Biomedical subjects

M R Hellmich

Publications and source records attributed to M R Hellmich.

At least 37 records · Page 2Linked to original sources

PubMed1998

Signal transduction mechanisms in neurotensin-mediated cellular regulation.

BACKGROUND: Neurotensin, an important gut hormone, binds its receptor (NTR) to stimulate proliferation of intestinal cells; the molecular mechanisms remain largely undefined. Mitogen-activated protein kinases (MAPKs) translocate to the nucleus and induce transcription factors (eg, c-Fos) in response to certain trophic agents. The purpose of this study was (1) to define the signaling mechanisms regulating neurotensin and (2) to determine whether the AP-1 transcription factor c-Fos is induced. METHODS: Expression of the NTR gene was determined in the human colon cancer cell lines KM12C, KML4A, and KM20 by Northern blot analysis and ribonuclease-protection experiments. To assess whether NTR was functionally coupled to the CA(2+)-signaling pathway, intracellular CA2+ ([Ca2+]i) mobilization was assessed by fura-2 spectrofluorometry. To determine whether the MAPK signaling pathway was activated in KM20 cells by neurotensin, Western blots for the phosphorylated forms of MAPK (p42 and p44) and in vitro kinase assays were performed. In addition, Western blots were performed to assess levels of c-Fos after neurotensin treatment. RESULTS: The NTR gene was expressed in the KM20 cell line but not in KM12C or KM12LA. The NTR in KM20 cells was functionally coupled to the CA(2+)-signaling pathway as noted by a rapid (30 seconds) mobilization of [Ca2+]i after addition of neurotensin; the neurotensin-mediated response was blocked by the NTR antagonist SR48692. Both p42MAPK and p44MAPK were stimulated by neurotensin approximately 3 to 6 minutes after treatment. Increased levels of c-Fos were demonstrated, with peak levels occurring 2 hours after addition of neurotensin. CONCLUSIONS: Our results demonstrate that neurotensin binds to its endogenous NTR in KM20 cells with stimulation of the Ca(2+)- and MAPK-signaling pathways and an increase in the levels of the AP-1 protein c-Fos. Delineating the signal transduction mechanisms regulating the cellular effects of neurotensin will provide important insights into the molecular pathways responsible for gut hormone-mediated proliferation.

Blotting, Northern↗

PubMed1997

Inhibition of neurotensin-induced pancreatic carcinoma growth by a nonpeptide neurotensin receptor antagonist, SR48692.

BACKGROUND: Recently, a nonpeptide neurotensin (NT) receptor antagonist, SR48692, was developed that selectively antagonizes the high affinity, biologically active NT binding site. The effect of SR48692 on NT-mediated growth of a human pancreatic carcinoma, MIA PaCa-2, was determined both in vitro and in vivo. METHODS: (125)I-NT binding and Northern blot analyses were performed for evaluation of the NT receptor in MIA PaCa-2 cells. Intracellular calcium ([Ca2+]i) mobilization and inositol phosphate (IP3) levels were measured. Cell growth studies were performed by counting cell numbers. Athymic nude mice were inoculated with MIA PaCa-2 cells and randomized into four groups to receive either vehicle (NT or SR48692) or NT + SR48692. RESULTS: MIA PaCa-2 cells possess both a high affinity, SR48692-sensitive and a levocabastine-insensitive NT binding site; Northern blot analysis demonstrated expression of the NT receptor. SR48692 inhibited [Ca2+]i mobilization, IP3 turnover, and MIA PaCa-2 cell growth induced by NT in a dose-dependent fashion. In in vivo experiments, NT significantly increased the size, weight, and DNA and protein content of xenografted MIA PaCa-2 tumors; SR48692 inhibited the effect of NT. CONCLUSIONS: The novel NT receptor antagonist SR48692 will be a valuable agent to delineate further the cellular mechanisms responsible for peptide-mediated growth of normal and neoplastic gut tissues.

Animals↗

PubMed1997

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q.

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.

3T3 Cells↗

PubMed1997

Regulation of growth of human gastric cancer by gastrin and glycine-extended progastrin.

