Identification and Characterization of a Specific Receptor for Cholecystokinin on Isolated Fundic Glands from Guinea Pig Gastric Mucosa Using a Biologically Active 125I-CCK-8 Probe

Abstract

Specific binding sites for cholecystokinin (CCK) have been identified and characterized in fundic glands isolated by collagenase treatment from guinea pig gastric mucosa using a biologically active ¹²⁵I-labeled derivative of the C-terminal octa-peptide of CCK (¹²⁵IIE-CCK-8). The time course of binding to these glands was rapid, temperature dependent and saturable. At 24, 30 and 37° C, half-maximal binding was reached at 5 min and full binding at 30 min. The addition of a large excess of CCK-8 after 15 and 30 min of binding at 24° C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of ¹²⁵IIE-CCK-8 bound to fundic glands with increasing concentrations of CCK-8 and other structurally related peptides. Gastrin II displaced 50% of the radioligand at 1.6nM, CCK-8 at 3.2nM, gastrin I at 16nM, and desulfated-CCK-8 and pentagastrin at 59nM. Secretin did not displace the radioligand from fundic glands at 1.0uM. The binding was also tissue specific as glands isolated from the antral mucosa did not contain specific binding sites for ¹²⁵IIE-CCK-8. This data provides evidence for specific receptors for CCK on gastric fundic glands that may be involved in the control of acid and pepsinogen secretion.     

Solubilization and characterization of CCK receptors from mouse pancreas

To study the characteristics of the CCK receptor, plasma membranes were prepared from mouse pancreatic acini, and CCK receptors solubilized with 1% digitonin. To measure hormone binding, the solubilized receptors were incubated with 125I-CCK at 4 degrees C and the hormone-receptor complex was precipitated with 10% polyethylene glycol. Specific 125I-CCK binding by the solubilized CCK receptor was compared to that by the plasma membrane-bound CCK receptor. Both the solubilized and the membrane-bound receptor displayed optimal binding at an acidic pH (between 6.0 and 7.0) and showed a similar sensitivity to monovalent and divalent cations. The solubilized receptors preserved their relative specificity for CCK molecules: CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. However, the soluble CCK receptor had a lower binding affinity than plasma membrane-bound receptor. Solubilized receptors preserved their relative specificity for inhibitors of CCK binding and action: dibutyryl cyclic GMP greater than N-CBZ-tryptophan greater than proglumide. Solubilized receptors had affinities for these antagonists that were similar to receptors on intact plasma membranes. These data indicate, therefore, that the specific binding properties of the CCK receptor are inherent to the solubilized glycoprotein molecules.     

Actions of cholera toxin on dispersed Chief cells from guinea pig stomach

Although much is known about the actions of cholera toxin on intestinal and extra-gastrointestinal tissues, almost nothing is known about the interaction of this toxin with cells in the stomach. In the present study, we prepared 125I-labeled cholera toxin (1900 Ci/mmol) and examined the binding of this radioligand to dispersed Chief cells from guinea pig stomach. Moreover, we examined the actions of cholera toxin on cellular cAMP and pepsinogen secretion from Chief cells. Binding of 125I-labeled cholera toxin could be detected within 5 min, was maximal by 60 min, and was increased by increasing the radioligand or cell concentrations. Inhibition of binding by unlabeled toxin indicated a dissociation constant of 3 nM and 8.7 X 10(5) cholera toxin receptors per Chief cell. In contrast to the rapidity of binding, a cholera toxin-induced increase in cAMP and pepsinogen secretion was not detected until 30-45 min of incubation. A 3 to 6-fold increase in cAMP and pepsinogen secretion was observed with maximal concentrations of cholera toxin. Binding of 125I-labeled cholera toxin and the toxin's actions on cAMP and pepsinogen secretion were inhibited by the B subunit of the toxin. Binding was not altered by other agents that have been shown to stimulate pepsinogen secretion (carbachol, CCK-8, secretin, vasoactive intestinal peptide, prostaglandin E1, or forskolin). These data indicate that Chief cells from guinea pig stomach possess a specific class of cholera toxin receptors. Binding of cholera toxin to these receptors causes an increase in cellular cAMP that stimulates pepsinogen secretion.     

