cells cultured on CEL and tPS were similar, but those on CEL contained less beta-catenin protein. Immunoprecipitation and immunostaining showed that both N-cadherins and beta-catenins were present at the membranes of cells on CEL. Cells proliferated significantly more slowly after 48 h on CEL and the cellulose coating caused most of them to arrest in G1. We also compared the melanin contents and tyrosinase activity of cells on CEL and controls grown on tPS. Melanogenesis was induced in cells aggregated on CEL. A cellulose substrate thus appears to be an outstanding tool for studying cell-cell interactions and cell functions in 3D cultures.

Animals↗

PubMed2006

Cholecystokinin-2 receptor modulates cell adhesion through beta 1-integrin in human pancreatic cancer cells.

Several lines of evidence suggest that gastrin and the CCK-2 receptor (CCK2R) could contribute to pancreatic carcinogenesis by modulating processes such as proliferation, cell adhesion or migration. In the current study, we used a 'cancer gene array' and identified beta1-integrin subunit as a new gastrin-regulated gene in human pancreatic cancer cells. We also demonstrated that Src family kinases and the phosphatidylinositol-3-kinase (PI-3-kinase) pathway play a crucial role in the expression of beta1-integrin induced by gastrin. Our results also showed that gastrin modulates cell-substrate adhesion via beta1-integrin. Indeed, using blocking anti-beta1-integrin monoclonal antibodies, we completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we observed that in response to gastrin, beta1-integrin is tyrosine phosphorylated by Src family kinases and associates with paxillin, a scaffold protein involved in focal adhesion and integrin signalling. This mechanism might be involved in gastrin-induced cell adhesion. Moreover, we showed in vivo that targeted CCK2R expression in the pancreas of Elas-CCK2 mice leads to the overexpression of beta1-integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals.

Animals↗

PubMed2005

Renal allograft tolerance in DLA-identical and haploidentical dogs after nonmyeloablative conditioning and transient immunosuppression with cyclosporine and mycophenolate mofetil.

BACKGROUND: Canine models of bone marrow and renal transplantation have provided important preclinical data relevant to developing novel therapeutic protocols for hematopoietic and solid organ transplantation in human beings. Nonmyeloablative transplantation has been shown to induce stable mixed hematopoietic chimerism in normal dogs and correct the phenotype of canine pyruvate kinase deficiency and Glanzman's thrombasthenia. In this study, we investigated the potential for inducing renal allograft tolerance using a nonmyeloablative bone marrow transplantation strategy that induces mixed chimerism in DLA-identical dogs. METHODS: Reciprocal renal allografts were performed in 4 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy total body irradiation [TBI]) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with and without simultaneous bone marrow transplantation. Two DLA-identical control dogs received reciprocal renal allografts without TBI or immunosuppression with CSP and MMF. Serum creatinine (Cr) concentration was monitored to assess renal allograft function. RESULTS: The renal allografts were acutely rejected in the 2 DLA-identical dogs without TBI or immunosuppression. There was long-term (>1 year) renal allograft survival as evidenced by a normal (<2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous bone marrow transplantation. CONCLUSIONS: Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal allograft tolerance in DLA-identical and DLA-haploidentical dogs without donor/host mixed hematopoietic chimerism. These findings suggest it may be possible to induce tolerance to solid organ transplants without the need for chronic immunosuppressive therapy or stable hematopoietic chimerism in the setting of both DLA-matched and haploidentical transplants.

Animals↗

PubMed2002

clap1, a gene encoding a copper-transporting ATPase involved in the process of infection by the phytopathogenic fungus Colletotrichum lindemuthianum.