BACKGROUND & AIMS: Gastrin (G-17) stimulates the growth of certain gastric and colon cancers mostly through gastrin/cholecystokinin (CCK)-B receptors. Glycine-extended gastrin (Gly-G) stimulates growth of a rat pancreatic acinar cell line; however, the effect of Gly-G on human gastric cancers is not known. The purpose of this study was to characterize the trophic effect of G-17 and Gly-G on two human gastric cancer cell lines, AGS and SIIA. METHODS: Binding analyses were performed, and cell growth was assessed by counting cells over a time course. RESULTS: G-17 stimulated growth of both AGS and SIIA cells. In AGS cells, gastrin/CCK-B receptor antagonists inhibited the effect of G-17 and competitively antagonized 125I-G-17 binding, whereas the CCK-preferring (CCK-A) receptor antagonists had no effect. In contrast, CCK-A receptor antagonists inhibited the stimulatory effect of G-17 in SIIA cells, whereas CCK-B receptor antagonists had no effect. Gly-G stimulated the growth of AGS and SIIA cells; neither the CCK-B nor the CCK-A receptor antagonists blocked this effect. CONCLUSIONS: G-17 stimulates proliferation of AGS cells through the CCK-B receptor; however, G-17-mediated growth of SIIA acts through a CCK-A-like receptor. Furthermore, Gly-G stimulates growth of human gastric cancer cell lines, possibly through a receptor other than the CCK-B or CCK-A receptor.

Cell Division↗

PubMed1997

Src-mediated activation of the human neurotensin/neuromedin N promoter.

BACKGROUND: Expression of the gene encoding the neurotensin/neuromedin N (NT/N) is developmentally regulated in the gut in a distinctive temporal and spatial fashion. Src kinase, a nonreceptor tyrosine kinase, has been implicated in the growth and differentiation of various tissues; its role in gut differentiation is not known. The purpose of this study was to determine whether the Src signaling pathway plays a role in the activation of the human NT/N promoter. METHODS: Caco-2 cells, a human colon cancer cell line that can differentiate to a small bowel phenotype, were transiently transfected with human NT/N promoter fragments linked to luciferase and various amounts of Src expression plasmids or dominant negative Raf; luciferase and beta-galactosidase activities were measured after 48 hours. RESULTS: Cotransfection of Src resulted in an approximate eightfold increase of NT/N promoter activity; mutation of a proximal activating protein-1/cyclic adenosine monophosphate responsive element site resulted in a dramatic decrease of Src-mediated NT/N induction. Cotransfection with a dominant negative Raf plasmid partially blocked Src-mediated NT/N activation. CONCLUSIONS: Src increases NT/N promoter activity in Caco-2 cells acting, in part, through a proximal AP-1/CRE promoter element. In addition, Src regulation of the NT/N promoter appears to be mediated through a Raf-dependent pathway. We propose that Src may play a role in tissue-specific gene expression in the gut.

CSK Tyrosine-Protein Kinase↗

PubMed1997

Cyclin-dependent kinase inhibitors block proliferation of human gastric cancer cells.

BACKGROUND: Olomoucine and roscovitine are novel compounds that are designed to inhibit cyclin-dependent kinases (e.g., Cdk2 and cdc2). Cdks regulate progression through key checkpoints of the cell cycle. The purpose of this study was to determine (1) whether olomoucine and roscovitine inhibit Cdk2 and cdc2 kinase activities of the human gastric cancer cell line SIIA and (2) whether olomoucine and roscovitine block cell proliferation and cell cycle progression. METHODS: SIIA cells were treated with olomoucine or roscovitine and examined for Cdk2 and cdc2 activities by using histone H1 as the substrate. Cell numbers were counted with a Coulter counter. Cell cycle distribution was analyzed by DNA flow cytometry. RESULTS: Olomoucine and roscovitine completely blocked Cdk2 and cdc2 activities in SIIA cells. Both compounds were also able to inhibit proliferation of SIIA cells, as well as three other human gastric cancer cell lines (AGS, MKN45-630, and SNU-1). Cell cycle analysis showed that treatment with olomoucine or roscovitine for 24 hours led to a decrease in the S phase population and an increase in the G2/M population. CONCLUSIONS: We have shown that Cdk inhibitors, olomoucine and roscovitine, are a new class of antineoplastic molecules with potential therapeutic benefits for gastric cancers.

