Adenosine A(2A) and A(2B) receptors in cultured human and porcine coronary artery endothelial cells.

We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-¿2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbon yl¿eth yl]phenyl]ethylamino-5'- ethylcarboxamidoadenosine ((125)I-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B(max)) of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K(d)) of 1. 17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC(50)): 5'-(N-ethylcarboxamido)adenosine (NECA) >/= CGS-21680 > 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A(2) receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A(2A) adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.

Genomic organization of the Hoxa4-Hoxa10 region from Morone saxatilis: implications for Hox gene evolution among vertebrates.

The physical mapping of Hox gene clusters from a limited number of vertebrates has shown an overall conservation in gene organization in which major evolutionary changes appear to be primarily restricted to the deletion of one or more genes, with the exception of the amplification of additional clusters as postulated from zebrafish. We have sequenced a 31 kb region of the HoxA cluster from the teleost Morone saxatilis (striped bass), both to provide a detailed physical map of this region and to better understand the nature of Hox cluster evolution among vertebrate taxa. We identified five linked Hox genes: Hoxa4, Hoxa5, Hoxa7, Hoxa9, and Hoxa10, which are organized similarly to those of other vertebrates. Furthermore, we have documented the absence of the Hoxa6 and Hoxa8 genes within the 31 kb contig. Comparison of our results to those published for other vertebrates suggests that the absence of Hoxa6 is a common characteristic of teleosts, whereas the absence of Hoxa8 is common to vertebrates in general, with the possible exception of zebrafish. Further comparisons between the HoxA genes from Morone with those from the pufferfish, Fugu rubripes, revealed the likely presence of a previously unreported Hoxa7 gene, or gene fragment, in the Fugu genome, which suggests that the Hoxa7 gene, unlike Hoxa6 or Hoxa8, is present in teleosts. In addition to these differences in vertebrate Hox cluster structure, we also observed a marked reduction in the length of the Hoxa4--a10 region between vertebrate lineages representative of teleosts and mammals. Comparative analysis of HoxA cluster organization among teleosts and mammals suggests that cluster length reduction and lineage-specific gene loss events are hallmarks of Hox cluster evolution.

Coupling of pancreatic gastrin/cholecystokinin-B (G/CCKB) receptors to phospholipase C and protein kinase C in AR4-2J tumoral cells.

Gastrin and cholecystokinin (CCK) have proven trophic effects on the gut. We have previously demonstrated that these peptides stimulate an early event in cellular proliferation, namely ornithine decarboxylase activity (ODC), in a rat exocrine pancreatic cell line AR4-2J. Furthermore, this effect is mediated through a G/CCKB receptor. Thus, in the present study we sought to examine the signal transduction mechanisms linked to the G/CCKB receptor occupancy. Both gastrin and CCK induced a rapid (maximum at 40 s) increase in inositol triphosphates (InsP3) and diacylglycerol (DAG) formation in a dose-dependent manner (EC50 = 5.6 nM) that quickly returned to baseline. Although InsP3 levels remained at baseline, DAG levels demonstrated a second gradual increase that was maximal at 15 min. CCK/gastrin efficiency to stimulate DAG and InsP3 formation (EC50 = 5.6 nM) could be correlated to the G/CCKB receptor occupancy, suggesting a coupling of this receptor to phospholipase C. To examine the involvement of protein kinase C (PKC) activation in the increase in ODC activity, we stimulated the AR4-2J cells with the phorbol ester TPA and observed an increase in ODC activity with a maximal effect at 100 nM. TPA stimulation of ODC activity was completely abolished by the PKC inhibitor staurosporine (50 nM). However, 50 nM staurosporine inhibited only 65% of the gastrin and CCK induced increase in ODC activity suggesting that a portion of the G/CCKB receptor-mediated increase in ODC activity is PKC independent.

Characterization of univectorial polyamine transport in duodenal crypt cell line.