A screen for insertional mutants of Colletrichum lindemuthianum, the causative agent of common bean anthracnose, led to the identification of a non-pathogenic, lightly colored transformant. This mutant is unable to induce disease symptoms on intact or wounded primary leaves of seedlings and plantlets of Phaseolus vulgaris. In vitro, it exhibits normal vegetative growth, sporulation and conidial germination, but the cultures remain beige instead of becoming black. Microscopic examination revealed that this mutant forms fewer appressoria than the wild-type strain, and these are misshapen and poorly melanized. Molecular analyses indicated that the mutagenic plasmid had targeted clap1, a gene encoding a putative copper-transporting ATPase sharing 35% identity with the human Menkes and Wilson proteins and the product of the CCC2 gene of Saccharomyces cerevisiae. Complementation of the non-pathogenic beige mutant with a wild-type allele of clap1 restored both pathogenicity and pigmentation. Conversely, replacement of the wild-type allele with a disrupted clap1 gene gave rise to non-pathogenic beige transformants. Compared with the wild-type strain, extracts from clap1 mutants were found to have very low levels of phenol oxidase activity. These observations suggest that the clap1 gene product may be involved in the pathogenicity of C. lindemuthianum strains because of its role in delivering copper to secreted cuproenzymes, such as the phenol oxidases that mediate the polymerization of 1,8-dihydroxynaphthalene to melanin.

Adenosine Triphosphatases↗

PubMed2002

Organization of cyclic AMP-dependent connexin 43 in Swiss 3T3 cells attached to a cellulose substratum.

We have previously shown that the adenylyl cyclase, which produces cyclic AMP (cAMP) in Swiss 3T3 cells, is activated by their attachment to a cellulose substratum (Cuprophan, CU). This substratum adsorbs vitronectin poorly, prevents cell spreading and causes them to aggregate. By contrast, cells spread out on polystyrene and contain low concentrations of cAMP. We have found that Connexin 43 (Cx 43) gap junction plaques are involved in this cell aggregation. MDL 12330 A, a specific inhibitor of adenylyl cyclase, prevented cell aggregation on CU and abolished Cx 43 channel clustering. But forskolin, a direct activator of adenylyl cyclase, and SBr cAMP, a cell-permeable analogue of cAMP, caused Cx 43 channel clustering in cells attached to polystyrene. Hence, Cx 43 channel clustering is regulated by cAMP in Swiss 3T3 cells. In addition, neither brefeldin A nor monensin (inhibitors of transit through the endoplasmic reticulum and Golgi apparatus), abolished Cx 43 channel clustering in cells aggregated on CU. Thus, the Cx 43 that form clusters in cells attached to CU are not dependent upon the trafficking of Cx 43 from intracellular storage sites, but are probably reorganised from the plasma membrane.

3T3 Cells↗

PubMed2001

Severe canine hereditary hemolytic anemia treated by nonmyeloablative marrow transplantation.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase (PK) deficiency can be corrected by marrow allografts from healthy littermates after a conventional high-dose myeloablative conditioning regimen. The nonmyeloablative conditioning regimen used here, which consisted of a sublethal dose of 200 cGy total body irradiation before and immunosuppression with mycophenolate mofetil and cyclosporine after a dog leukocyte antigen (DLA)-identical littermate allograft, has been found to be effective in establishing stable mixed donor/host hematopoietic chimerism in normal dogs. We explored the feasibility of nonmyeloablative marrow allografts for the treatment of canine PK deficiency and studied the effect of stable allogeneic mixed hematopoietic chimerism on the natural course of the disease. Five affected dogs received transplants, of which 3 dogs had advanced liver cirrhosis and myelofibrosis. Both complications were presumed to be due to iron overload. All 5 dogs showed initial engraftment. Two rejected their grafts after 6 weeks but survived with completeautologous marrow recovery and return of the disease. One died from liver failure on day 27 with 60% donor engraftment. Two dogs have shown sustained mixed donor/host chimerism for more than a year with 85% and 12% donor hematopoietic cells, respectively. Overall clinical response correlated with the degree of donor chimerism. The dog with the low degree of chimerism achieved partial resolution of hemolysis, but the disease symptoms persisted as manifested by increasing iron overload resulting in progression of marrow and liver fibrosis. The dog with the high degree of donor chimerism achieved almost complete resolution of hemolysis with a decrease of marrow iron content and resolution of marrow fibrosis. These observations suggest that mixed hematopoietic chimerism can be relatively safely established in dogs with PK deficiency even in the presence of advanced liver cirrhosis. However, although effective in correcting or delaying the development of myelofibrosis, a low degree of mixed chimerism was not sufficient to prevent continued hemolysis of red blood cells of host origin. Complete donor chimerism appears necessary to achieve a long-term cure.