Antineoplastic Agents↗

PubMed1997

Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract.

The purpose of this study was to examine the distribution and localization of an intestinal cholecystokinin (CCK)-releasing factor, called luminal CCK-releasing factor (LCRF), in the gastrointestinal tract and pancreas of the rat. RIA analysis indicates that LCRF immunoreactivity is found throughout the gut including the pancreas, stomach, duodenum, jejunum, ileum, and colon with the highest levels in the small intestine. Immunohistochemistry analysis shows LCRF immunoreactivity staining in intestinal villi, Brunner's glands of the duodenum, the duodenal myenteric plexus, gastric pits, pancreatic ductules, and pancreatic islets. These results indicate potential sources for secretagogue-stimulated release of luminal LCRF and support the hypothesis that LCRF is secreted into the intestinal lumen to stimulate CCK release from mucosal CCK cells.

Animals↗

PubMed1996

Bombesin-mediated AP-1 activation in a human gastric cancer (SIIA).

BACKGROUND: Bombesin, a gut tetradecapeptide homologous to the mammalian gastrin-releasing peptide (GRP), stimulates the growth of the human gastric cancer line SIIA through specific GRP receptors (GRP-Rs); the cellular mechanisms are not known. The purpose of our study was to (1) confirm functional GRP-R in SIIA and (2) determine whether bombesin alters the expression and binding activity of the AP-1 transcription factors, c-jun and jun-B. METHODS: SIIA cells were treated with bombesin, and intracellular calcium mobilization was measured by means of fura-2 spectrofluorometry. To assess changes in c-jun and jun-B, RNA and protein were extracted for Northern and Western blots, respectively; nuclear protein was extracted for gel mobility shifts to determine AP-1 binding activity. RESULTS: SIIA cells mobilized intracellular calcium in response to bombesin, exhibiting a functional cell-surface GRP-R. Bombesin treatment increased expression of both c-jun and jun-B mRNA by 0.5 hours, with maximal expression at 1 hour; concomitant increases in steady-state levels of c-Jun and JunB protein were identified. Moreover, bombesin increased binding of the AP-1 proteins as shown by gel shifts. CONCLUSIONS: The SIIA human gastric cancer possesses functional GRP-R coupled to the calcium second messenger pathway. Further, bombesin stimulates expression of c-jun and jun-B mRNA and protein and increases binding activity of AP-1 proteins. Delineating the cellular pathways involved in bombesin-mediated gene activation will provide important insights into the mechanisms responsible for normal and neoplastic gut growth.

Adenocarcinoma↗

PubMed1996

Molecular cloning and functional characterization of the oxytocin receptor from a rat pancreatic cell line (RINm5F).

Oxytocin (OT) and vasopressin (AVP) stimulate insulin and glucagon release from the pancreas, and evoke insulin secretion from the rat insulinoma cell line, RINm5F. To determine which AVP/OT receptor subtype is expressed in RINm5F cells, we used PCR with degenerate primers to two transmembrane domains of the AVP (V1a, V1b (or V3), V2) and OT receptors (OTRs). The single PCR fragment identified was used to obtain a full length cDNA from a RINm5F cDNA library. Comparison of the deduced amino acid sequence of this clone with uterine OTR sequences from several species (human, sheep, bovine) and to the pig kidney epithelial cell (LLC-PK1) OTR reveals a very high degree of homology. After the RIN cell OTR cDNA was stably transfected into CHO cells (CHO-OTR), the cell membranes bound iodinated oxytocin antagonist with an apparent Kd comparable to that of RIN cell membranes and those from other OT target cells. Comparison of the ligand specificities of CHO-OTR and RIN cells membranes showed that the relative Ki values of a series of OT analogues were approximately equivalent in both preparations. The rank order of apparent Ki values also corresponded to published values for the rat myometrium, where OT elicits intracellular calcium transients, and increases inositol phosphate production. In uterin endometrium and amnion cells, OT stimulates prostaglandin release. Stimulation of CHO-OTR cells with OT caused an increase in cytosolic calcium concentration originating from both intracellular and extracellular sources, and a dose-dependent increase in inositol phosphate levels. Arachidonic acid release and PGE2 synthesis were also stimulated by OT. These findings (amino acid sequence homology, binding specificity, and signal transduction/second messenger production) suggest that OTRs from RINm5F cells are indistinguishable from OTRs that have been described in other tissues. The expression of OTR in pancreatic cells implies that OT plays a role in pancreatic function.