High levels of polyamines have been identified in the lumen of the intestines. Luminal polyamines are involved in normal mucosal growth and may be the primary source of extracellular polyamines for tumors grown in animals undergoing polyamine antimetabolite therapy. The vectorial movement of polyamines across an in vitro model of the gut was studied in epithelial cells grown in culture. IEC-6 cells were plated on either plastic or raised inserts. Cells grown on plastic were employed to define the kinetic constants for putrescine and spermidine uptake. Eadie-Hofstee plot analysis of putrescine uptake was characteristic of a single class of transporter with a Michaelis constant (Km) of 4.85 +/- 0.57 microM and a maximal velocity (Vmax) of 627 +/- 85 pmol x 15 min-1 x 10(6) cells-1. The plot for spermidine uptake was curvilinear and representative of the interaction of spermidine with two sites: Km 1 and 2 are 0.26 +/- 0.13 and 2.1 +/- 0.77 microM; Vmax 1 and 2 are 177 +/- 50 and 429.5 +/- 70 pmol x 15 min-1 x 10(6) cells-1, respectively. Calmodulin antagonism blocked the uptake of putrescine and the low-affinity but not high-affinity spermidine uptake system. Seven-day postconfluent cells grown on plastic inserts were used to study the vectorial movement of polyamines in a polarized epithelium. The apical membrane domain expressed two sites with similar kinetic constants to those observed when cells were grown on plastic. In contrast, however, the basolateral membrane did not transport polyamines. Spermidine uptake through this membrane was only a fraction of that in the apical membrane and was completely nonspecific.(ABSTRACT TRUNCATED AT 250 WORDS)

Paraquat uptake in the cultured gastrointestinal epithelial cell line, IEC-6.

Although the major target organ of paraquat toxicity is the lung, the primary route of absorption of the herbicide is across the gastrointestinal epithelium. Thus, uptake of paraquat was investigated in the rat duodenal crypt cell line, IEC-6. The herbicide was toxic; as measured by cell number, the LD50 was 75 microM. Using 2 microM [14C]paraquat as tracer, uptake of the herbicide was found to be slow but linear over 24 hr at 37 degrees C. No accumulation was observed at 4 degrees C. Inhibition studies showed paraquat inhibited both putrescine and spermidine uptake after only a 15-min incubation. The Km for putrescine was increased when incubated in the presence of paraquat whereas Vmax was not changed, suggesting a competitive mode of inhibition. In the reverse situation, putrescine also inhibited paraquat uptake (IC50 10 microM). Paraquat uptake was reduced in a dose-dependent manner by the putative calmodulin antagonist W-7, but was not affected by KN-62, a Ca2+/calmodulin kinase II specific inhibitor. These results provide evidence that paraquat uptake occurs through the polyamine transport system in IEC-6 cells. Furthermore, this process is modulated by intracellular events involving a major signaling protein, Ca2+/calmodulin.

Stimulation of rat pancreatic tumoral AR4-2J cell proliferation by pituitary adenylate cyclase-activating peptide.

In the present work the effects of the novel neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) on both AR4-2J cell growth and the modulation of ornithine decarboxylase activity were investigated. Both PACAP38 and the amidated form PACAP27 caused a concentration-dependent stimulation of AR4-2J cell growth; the maximal increase was seen at 1 nmol/L (30% above control, P less than 0.01) with a half-maximal effect at 0.01 nmol/L. Ornithine decarboxylase activity was also increased by PACAP in a dose-dependent manner, reaching half-maximal stimulation at 0.5 nmol/L. The addition of 1 nmol/L of somatostatin analog SMS 201-995 totally suppressed PACAP-stimulated AR4-2J cell growth. Vasoactive intestinal polypeptide (3 mumol/L) and 8-bromo-cyclic adenosine monophosphate (1 mmol/L) had no effect on cell proliferation. Treatment of cells by pertussis toxin (25 ng.mL-1.day-1) suppressed PACAP-stimulated AR4-2J cell growth but enhanced PACAP-induced stimulation of adenylate cyclase activity. It was concluded that PACAP stimulates AR4-2J cell proliferation by a mechanism that seems independent of cyclic adenosine monophosphate production. The mitogenic effect of PACAP depends on a pertussis toxin-sensitive G protein and is associated with an increase of ornithine decarboxylase activity.

Effect of basic fibroblast growth factor on ornithine decarboxylase activity and mRNA expression in a pancreatic tumoral cell line (AR4-2J).

Basic fibroblast growth factor (bFGF) is a potent mitogen for various cell types. Induction of ornithine decarboxylase (ODC) activity is one of the early events triggered in proliferating cells. Our aim was to study the effect of bFGF on ODC activity and ODC mRNA expression in a pancreatic tumoral cell line, AR4-2J. Following kinetic and dose-response studies, we found that maximal stimulation (150% over control) of ODC activity occurred after 3 h of bFGF treatment (10(-9) M), the EC50 being 20 pM. To elucidate the mechanism by which bFGF stimulates ODC activity, we measured the ODC mRNA levels by Northern blot hybridization using a 32P-labeled rat cDNA probe. In AR4-2J cells treated with bFGF at 10(-9) M over 120 min, ODC mRNA expression was transiently increased by 71.6% at 60 min. Furthermore, bFGF was also able to stimulate ODC mRNA synthesis in the presence of cycloheximide. In conclusion, in AR4-2J cells of pancreatic origin, bFGF stimulates ODC gene transcription. This effect contributes to the stimulation of ODC enzymatic activity and to the proliferative effect of bFGF on this cell line.