Anemia, Hemolytic, Congenital↗

PubMed2001

Definition of tissue-specific and general requirements for plant infection in a phytopathogenic fungus.

Although plant diseases are usually characterized by the part of the plant that is affected (e.g., leaf spots, root rots, wilts), surprisingly little is known about the factors that condition the ability of pathogens to colonize different plant tissues. Here we demonstrate that the leaf blast pathogen Magnaporthe grisea also can infect plant roots, and we exploit this finding to distinguish tissue-specific and general requirements for plant infection. Tests of a M. grisea mutant collection identified some mutants that were defective specifically in infection of either leaves or roots, and others such as the map kinase mutant pmk1 that were generally defective in pathogenicity. Conservation of a functional PMK1-related MAP kinase in the root pathogen Gaeumannomyces graminis was also demonstrated. Exploitation of the ability of M. grisea to infect distinct plant tissues thus represents a powerful tool for the comprehensive dissection of genetic determinants of tissue specificity and global requirements for plant infection.

Amino Acid Sequence↗

PubMed2000

Effects of targeted replacement of the tomatinase gene on the interaction of Septoria lycopersici with tomato plants.

Many plants produce constitutive antifungal molecules belonging to the saponin family of secondary metabolites, which have been implicated in plant defense. Successful pathogens of these plants must presumably have some means of combating the chemical defenses of their hosts. In the oat root pathogen Gaeumannomyces graminis, the saponin-detoxifying enzyme avenacinase has been shown to be essential for pathogenicity. A number of other phytopathogenic fungi also produce saponin-degrading enzymes, although the significance of these for saponin resistance and pathogenicity has not yet been established. The tomato leaf spot pathogen Septoria lycopersici secretes the enzyme tomatinase, which degrades the tomato steroidal glycoalkaloid alpha-tomatine. Here we report the isolation and characterization of tomatinase-deficient mutants of S. lycopersici following targeted gene disruption. Tomatinase-minus mutants were more sensitive to alpha-tomatine than the wild-type strain. They could, however, still grow in the presence of 1 mM alpha-tomatine, suggesting that nondegradative mechanisms of tolerance are also important. There were no obvious effects of loss of tomatinase on macroscopic lesion formation on tomato leaves, but trypan blue staining of infected tissue during the early stages of infection revealed more dying mesophyll cells in leaves that had been inoculated with tomatinase-minus mutants. Expression of a defense-related basic beta-1,3 glucanase gene was also enhanced in these leaves. These differences in plant response may be associated with subtle differences in the growth of the wild-type and mutant strains during infection. Alternatively, tomatinase may be involved in suppression of plant defense mechanisms.

Avena↗

PubMed2000

A GAL4-like protein is involved in the switch between biotrophic and necrotrophic phases of the infection process of Colletotrichum lindemuthianum on common bean. .

Random insertional mutagenesis was conducted with the hemibiotrophic fungus Colletotrichum lindemuthianum, causal agent of common bean anthracnose. Nine mutants that were altered in their infection process on the host plant were generated. One of these, H433 is a nonpathogenic mutant able to induce necrotic spots on infected leaves rapidly. These spots are similar to those observed during the hypersensitive reaction. Cytological observations showed that the development of the mutant H433 is stopped at the switch between the biotrophic and the necrotrophic phases. This mutant carries two independent insertions of the transforming plasmid pAN7-1. Complementation studies using the wild-type genomic regions corresponding to the two insertions showed that one is responsible for the H433 phenotype. Sequencing analysis identified a single open reading frame that encoded a putative transcriptional activator belonging to the fungal zinc cluster (Zn[II](2)Cys(6)) family. The corresponding gene was designated CLTA1 (for C. lindemuthianum transcriptional activator 1). Expression studies showed that CLTA1 is expressed in low amounts during in vitro culture. Targeted disrupted strains were generated, and they exhibited the same phenotype as the original mutant H433. Complementation of these disrupted strains by the CLTA1 gene led to full restoration of pathogenicity. This study demonstrates that CLTA1 is both a pathogenicity gene and a regulatory gene involved in the switch between biotrophy and necrotrophy of the infection process of a hemibiotrophic fungus.