Amino Acid Sequence↗

PubMed1995

Characterization of gastrin-releasing peptide receptor expressed in Sf9 insect cells by baculovirus.

Whereas baculovirus expression systems have been extensively used for high-level expression of steroid receptors and receptors coupled to adenylate cyclase, there are few studies on peptide receptors coupled to phospholipase C (PLC). In the present study we have expressed the murine gastrin-releasing peptide receptor (mGRP-R) in Sf9 cells using a recombinant baculovirus and characterized it structurally and functionally. mGRP-R was detectible 12 h post infection with recombinant baculovirus carrying mGRP-R cDNA and became maximal at 60 h post infection (Bmax = 6 pmol/mg protein), which is a 4-60-fold greater density than is found in native tissues. The mGRP-R in Sf9 cells assessed by affinity labeling or immunoblotting was smaller than that in native tissues (M(r) = 51 kD vs 82 kD), and the difference was due to the extent of glycosylation. In Sf9 cells the mGRP-R had at least two of the four potential extracellular glycosylation sites glycosylated, whereas in the native receptor all four were approximately equally glycosylated. In Sf9 cells the glycosylation was entirely biantennary complex, in contrast to the native mGRP-R, where it was entirely tri- and tetraantennary complex N-linked oligosaccharides. Affinity labeling studies revealed a band with an apparent molecular mass about 40 kDa higher than the 51-kDa mGRP-R band. The intensity of this band correlated with the extent of functional G protein coupling, suggesting that it may represent an mGRP-R-G protein complex. In binding studies the affinity of the mGRP-r in Sf9 cells for the agonists bombesin (Bn), GRP, and neuromedin B (NMB) varied differently with infection time: with Bn the affinity decreased 3-fold with longer infection times, with GRP it remained unchanged, and with NMB it decreased 10-fold. GPP(NH)p inhibited binding of either [125I]Tyr4Bn or [125I]GRP at 24 h post infection, but not at 96 h post infection. Agonists activated PLC, increasing both [3H]IP and [Ca2+]i; however, the efficacy of each agonist decreased with infection time. These results demonstrate that by the use of recombinant baculovirus infected Sf9 cells the PLC-linked receptor mGRP-R can be expressed in amounts significantly greater than those in native tissues. The mGRP-R expressed in these Sf9 cells is incompletely glycosylated and has less complex N-linked oligosaccharide chains, yet it is fully coupled to G proteins and activates phospholipase C, similar to the native receptor, if short infection times are used.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗

PubMed1994

Cloning and characterization of the Drosophila melanogaster CDK5 homolog.

The D. melanogaster homolog of mammalian CDK5 has been cloned and its chromosomal location determined. The gene for Cdk5 consists of 4 exons separated by 3 short introns ranging in size from 61-160 bp. Northern blot analysis revealed a single mRNA of approximately 1.6 kb that is expressed at highest levels in the adult fly. The putative amino acid sequence for Drosophila Cdk5 predicts a protein with a mass of approximately 32 kDa that is 77% identical to its mammalian counter-parts. Drosophila Cdk5 gene is located in polytene chromosomal region 52BC of the right arm of chromosome 2. This study provides the framework for a molecular genetic analysis of CDK5 function.

Amino Acid Sequence↗

PubMed1992

Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.