Putrescine and spermidine uptake is regulated by proliferation and dexamethasone treatment in AR4-2J cells.

Polyamines are essential for cell growth and differentiation. Their specific uptake contributes to the regulation of intracellular polyamine levels. In this study, we describe the modulation of this transport mechanism in a rat tumoral pancreatic acinar cell line (AR4-2J) and analyze the transport system characteristics of the normal rat pancreatic acini. Normal acini had a common carrier for spermidine and spermine, like AR4-2J cells, but not a specific putrescine carrier. Intracellular polyamine deprivation enhanced putrescine and spermidine uptake of AR4-2J cells with no modification of polyamine carrier affinity. Uptake was modulated during growth and decreased for both polymaines at confluence. AR4-2J cell differentiation with dexamethasone prevented cell proliferation and diminished uptake of both putrescine and spermidine without affecting their respective carrier affinities. Our data show, first, that the polyamine transport system could be modulated by polyamine metabolism with no change in its affinity characteristics. Second, in rat pancreatic acinar cells, neoplastic transformation was partly characterized by induction of a high-affinity putrescine carrier. This phenotype was not reversed by dexamethasone-induced cell differentiation.

Lorglumide and loxiglumide inhibit gastrin-stimulated DNA synthesis in a rat tumoral acinar pancreatic cell line (AR42J).

Many reports emphasized the role of gastrin as growth factor on normal gastrointestinal mucosa and pancreas. In the present study, we analyzed the proliferative effects of cholecystokinin (CCK) and gastrin peptides on a rat tumoral pancreatic cell line, AR42J, which possesses both CCKA and CCKB receptor subtypes. The results showed a good correlation between the binding of gastrin to CCKB receptor [Kd 1.125 +/- 0.3 (SD) nM] and its ability to either induce ornithine decarboxylase activity [50% effective concentration, 0.6 +/- 0.3 nM] and [3H]-thymidine incorporation [50% effective concentration, 2 +/- 0.4 nM]. Furthermore, the ability of different cholecystokinin and gastrin antagonists such as proglumide and asperlicin derivatives (respectively, CR1409, CR1505, and L364,718) were tested. We found that all antagonists displaced 125I-labeled gastrin binding, with the following order of potencies: L364,718 greater than CR1409 greater than CR1505 greater than proglumide. Furthermore, the 50% inhibitory concentration of CR1409 and CR1505 to inhibit gastrin stimulated ornithine decarboxylase activity (an early event involved in cell proliferation) and [3H]thymidine incorporation were in agreement with their constants of inhibition (Ki) on gastrin binding. The L364,718 compound, at a concentration which fully occupied the CCKA without affecting the CCKB, had no effect on gastrin stimulated ornithine decarboxylase activity and [3H]thymidine incorporation. In addition, this compound appeared to be a full agonist on CCKB receptor. These results confirm the implication of the CCKB receptor in the proliferative response of AR42J cells to gastrin.

Characterization of putrescine- and spermidine-transport systems of a rat pancreatic acinar tumoral cell line (AR4-2J).

Polyamines are polycationic molecules essential for cell growth and differentiation. Recent work has focused on cell polyamine-transport systems as a way to regulate intracellular polyamine levels. In this study, we demonstrate the presence of two different active transporters for putrescine and spermidine in a rat tumoral cell line (AR4-2J). The first has a Km of 3.1 microM and a Vmax of 3.7 pmol/15 min per micrograms of DNA for putrescine and the second a Km of 0.42 microM and a Vmax of 4.7 pmol/15 min per micrograms of DNA for spermidine. Competition studies performed between the polyamines confirm the difference between these two carriers; one has an equal affinity for the three main polyamines, and the other has a lower affinity for putrescine. Amino acids do not share this transport system, which is Na(+)-independent. Choline chloride inhibits selectively and in a dose-responsive manner the uptake of putrescine without affecting that of spermidine. These data demonstrate that AR4-2J cells possess two polyamine transporters; one is specific for aminopropyl groups (spermidine and spermine), and the other is choline-sensitive, but cannot discriminate between aminobutyl (putrescine) and aminopropyl groups.