Amino Acid Sequence↗

PubMed1999

Exogenous CCK and gastrin stimulate pancreatic exocrine secretion via CCK-A but also via CCK-B/gastrin receptors in the calf.

A predominance of the pancreatic cholecystokinin (CCK) receptor of the B/gastrin subtype (CCK-B/G) was reported in calves older than 1 month. Specific CCK-A and CCK-B/G receptor antagonists (SR 27897 and PD 135158, respectively) were used to identify the CCK receptor subtype involved in exogenous CCK- and gastrin-induced exocrine pancreatic responses. Conscious calves (2 months old) with catheterized pancreas, jugular vein and duodenum were used; the pancreatic juice was continuously reinfused. CCK (30 pmol kg-1 min-1, 40 min) evoked an increase in pancreatic juice flow and enzyme secretion, while the same dose of gastrin increased enzyme secretion alone. CCK-induced pancreatic secretion was abolished by SR 27897 (15 nmol kg-1 min-1, 55 min) and reduced by PD 135158 (0.15 nmol kg-1 min-1, 55 min). Gastrin-induced enzyme secretion was reduced by PD 135158 (50% to 90%) and to a lesser extent by SR 27897 (50% to 60%). These results demonstrate that CCK and gastrin in the physiological range stimulate pancreatic exocrine secretion in calves and that these effects are partly mediated by CCK-B/G receptors. Although CCK-A receptors are not predominantly expressed, they seem to play a major role in the response of pancreatic exocrine secretion to CCK.

Animals↗

PubMed1999

The CCKB/gastrin receptor is coupled to the regulation of enzyme secretion, protein synthesis and p70 S6 kinase activity in acinar cells from ElasCCKB transgenic mice.

In order to determine which physiological functions can be regulated by the pancreatic CCKB/gastrin receptor, studies were carried out on pancreatic acini from mice expressing transgenic CCKB/gastrin receptors in the exocrine pancreas (ElasCCKB mice). Acini were stimulated by sulfated gastrin in the presence of SR 27897 (1.8 microM), blocking endogenous CCKA receptors. After 30 min incubation with gastrin, the secretion of chymotrypsinogen and amylase showed superimposable monophasic dose-response curves. Enzyme secretion was detectable and maximal at 100 pM and 1 nM of gastrin, respectively. No increase in chymotrypsinogen and amylase mRNAs was detected for doses of gastrin which specifically occupy the CCKB/gastrin receptor. In contrast, gastrin stimulated total protein synthesis in isolated acini from ElasCCKB mice. [35S]Methionine incorporation into total proteins was increased dose-dependently to a maximum for 30 pM gastrin and inhibited with higher doses (> 300 pM). Gastrin stimulated p70 S6 kinase activity for concentrations ranging from 10 pM to 1 nM. Gastrin-stimulated p70 S6 kinase activity and protein synthesis were blocked by rapamycin and wortmannin. Therefore, in ElasCCKB mice acinar cells, the CCKB/gastrin receptor mediates enzyme release and protein synthesis. However, a more efficient coupling of the CCKB/gastrin receptor to protein synthesis than to enzyme secretion was demonstrated. CCKB/gastrin receptor-stimulated protein synthesis likely results from an enhancement of mRNA translation and involves phosphatidyl inositol 3-kinase and p70 S6 kinase.

Amino Acid Sequence↗

PubMed1999

Evidence for a functional role of the cholecystokinin-B/gastrin receptor in the human fetal and adult pancreas.

Gastrin (G) and cholecystokinin (CCK) are gastrointestinal neuropeptides that are released into circulation during a meal. G is also transiently expressed during embryogenic and early ontogenic development of the pancreas and is believed to act on islet-cell development. Both peptides act on pancreatic endocrine function; however, the effects are dependent on the species and on cellular and molecular underlying mechanisms that remain poorly characterized. Since CCK-B/G subtype receptor is predominant over the CCK-A subtype in the human pancreas, we hypothesized that it could be expressed by islet cells. Here we present reverse transcription-polymerase chain reaction and immunohistochemistry data demonstrating that the CCK-B/G receptor is expressed in islet cells and that islet glucagon-producing cells are the major site of CCK-B/G receptor expression in adult and fetal pancreas. Moreover, G immunoreactivity was detected in the fetal human pancreas at embryogenic week 22. G- and CCK-stimulated glucagon are released from purified human islets. Concentration of CCK and G eliciting a half-maximal level of glucagon secretion were 13 +/- 6 and 8 +/- 5 pmol/l, respectively. Maximal glucagon secretion was achieved in the presence of 30 pmol/l peptides and was similar to that obtained in the presence of 10 mmol/l L-arginine (1.6 pmol x ml(-1) x 90 min(-1)). The nonpeptide antagonist of the CCK-B/G receptor, RPR-101048, fully inhibited CCK- and G-stimulated glucagon secretion at 100 nmol/l concentration. These data are consistent with the view that the CCK-B/G receptor is involved in glucose homeostasis in adult humans and mediates the autocrine effects of G on islet differentiation and growth in the fetal pancreas.