Characterization of cholecystokinin receptors on guinea pig gastric chief cell membranes

The binding of cholecystokinin (CCK) to its receptors on guinea pig gastric chief cell membranes were characterized by the use of 125I-CCK-octapeptide (CCK8). At 30 degrees C optimal binding was obtained at acidic pH in the presence of Mg2+, while Na+ reduced the binding. In contrast to reports on pancreatic and brain CCK receptors, scatchard analysis of CCK binding to chief cell membranes revealed two classes of binding sites. Whereas, in the presence of a non-hydrolyzable GTP analog, GTP gamma S, only a low affinity site of CCK binding was observed. Chief cell receptors recognized CCK analogs, with an order of potency of: CCK8 greater than gastrin-I greater than CCK4. Although all CCK receptor antagonists tested (dibutyryl cyclic GMP, L-364718 and CR1409) inhibited labeled CCK binding to chief cell membranes, the relative potencies of these antagonists in terms of inhibiting labeled CCK binding were different from those observed in either pancreatic membranes or brain membranes. The results indicate, therefore, that on gastric chief cell membranes there exist specific CCK receptors, which are coupled to G protein. Furthermore, chief cell CCK receptors may be distinct from pancreatic or brain type CCK receptors.     

Regulation of free cytosolic Ca2+ in the isolated guinea pig gastric chief cells

Stimulation with COOH-terminal octapeptide of cholecystokinin (CCK8) or carbachol resulted in a rapid increase in Quin-2 fluorescence of isolated guinea pig gastric chief cells, whereas histamine, vasoactive intestinal peptide, secretin or forskolin had no effect. The minimum effective dose of CCK8 or carbachol to elicit the rise in Quin-2 fluorescence was almost similar to that for pepsinogen secretion. Removal of Ca2+ from extracellular medium or Ca2+ channel blockers did not affect CCK8- or carbachol-induced increase in Quin-2 fluorescence. Moreover, following addition of CCK8, carbachol was unable to stimulate a second increase in Quin-2 fluorescence. These results suggest that CCK8 and carbachol share common Ca2+ pools and an increase in free cytosolic Ca2+ concentration may mediate CCK8- or carbachol-induced pepsinogen secretion from gastric chief cells.     

Difference in effects of sodium fluoride and cholecystokinin on diacylglycerol accumulation and calcium increase in guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, sodium fluoride (NaF) stimulated a monophasic increase in diacylglycerol accumulation, while cholecystokinin (CCK) strongly stimulated its biphasic accumulation. NaF evoked an increase in initial Ca2+ influx rate with a slow increase in intracellular free Ca2+ concentration [( Ca2+]i), while CCK stimulated a rapid increase in [Ca2+]i followed by a late sustained phase of the [Ca2+]i increase. Lanthanum chloride (La3+) effectively blocked NaF-stimulated increase in [Ca2+]i, but it blocked only CCK-stimulated late sustained phase of [Ca2+]i increase. The effect of NaF on pepsinogen secretion was enhanced in the presence of 100 microM AlCl3. Furthermore, pertussis toxin did not affect NaF-evoked diacylglycerol accumulation at all. These results suggest that NaF may activate a pertussis-toxin insensitive guanine nucleotide regulatory protein (G protein) coupled to a signal transducing mechanism which seems to be distinct from that activated by CCK, thereby inducing increases in diacylglycerol accumulation, Ca2+ influx and pepsinogen secretion in guinea pig gastric chief cells.     

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.     

Brain CCK receptors: species differences in regional distribution and selectivity

The binding of 125I-CCK-33 to its receptors prepared from cerebral cortex and cerebellum was studied in four species: mouse, rat, hamster, and guinea pig. Only the guinea pig showed significant binding to membranes from cerebellum and this binding was comparable to that observed for cerebral cortex. In all four species, the order of potency of unlabeled analogs to compete for the binding site was CCK-8 greater than CCK-33 greater than desulfated CCK-8 greater than CCK-4. While the affinity for CCK-8 and CCK-33 was similar in the various species, the relative affinity for desulfated CCK-8 and CCK-4 was less for hamster and guinea pig, indicating species differences in receptor specificity, as well as in regional localization.     

Involvement of Ca2+ influx in F(-)-stimulated pepsinogen release from guinea pig gastric chief cells

In isolated guinea pig gastric chief cells, pepsinogen release was stimulated by NaF in a dose-dependent manner. Cholecystokinin (CCK) and Ca2+ ionophore A23187 had no additional effect on NaF-stimulated pepsinogen release. CCK caused a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) monitored by Quin-2 and markedly stimulated inositol phosphate accumulation in chief cells. By contrast, NaF did not cause any change in [Ca2+]i. NaF, even at a maximal concentration for pepsinogen release, appeared to be relatively ineffective on inositol phosphate accumulation. On the other hand, NaF markedly stimulated Ca2+ influx into chief cells. These results suggest that F- stimulates pepsinogen release probably by increasing Ca2+ influx into chief cells. Since F- is a well known activator of guanine nucleotide regulatory proteins (G proteins), it is proposed that there may exist a G protein regulating the opening of Ca2+ channel in gastric chief cells.     