Recent studies have shown that there exists a family of protein kinases structurally and functionally related to the yeast cell cycle regulatory kinase cdc2 [Meyerson, M., Faha, B., Su, L.-K., Harlow, E. & Tsai, L.-H. (1991) Cold Spring Harbor Symp. Quant. Biol. 56, 177-186 and Meyerson, M., Enders, G. H., Wu, C.-L., Su, L.-K., Gorka, C., Nelson, C., Harlow, E. & Tsai, L.-H. (1992) EMBO J. 11, 2909-2917]. Two members of cdc2 family, p34cdc2 (also named cdk1) and cdk2, have been identified in mammalian cells. cdk1 kinase regulates the progression from G2 to M phase, and cdk2 kinase has been proposed to regulate the progression from G1 to S phase. In this work, we have cloned and structurally characterized a third member of the cdc2 kinase family with 58% amino acid sequence identity to mouse cdk1 and 61% identity to human cdk2. We call this kinase neuronal cdc2-like kinase (nclk) because, in contrast to either cdk1 or cdk2, nclk is expressed at high levels in terminally differentiated neurons no longer in the cell cycle. Previous studies have shown [Hisanaga, S., Kusubata, M., Okumura, E. & Kishimoto, T. (1991) J. Biol. Chem. 266, 21798-21803 and Guan, R. J., Hall, F. L. & Cohlberg, J. A. (1992) J. Neurochem. 58, 1365-1371] that cdk1 kinase, but not other structurally defined protein kinases, could phosphorylate the repeated Lys-Ser-Pro (KSP) motifs found in mammalian high and middle molecular mass neurofilament subunits in vitro, but the precise molecular nature of the endogenous neuronal KSP kinase has remained undefined. The structural similarity of nclk to cdk1 kinase and its high level of expression in terminally differentiated neurons suggest that nclk may play a role in the phosphorylation of the neurofilament KSP repeats in vivo, a function distinct from cell cycle regulation.

Amino Acid Sequence↗

PubMed1992

A high-molecular-weight squid neurofilament protein contains a lamin-like rod domain and a tail domain with Lys-Ser-Pro repeats.

Previous studies have shown that two low molecular-weight neurofilament (NF) proteins (NF-60 and NF-70) from the squid Loligo pealei are translated from mRNAs that are splice variants of a single squid NF gene. In this study, we report the isolation and characterization of cDNA clones encoding a high-molecular-weight squid NF protein (NF-220), the mRNA of which derives from the same squid NF gene. All three proteins are identical in their amino-terminal and lamin-like rod domains but differ in their carboxyl-terminal tail regions. In contrast to the short tail domains of NF-60 and NF-70, the NF-220 protein has a longer tail domain containing an acidic cluster of amino acids immediately followed by repeated copies of the sequence motif Lys-Ser-Pro. The Lys-Ser-Pro domain is similar to that of mammalian medium NF (NF-M) and high NF (NF-H) proteins, where the serines are highly phosphorylated. Except for these Lys-Ser-Pro motifs, there is surprisingly little structural similarity between the squid NF-220 protein and mammalian NF-M and NF-H proteins. Furthermore, the location of introns in squid NF-220 protein shows that it is more closely related to nuclear lamins and type III intermediate-filament proteins than to vertebrate NF proteins.

Amino Acid Sequence↗

PubMed1991

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.

Adenosine Diphosphate Ribose↗

PubMed1991

Primary structure of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified from the ovotestis of the marine mollusk Aplysia californica. The enzyme converts NAD to cyclic ADP-ribose (cADPR), which is a potent mobilizer of Ca2+. It is likely that the NADase serves to raise Ca2+ levels in the ova at appropriate times. A 1.2-kb cDNA clone containing the complete coding sequence of the native NADase protein was isolated from an unamplified ovotestis cDNA library and represents the first cloning of an NADase that generates cADPR. In vitro translation studies indicate that the protein initially has a signal sequence that may help to target it to discrete vesicles of the ova in which it is found. There are 12 cysteines in the open reading frame, two of these being in the signal sequence. No part of the sequence has significant similarity to other proteins or known nucleotide binding site consensus sequences. Northern blot analysis of poly(A)+ selected ovotestis RNA has identified an NADase mRNA of 1.85 kb. In situ hybridization analysis of cryostat sections from ovotestis has shown that the NADase mRNA is restricted to the immature ova, although the NADase protein is present in both immature and mature eggs.

Amino Acid Sequence↗

PubMed1986

Development of muscarinic cholinergic inhibition of adenylate cyclase in embryonic chick heart. Its relationship to changes in the inhibitory guanine nucleotide regulatory protein.