Adult↗

PubMed1998

Transgenic CCK-B/gastrin receptor mediates murine exocrine pancreatic secretion.

BACKGROUND & AIMS: The presence of cholecystokinin (CCK)-B/gastrin receptors in the pancreas of higher mammals including humans has been shown, but their physiological function in the normal pancreas is unknown. The aim of this study was to investigate whether they couple to the secretory machinery of normal acinar cells. METHODS: A transgenic mouse strain expressing the human CCK-B/gastrin receptor in the exocrine pancreas was created. The transgenic construction used the promoter region of the elastase I gene and the human CCK-B/gastrin receptor gene. Analysis of ElasCCKB mice included polymerase chain reaction and receptor autoradiography. Molecular and binding features of the CCK-B/gastrin receptor were determined by Western blot and radioligand binding studies. Amylase secretion and inositol phosphate production assays were used in functional characterization. RESULTS: The CCK-B/gastrin receptor was expressed in the exocrine pancreas and had typical molecular and binding features. CCK and sulfated gastrin stimulated enzyme secretion with identical potencies and efficacies. They activated phospholipase C, but CCK was 60-fold less potent than sulfated gastrin. CONCLUSIONS: The data show that the CCK-B/gastrin receptor mediates exocytosis in acinar cells and can differentially couple to phospholipase C depending on the agonist. The ElasCCKB mice provide a useful model to study phospholipase C-dependent and -independent intracellular transduction pathways leading to pancreatic exocrine secretion.

Amylases↗

PubMed1998

Differential tissular expression of the CCK(A) and CCK(B) gastrin receptor genes during postnatal development in the calf.

Local and temporal expression of CCK(A) and CCK(B)/gastrin receptor genes was studied in the calf with a quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method. Cerebral cortex, antrum, fundus, gall bladder, pancreas and liver were analyzed in calves at 0, 2, 7, 21, 28 and 150 days of age. Cerebral cortex and pancreas expressed both receptor genes with a ratio between CCK(A) and CCK(B)/gastrin receptor transcripts varying according to the age. Gall bladder and fundus showed an exclusive expression of CCK(A) and CCK(B)/gastrin receptor mRNAs, respectively, with the highest levels of transcripts in newborn and 28-day-old calves. The rank order for CCK(A) receptor mRNA expression was gall bladder > pancreas > cerebral cortex >>> antrum and that for CCK(B)/gastrin receptor mRNA expression was cerebral cortex / pancreas / fundus >> antrum. No CCK(A) and CCK(B)/gastrin receptor mRNA was detected in liver, regardless of the age of calves. The present data represent a basis for a better understanding of the ontogeny of physiological functions linked to the CCK(A) and CCK(B)/gastrin receptors.

Aging↗

PubMed1998

clk1, a serine/threonine protein kinase-encoding gene, is involved in pathogenicity of Colletotrichum lindemuthianum on common bean.