Difference in effects of pirenzepine and atropine on carbachol-induced pepsinogen secretion from isolated gastric glands

The effect of pirenzepine on carbamylcholine (carbachol)-stimulated pepsinogen secretion was compared with that of atropine in the isolated guinea pig gastric glands. Pirenzepine and atropine caused a dose dependent inhibition of carbachol-stimulated pepsinogen secretion. Moreover, pirenzepine as well as atropine produced a rightward shift in the dose response curve of carbachol-stimulated pepsinogen secretion but did not alter the maximum increase in pepsinogen secretion. Results therefore demonstrate that pirenzepine acts as a specific receptor antagonist in the interaction of carbachol with its receptor on gastric chief cells. However, pirenzepine was 50 times less potent than atropine in inhibiting pepsinogen secretion. Half maximal inhibitory concentration of pirenzepine was 2 X 10(-5) M when a maximally effective concentration of carbachol was used, while that of atropine was 4 X 10(-7) M. Results, therefore, suggest that muscarinic receptor on gastric chief cells to which pirenzepine binds may be an intermediate affinity type.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Porcine ileal polypeptide causes an increase in cytoplasmic Ca2+ in both parietal and chief cells resulting in acid and pepsinogen secretion

Porcine ileal polypeptide, an enterooxyntin isolated from distal small intestinal mucosal epithelium, has been observed to stimulate gastric acid secretion in vivo as well as in vitro (Wider, M.D. et al. (1984) Endocrinology 115, 1484-1491, Wider M.D. et al. (1986) Endocrinology 118, 1546-1550). We report here that porcine ileal polypeptide stimulates both acid (aminopyrine accumulation) and pepsinogen secretion in isolated, enriched populations of guinea pig parietal and chief cells in a dose-dependent manner. Further, 10(-9) M porcine ileal polypeptide caused an increase in cytoplasmic Ca2+ concentration in both parietal and chief cells similar in magnitude to that observed with gastrin-17 (10(-8) M) (as measured by both fura-2 and aequorin) and cholecystokinin octapeptide (CCK-OP) (10(-8) M), respectively. Porcine ileal polypeptide has been observed to cause no stimulation of cAMP production in gastric glands from guinea pigs (Gespach, C., personal communication) nor is there any effect of medium Ca2+ depletion on acid production observed with guinea pig gastric mucosal sections. It is concluded that porcine ileal polypeptide, at concentrations similar to circulating levels observed in plasma of normal pigs (5 x 10(-9) M), acts directly on the parietal and chief cells to cause the mobilization of intracellular Ca2+ from the stores resulting in acid and pepsinogen secretion. These experiments demonstrate that this peptide is a potent enterooxyntin and chief cell secretagogue which acts via the same signal transduction mechanisms as gastrin and cholecystokinin.     

Cellular distribution of gastric chief cell protein kinase C activity: differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin.

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose-dependent, 6-fold increase in pepsinogen secretion and membrane-associated PKC activity. Similarly, dose-response curves for pepsinogen secretion and the increase in membrane-associated PKC activity induced by a membrane-permeant DAG (1-oleoyl-2-acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 microM) and carbachol (0.1 microM to 1.0 mM) caused a 4-fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA- and DAG-induced pepsinogen secretion is accompanied by increased membrane-associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.     

Characterization of [3H]pentagastrin binding in guinea pig gastric glands--an alternative convenient ligand for receptor binding assay

The binding of [3H]pentagastrin to guinea pig gastric glands was specific, saturable and of high affinity (Kd = 5 nM). The relative order of potencies for gastrin and CCK analogs in displacing [3H]pentagastrin binding correlated well with those obtained using [125I]gastrin and their reported biological potencies for stimulating acid secretion. Nonselective CCK/gastrin antagonists including carbobenzoxy-CCK (26-32), proglumide and benzotript, but not the selective peripheral CCK antagonist, asperlicin, inhibited specific [3H]pentagastrin binding. The results indicate that [3H]pentagastrin labels physiologically relevant gastrin receptors in guinea pig gastric glands.     