Regulation of proliferation by fibroblast growth factor in a pancreatic cancer cell line.

Basic fibroblast growth factor (bFGF) is a potent mitogen for various cell types. We report here the first study of the effects of bFGF on a digestive tract-derived cell line. The effect of bFGF on the proliferation of AR4-2J cells, tumor cells of acinar pancreatic origin, was investigated together with modulation of ornithine decarboxylase (ODC) activity, an intracellular event involved in cell proliferation. bFGF caused a concentration-dependent stimulation of AR4-2J cell growth, with a half maximal effect (EC50) at 22 +/- 2 pM. ODC activity, assayed by the CO2-trapping method, was also increased by bFGF in a dose-dependent manner, reaching half-maximal stimulation at 20 pM. We conclude that bFGF is a very potent growth promoting factor for cells of pancreatic origin, already effective at picomolar concentrations. The parallelism between the growth assay and the ODC activity assay implicates the involvement of ODC activity in the pathway of the mitogenic effect of bFGF. The stimulation of ODC activity therefore seems to be a reliable early marker for cell proliferation in this model.

Gastrin modulates growth of a rat acinar pancreatic cell line: receptor analysis and signal transduction.

Recent studies have demonstrated the presence of both CCKA and CCKB receptors on dog and guinea pig pancreas. Although CCKA receptors are implicated in enzymatic secretion, biological effects of CCKB receptors are still unknown. We have previously found that a rat acinar pancreatic cell line (AR4-2J) possesses both receptor subtypes. In this work we report the ability of various CCK/gastrin agonists and antagonists to bind with these receptors. We found that gastrin, pentagastrin and Gastrin/CCK4 induce ornithine decarboxylase activity, an early event involved in cell proliferation, as well as 3H-thymidine incorporation. Furthermore, these effects occur at doses at which these peptides interact only with the CCKB receptor subtype. In view of these data we propose that modulation of AR4-2J cell growth by gastrin agonists specifically involve occupation of the CCKB receptor.

Cholecystokinin and gastrin peptides stimulate ODC activity in a rat pancreatic cell line.

Recent studies have demonstrated that cholecystokinin (CCK) receptors are heterologous in peripheral tissues and in the central nervous system and that CCK-gastrin (CCK-G) peptides are potent trophic factors for the gastrointestinal tract. In the present study we used 125I-labeled gastrin and 125I-labeled CCK to demonstrate the heterogeneity of CCK receptors on a rat pancreatic acinar cell line (AR4-2J) and analyze the role of these receptors in increasing the activity of ornithine decarboxylase. Pharmacological analysis of radioligand binding fit well with the presence of two different receptors: 1) a CCK-selective receptor having the characteristics of the CCK receptor present on normal pancreatic cells and 2) a high-affinity, low-selectivity CCK-G binding site that interacts with all CCK-G peptides sulfated and nonsulfated. CCK-G peptides stimulate ornithine decarboxylase activity with the following order of potencies (EC50): G-(2-17)-ds (0.1 nM) greater than or equal to CCK-9 (0.25 nM) greater than or equal to pentagastrin (0.4 nM) greater than CCK-4 (6 nM). This stimulation was not inhibited by CCK antagonist (asperlicin) at a concentration range that blocks the CCK receptor but does not compete with 125I-labeled gastrin binding to the CCK-G receptor. These results, obtained with CCK-G agonists and antagonists, demonstrate that ornithine decarboxylase stimulation in these cells is mediated via the CCK-G receptor.

Direct inhibitory effects of a somatostatin analog, SMS 201-995, on AR4-2J cell proliferation via pertussis toxin-sensitive guanosine triphosphate-binding protein-independent mechanism.