A random insertional mutagenesis in Colletotrichum lindemuthianum, the causal agent of common bean anthracnose, generated four mutants that showed altered pathogenicity when tested on intact seedlings, excised leaves, and/or excised hypocotyls. One of these mutants, H290, produced very few lesions on bean leaves and appeared affected in its ability to penetrate the leaf cuticle. Molecular analyses showed that the border sequences of the unique integration site of the disrupting pAN7-1 plasmid in the mutant exhibited homology with conserved domains of serine/threonine protein kinases. The corresponding wild-type sequences were cloned and a gene replacement vector with a mutated copy harboring a selection marker constructed. Transformation of the wild-type pathogen produced a strain with a phenotype identical to the original mutant. Genomic and cDNA sequences indicated that the disrupted gene is a member of the serine/threonine protein kinase family. The gene, called clk1 (Colletotrichum lindemuthianum kinase 1), was weakly expressed in the mycelium of the wild-type strain grown on rich and minimal synthetic media but was undetectable during the infection even when a sensitive reverse transcriptase-polymerase chain reaction methodology was used. This study represents the first characterization of altered pathogenicity mutants in C. lindemuthianum produced by random mutagenesis and demonstrates the involvement of a member of the serine/threonine kinase gene family in the early steps of the infection process.

Amino Acid Sequence↗

PubMed1997

Differential expression of the CCK-A and CCK-B/gastrin receptor genes in human cancers of the esophagus, stomach and colon.

The expression of cholecystokinin (CCK) and gastrin (G) receptors in human gastrointestinal cancers remains poorly documented and is still of a controversial nature. We have measured the levels of mRNA for CCK-A and CCK-B/gastrin receptors using quantitative reverse transcription-polymerase chain reaction (RT-PCR) in primary digestive cancers and hepatic metastases. CCK-A-receptor mRNA was detected in 5 out of 8 esophageal cancers (0.1-1 fg/microg), in 5 out of 8 gastric cancers (0.05-4.2 fg/microg) and in 5 out of 12 colon cancers (0.1-1 fg/microg RNA). CCK-B/gastrin mRNA was not detected in esophageal cancers but was detected in 7 out of 8 gastric cancers (0.05-5.2 fg/microg), and in only 2 out of 12 colon adenocarcinomas (0.05-1 fg/microg RNA). The expression of the CCK-A receptor in esophageal, gastric and colon cancers and of the CCK-B/gastrin receptor in the majority of gastric adenocarcinomas screened may be an important indicator of the influence of CCK and gastrin of local or systemic origin on the growth of these cancers.

Adenocarcinoma↗

PubMed1997

Pharmacological and biochemical evidence for the simultaneous expression of CCKB/gastrin and CCKA receptors in the pig pancreas.

1. In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved. 2. Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors. 3. The binding of [125I]-BH-[Thr, Nle]CCK-9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 +/- 0.02 nM) and a binding capacity, Bmax = 110.64 +/- 12.50 fmol mg-1 protein. 4. Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK-9, gastrin, PD 135158, L-365, 260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK-9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L-365,260. 5. These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant. 6. Two distinct membrane proteins (50 and 85-100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors. 7. In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.

Affinity Labels↗

PubMed1996

Molecular cloning, developmental expression and pharmacological characterization of the CCKB/gastrin receptor in the calf pancreas.

We have cloned the calf predominant pancreatic cholecystokinin B (CCKB)/gastrin receptor cDNA. It encodes a 454 amino acid protein with 90% identity with the CCKB/gastrin receptor cloned in other species and tissues. However, the calf pancreatic CCKB/gastrin receptor contains a pentapeptide cassette within the third intracellular loop which is absent in the cloned human brain and stomach receptor. Quantification of the CCKB/gastrin receptor mRNA levels by reverse transcription polymerase chain reaction demonstrated the same level of transcripts at birth, +7 and +28 days. On the other hand, binding study with pancreatic membranes showing a dramatic increase (600-fold) in the number of CCKB/gastrin receptor sites between at birth and +28 days indicates that the development of the calf pancreatic CCKB/gastrin receptor occurs during the first 4 weeks of post-natal life. COS monkey cells (COS-7 cells) transiently transfected by the cloned cDNA exhibit binding of 125I-Bolton-Hunter-[Thr28,Ahx31]CCK-(25-33) and 125I-Bolton-Hunter-[Leu15]human gastrin-(2-17) to two affinity classes of sites. Kd values of the high affinity binding components indicate a 4-fold higher affinity of the receptor for sulfated gastrin than for CCK. Finally, the recombinant receptor is coupled to G proteins and [Ca2+]i mobilization, and is expressed as a glycoprotein of 82 kDa.

Amino Acid Sequence↗