Combined effect of phorbol ester and, A23187 or dibutyryl cyclic AMP on pepsinogen secretion from isolated gastric glands

In isolated guinea pig gastric glands, pepsinogen secretion was stimulated by the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in a dose dependent manner. Calcium-deprivation from the medium resulted in the decrease in TPA-induced pepsinogen secretion. The combination of 0.4 microM Ca2+ionophore A23187 and TPA stimulated pepsinogen secretion slightly higher than the calculated additive value for each agent. This synergistic effect of the agents supports a role of calcium-activated, phospholipid-dependent protein Kinase (protein Kinase C) in gastric pepsinogen secretion. Furthermore, pepsinogen secretion was also stimulated by dibutyryl cyclic AMP (dbc AMP) and dbc AMP slightly enhanced TPA-induced pepsinogen secretion. Results suggest that gastric chief cells possess at least two different secretory pathways for pepsinogen which are probably dependent on protein kinase C and cyclic AMP, respectively.     

Low-affinity CCK-1 receptors inhibit bombesin-stimulated secretion in rat pancreatic acini--implication of the actin cytoskeleton

EXPERIMENTAL OBJECTIVES: Stimulation of low-affinity CCK-1 receptors on pancreatic acini leads to inhibition of enzyme secretion. We studied signal transduction mechanisms to identify potential causes for the reduced secretion. RESULTS: Co-stimulation experiments with CCK, CCK-JMV-180, and bombesin revealed an inhibition of bombesin-stimulated enzyme secretion by low-affinity CCK-1 receptors. Binding of 125I-gastrin-releasing peptide (the mammalian analogue of bombesin) to acini after CCK preincubation was not altered. After a short preincubation of acini with high concentrations of CCK, intracellular calcium remained responsive to bombesin. In contrast to bombesin or CCK at concentrations of 10(-10) M or lower, high concentrations of CCK caused a strong activation of p125 focal adhesion kinase (p125(FAK)) and a marked reorganisation of the actin cytoskeleton. CONCLUSIONS: Inhibitory mechanisms triggered by low-affinity CCK-1 receptors interrupt enzyme secretion from pancreatic acini at late stages in the signal transduction cascades since bombesin receptor binding and early signalling events remained intact after CCK preincubation. A reorganisation of the actin cytoskeleton is suggested to be the mechanism by which low-affinity CCK-1 receptors actively interrupt enzyme secretion stimulated by other receptors.     

Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors

Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R-/-) or M3 (M3R-/-) receptor or that lacked both receptors (M(1/3)R-/-) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R-/- mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R-/- with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M(1/3)R-/- double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M(1/3)R-/- mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.     

Cholecystokinin induces the interaction of its receptor with a guanine nucleotide binding protein

To evaluate the relation between the pancreatic cholecystokinin (CCK) receptor and guanine nucleotide-binding protein(s) we studied the effects of nucleotides on 125I-CCK binding to pancreatic acinar plasma membranes, 125I-CCK binding to solubilized 125I-CCK receptors, and the stability of the solubilized 125I-CCK-receptor complex. In plasma membranes, guanine nucleotides both inhibited CCK binding and increased the dissociation of CCK from its receptor. The potency of the nucleotides studied was GTP gamma S = GMP-PNP greater than GTP much greater than ATP. When membranes were solubilized with digitonin, subsequent binding of CCK was insensitive to guanine nucleotides including GTP, GMP-PNP and GTP gamma S. However, if CCK binding occurred before solubilization of the membranes, guanine nucleotides increased dissociation at concentrations and with a specificity similar to that observed for effects on intact pancreatic membranes. It is concluded that guanine nucleotides act via a protein which is separable from the receptor to induce dissociation of bound CCK. Moreover, CCK binding induces an association in the plasma membrane of the CCK receptor with this guanine nucleotide binding protein.     

A synthetic peptide derivative that is a cholecystokinin receptor antagonist

So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.     

The binding characteristics of 125I-gastrin and 125I-CCK8 to guinea pig fundic gastric glands differ: Is there more than one binding site for peptides of the CCK-gastrin family?

The binding of biologically active 125I-labeled derivatives of the C-terminal octapeptide of cholecystokinin (125I-CCK8) and gastrin (125I-G) to dispersed guinea pig fundic glands were compared at 24 degrees C. Although both peptides share the same C-terminal pentapeptide sequence, differences were found in the amount of each radioligand bound to fundic glands, their dissociation behavior, and their Scatchard plots. However, each peptide was able to displace the other radioligand from the glands at nM concentrations which indicated that both peptides bound to the same site. The different binding characteristics observed for 125I-G and 125I-CCK8 most likely resulted from the different dissociation rates of each peptide.