Somatostatin has been demonstrated to negatively regulate pancreatic growth in vivo. In this study we used the AR4-2J rat pancreatic acinar tumor cell line to investigate the effect of a stable somatostatin analog, SMS 201-995 (SMS) on cell proliferation. SMS induced an antiproliferative effect on both serum or epidermal growth factor (EGF)-induced cell proliferation; exposure of the cells for 48 h to SMS caused a slight inhibition of serum-induced proliferation (maximal inhibition, 26%) and abolished the growth-promoting effect of EGF. Maximal effect was observed with 10 nM SMS, and half-maximal (IC50) effect with 0.06-0.1 nM SMS. Binding studies with an iodinated derivative of SMS, [125I-Tyr3]SMS, revealed the presence of a single class of high affinity binding sites on AR4-2J plasma membranes with an equilibrium dissociation constant of 0.2 +/- 0.03 nM and a binding site number of 1.1 +/- 0.07 pmol/mg protein. Addition of the nonhydrolyzable GTP analog, guanosine 5-[gamma-thio] triphosphate (GTP gamma S), increased the rate of dissociation of the specifically bound peptide in agreement with the coupling of somatostatin receptors with a GTP-binding regulatory protein. The good agreement between the IC50 for SMS inhibition of cell proliferation and the apparent Kd for binding indicates that the characterized binding sites are the somatostatin receptors that mediate the antiproliferative effect of SMS. When cells were grown in serum-free medium EGF stimulated AR4-2J cell proliferation with half-maximal (ED50) and maximal effects at 0.6 and 10 nM EGF, respectively. This stimulatory effect of EGF was mediated by specific receptors, since binding studies with [125I]EGF indicated that AR4-2J cells contained a single class of EGF receptors (13,000 sites/cell), with an affinity constant for [125I]EGF (Kd = 0.9 +/- 0.09 nM) close to the ED50 for EGF stimulation of cell growth. To examine if SMS-induced growth inhibition involved a cAMP-dependent mechanism we first studied the effect of SMS on cAMP production. SMS had no effect on basal cAMP, but completely inhibited VIP-stimulated cAMP production with an IC50 of 0.2 nM. Pertussis toxin, which is known to abolish the inhibitory effect of somatostatin on adenylate cyclase activity in AR4-2J cells, did not reverse the ability of SMS to inhibit cell proliferation as well as EGF-induced cell proliferation. These data indicate that the antiproliferative effect of SMS does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

Interrelation of secretory and trophic responses in the exocrine pancreas.

This paper was addressed to know whether early events in mitogenesis (activation of the Na+/H+, activation of ornithine decarboxylase and formation of cyclic AMP) are involved in pancreatic cell proliferation and mediate secretory process. The AR4-2J cell line was used. Analogues of amiloride inhibited cell proliferation but had no effect on amylase release. Activation of ornithine decarboxylase was triggered via a CCK B receptor type not involved in pancreatic secretion. Inhibition of cyclic AMP was not involved in inhibition of cell proliferation caused by somatostatin. Specific effectors might be related either to the secretory or to the trophic pathway. Another possibility is that multiple receptor sub-classes are linked to specific pathways.

CCK and gastrin inhibit adenylate cyclase activity through a pertussis toxin-sensitive mechanism in the tumoral rat pancreatic acinar cell line AR 4-2J.

(Thr28,Nle31)CCK(23-33) (CCK-9) and gastrin(1-17)I (gastrin) inhibited adenylate cyclase activity in membranes from the tumoral rat pancreatic acinar cell line AR 4-2J through a Bordetella pertussis toxin-sensitive mechanism. This contrasted with the stimulatory effect exerted by CCK-9 on adenylate cyclase activity in membranes from normal rat pancreas. The relative potency of CCK-9, gastrin, and related peptides in inhibiting adenylate cyclase, when confronted with previous evidence, suggests that 'non-selective CCK-gastrin CCK-B receptors' predominating over 'selective CCK-A receptors' in the AR 4-2J cell line, favored the coupling of the first receptors to adenylate cyclase through Gi, while CCK-A receptors capable of stimulating the enzyme through Gs were detected only after Bordetella pertussis toxin pretreatment.

Characterization of muscarinic receptors in human pancreatic membranes.

Crude membranes (27,000 x g pellets) from three normal human pancreata were prepared. Muscarinic receptors were investigated by the ability of three antagonists (atropine, pirenzepine, and AF-DX 116) and three agonists (carbamylcholine, oxotremorine, and pilocarpine) to inhibit [3H]NMS binding. These receptors showed for pirenzepine and AF-DX 116 a M2 beta specificity, typical of secretory glands and smooth muscle, that was comparable to that of rat pancreatic membranes, i.e., a low affinity for the two antagonists (Ki of 0.4 and 0.2 microM, respectively). In addition, these receptors were predominantly in a low affinity state for the agonist carbamylcholine (Ki of 100 microM).

Secretin receptors in human pancreatic membranes.