Parasympathetic and sympathetic innervation of the embryonic chick heart proceed non-coordinately. beta-Adrenergic agonists mediate an increase in beating rate in embryonic chick heart prior to ingrowth of the vagus nerve (Culver, N. G., and Fishman, D. A. (1977) Am. J. Physiol. 232, R116-R123) while muscarinic agonists exert relatively little effect on beating rate in hearts 2-4 days in ovo (Papanno, A. J. (1979) Pharmacol. Rev. 29, 3-33). Studies of developmental changes in the ability of muscarinic agonists to inhibit adenylate cyclase activity and their relationship to the development of a physiologic response of the embryonic chick heart to muscarinic stimulation have been inconclusive. In the current studies the ability of isoproterenol to stimulate adenylate cyclase activity did not change during development. Maximum stimulation above basal was 760 pmol of cAMP/10 min/mg of proterin with an IC50 of 1.5 X 10(-6) M for isoproterenol in homogenates of hearts 2 1/2, 3 1/2, and 10 days in ovo and 3 days posthatching. However, inhibition of isoproterenol-stimulated adenylate cyclase activity by carbamylcholine increased from 7.6% with a IC50 for carbamylcholine of 16 +/- 5.0 microM at day 2 1/2 in ovo to 29% with an IC50 of 0.4 +/- 0.1 microM at day 10 in ovo and to 43% with a IC50 of 0.6 +/- 0.1 microM at 3 days posthatching. Since previous data had demonstrated the presence of muscarinic receptors as early as 2 1/2 days in ovo (Galper, J. B., Klein, W., and Catterall, W. A. (1977) J. Biol. Chem. 252, 8692-8699), studies of developmental changes in guanine nucleotide-coupling proteins were carried out to determine whether early in development muscarinic receptors were uncoupled from a physiologic response. Studies of pertussis toxin-catalyzed ADP-ribosylation of homogenates of embryonic chick heart with [32P]NAD demonstrated the presence of two ADP-ribosylated proteins at 39,000 and 41,000 kDa, respectively. Both ADP-ribosylation and immunoblotting of homogenates with an antibody to the 39-kDa guanine nucleotide-binding protein in bovine brain demonstrated that the 39-kDa alpha protein increased 1.8-fold between days 2 1/2 and 3 1/2 in ovo and another 1.8-fold from day 3 1/2 to 10 in ovo in parallel with the increase in the extent of muscarinic inhibition of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗

PubMedDate not supplied

A functional in vitro model to examine signaling mechanisms in gastrin-mediated human cell growth.

Gastrin (G-17) is a trophic hormone with a high affinity for the cholecystokinin-B receptor (CCK-BR); the mechanisms linking receptor binding and activation of downstream events to cell growth are not known, and these studies have been hampered by the lack of a cell model. We have established a pancreatic carcinoid cell line, BON, which produces a number of gut hormones; however, these cells lack native CCK-BR. The purpose of our study was to develop a model cell line containing the CCK-BR and to characterize the cellular mechanisms involved in gastrin regulation of human cell growth. BON cells were transfected with an expression plasmid containing the human CCK-BR, and stable clones were selected using G418. Functional CCK-BR was confirmed by reverse transcriptase-polymerase chain reaction, 125I-gastrin binding, and mobilization of intracellular calcium ([Ca2+]i) in response to G-17. Stable transfectants were treated with G-17 (+/-) the CCK-BR antagonist, L365,260 (L-60); growth was assessed using a Coulter counter. G-17 stimulated the growth of the stable clones, whereas the selective CCK-BR antagonist, L-60, abolished this G-17-mediated trophic effect. We have shown that G-17, acting through the CCK-BR, mobilizes [Ca2+]i as a second messenger and stimulates cell growth. Our unique BON cell line, stably transfected with the human CCK-BR, provides a novel paradigm to further delineate signaling mechanisms in gastrin regulation of human cell growth.

Cell Division↗

PubMedDate not supplied

The role of Src family kinases in the normal and neoplastic gastrointestinal tract.

Src family kinases are a group of non-receptor tyrosine kinases that mediate signal transduction pathways involved in the growth and differentiation of normal tissues. Considerable evidence exists for a role of these proteins in neoplastic progression in various organ systems including the nervous, hematopoietic and skeletal systems. In addition, the role of the Src kinase family has been characterized for colon cancer, but only limited progress has been made in delineating the role of Src kinases in the normal gastrointestinal (GI) tract and extracolonic GI cancers. In this review, we provide an up-to-date assessment of the Src family kinases in the normal and neoplastic GI tract.

Biomarkers, Tumor↗