Crude membranes (27,000 g pellets) from five normal human pancreases were prepared. In the presence of GTP, the peptides of the secretin family stimulated adenylate cyclase activity, their order of potency being: secretin greater than helodermin greater than peptide histidine isoleucinamide (PHI) greater than or equal to vasoactive intestinal peptide (VIP) greater than growth hormone releasing factor (GRF) (1-29)-NH2. In addition, helodermin and PHI were more efficient than secretin. Secretin (3-27) inhibited fully the secretin stimulation and partially only the helodermin and PHI stimulation of the enzyme. Secretin receptors were investigated by the ability of secretin and related peptides to inhibit tracer binding. [125I]Secretin binding was fully inhibited by secretin (Kd 0.8 nM), helodermin (Kd 200 nM), and PHI (Kd 250 nM). VIP and GRF(1-29)-NH2 induced partial (20%) inhibition at a high 10 microM concentration. The fragments secretin (2-27), (3-27), (4-27), and (7-27) showed the same low potency and efficacy based on their ability to stimulate adenylate cyclase and to occupy secretin receptors. The analogues [Val5]secretin and [Ala2]secretin had a higher potency than secretin. Based on this comparison of adenylate cyclase stimulation and [125I]secretin binding inhibition, it is tempting to conclude that the human pancreas: (a) possesses highly specific secretin receptors and (b) such receptors could not fully account for the whole pattern of adenylate cyclase activation by related peptides, so that the presence of an added type of "helodermin-PHI-preferring" receptors is suggested.

Characterisation of gastrin receptors on a rat pancreatic acinar cell line (AR42J). A possible model for studying gastrin mediated cell growth and proliferation.

Trophic changes of the exocrine pancreas after in vivo gastrin (G)/CCK treatment are well documented but up to now the study of the mechanisms involved is restricted by the lack of a suitable in vitro model. Nevertheless the in vivo trophic effect induced by gastrin/CCK peptides has been associated with an increase of ornithine decarboxylase (ODC) activity. In the present work, using the AR42J cell line in which CCK receptors and stimulation of amylase release by CCK peptides has already been demonstrated, we investigated the presence of gastrin binding sites and the possible modulation of proliferation by an inhibitor of ODC activity. 125I-BH-G17ns binding is saturable, reversible and specific. Potencies of the different analogues tested are G17ns greater than CCK8 greater than CCK8ns greater than or equal to G6s greater than G/CCK4. Furthermore dBt cGMP, a non-peptide antagonist for CCK receptors, does not compete for gastrin binding. This indicates the existence of a subclass of gastrin binding sites. Difluoromethyl ornithine (DFMO) (1 mM), an irreversible inhibitor of ODC, inhibits cell growth from day 3 up to day 7. This growth inhibition is dose dependent and closely related to an intracellular polyamine modulation. Putrescine and spermidine levels fell under detectable values while spermine levels increased. All these data suggest that this cell line could be a useful in vitro model to study the mechanisms of gastrin induced growth control.

Molecular properties of solubilized CCK receptor from guinea-pig pancreas.

In order to characterize the CCK receptor in guinea-pig pancreas, iodinated CCK-39 was bound to pancreatic membranes and the reversible complex was solubilized using various non-denaturing detergents. In term of recovery of ligand stabilized receptors, the relative potencies were Zwittergent 3-14 greater than CHAPS = CHAPSO greater than digitonin greater than MEGA 10 greater than octyl beta-D-glucopyranoside. The stability of receptor complexes was increased by glycerol. Chromatographic analysis revealed that digitonin was the most efficient detergent for disaggregation of CCK receptor complex since it yielded a 76 kDa component in addition to the large components obtained after solubilization with CHAPS and Zwittergent. Furthermore, CCK receptors were covalently labelled using dissuccinimidyl suberate or UV irradiation of labelled membranes by photoactivable radioiodinated CCK-39 and subsequently solubilized by CHAPS + SDS or by SDS alone. A predominant molecule was characterized by chromatography (76 kDa) and SDS-PAGE (89 kDa). In addition to this component, other components having molecular masses of 130-150 kDa, 57 kDa and 40 kDa were detected by SDS-PAGE. They correspond to minor bands. These bands, except the 40 kDa band, were protected from covalent labelling by the presence of CCK-39 (10(-6) M) during initial incubation. Reduction under beta-mercaptoethanol mainly resulted in the decrease of high molecular weight aggregates (Mr greater than 200 kDa). We concluded that for a given detergent a specific molecular weight pattern of solubilized CCK receptor complex is achieved. The minimal component had a molecular mass of 71-84 kDa according to the method of biochemical analysis used.