Pituitary adenylate cyclase activating peptide receptors regulate the growth of non-small cell lung cancer cells.

We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.

Cibacron blue-induced enhancement of agonist binding to cholecystokinin (CCK) receptors in solubilized pancreatic membranes.

The pancreatic receptor for cholecystokinin (CCK) typifies many G protein-coupled receptors in that its ability to bind agonist can be reduced by GTP or the solubilization of membranes. We found, however, that a dye, cibacron blue, caused up to a 6-fold increase in binding of the CCK receptor agonist, 125I-CCK-8, to rat pancreatic membranes solubilized with digitonin. Binding optimally enhanced in this manner was comparable to binding of 125I-CCK-8 to native membranes with respect to time-course, maximal amount bound, reversibility, and sensitivity to inhibition by various CCK receptor ligands. Increases in affinity of the CCK receptor for CCK-8 accounted fully for the enhancement of binding of 125I-CCK-8. Cibacron blue did not enhance binding of 125I-CCK-8 to native membranes, and also failed to enhance binding of the CCK receptor antagonist, [3H]L-364,718, to solubilized or native membranes. The ability of cibacron blue to enhance binding of agonist but not that of antagonist suggests that this dye may mimic or perhaps stimulate the effects of G protein on CCK receptors. Such a phenomenon may provide new insights into the mechanisms by which receptors distinguish agonists from antagonists.

Biochemical regulation of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

We used rat pancreatic acini and measured binding of [125I]CCK-8 and [3H]L-364,718 to the three different states of the CCK receptor to examine potential biochemical regulation of ligand binding for each receptor state. Binding of [125I]CCK-8 to the high affinity state of the receptor was measured as carbachol-inhibitable binding of [125I]CCK-8, whereas binding of [125I]CCK-8 to the low affinity state was measured as carbachol-resistant binding of [125I]CCK-8. Interaction of CCK-8 with the very low affinity state of the CCK receptor was measured as CCK-8-inhibitable binding of [3H]L-364,718. [125I]CCK-8 that was bound to the high affinity state dissociated slowly at a rate of 0.20%/min and this dissociation was not altered by 30 mM NaF. Dissociation of [125I]CCK-8 bound to the low affinity state was biphasic--22% of the bound radioactivity dissociated completely within 3 min and the remaining 78% dissociated slowly at a rate of 0.19%/min. Dissociation of [125I]CCK-8 from the low affinity state was not altered by 30 mM NaF. The pattern of dissociation of bound [125I]CCK-8 from the pancreatic CCK receptor expressed in COS cells was also biphasic and closely resembled that observed in pancreatic acini. CCK-8 that was bound to the very low affinity state dissociated completely during a 20-min period of washing and resuspension of acini that had been first incubated with CCK-8. We found extensive biochemical regulation of the different states of the CCK receptor in pancreatic acini. Bombesin, TPA, NaF, CCCP and trifluoperazine each altered binding of [125I]CCK-8 to the high affinity state and to the low affinity state, and except for bombesin each agent was more potent in affecting the high affinity state than the low affinity state. No agent tested affected the low affinity state but not the high affinity state. In contrast, a number of agents affected the high affinity state but not the low affinity state. These included receptor-mediated agonists (carbachol, secretin, VIP), 8Br-cAMP, NEM, agents that affect microtubules or microfilaments (cytochalasin B, vinblastine), calmodulin inhibitors (W-7, chlorpromazine) and genistein. Experiments with EGTA, A23187 and thapsigargin indicated that none of the three receptor states was influenced by intracellular or extracellular calcium. No agent tested altered the interaction of CCK-8 with the very low affinity state of the CCK receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Interaction of CCK with pancreatic acinar cells.

Recent studies demonstrate that cholecystokinin-like peptides are widely distributed in the CNS as well as in the peripheral nervous system and gastrointestinal tract. Studies with agonists have demonstrated multiple classes of receptors and recently potent receptor antagonists have been described which will distinguish these classes and should allow a better understanding of the role of CCK in various physiological processes. One of the known peripheral physiological functions of CCK is the stimulation of digestive enzymes from pancreatic acinar cells. In recent years the interaction of CCK with pancreatic acinar cells has been extensively studied and significant advances have been made in understanding its cellular basis of action. Robert Jensen and colleagues report on each of these areas.

Polyethylene glycol-mediated infection of non-permissive mammalian cells with semliki forest virus: application to signal transduction studies.

Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.

Famotidine in the therapy of gastric hypersecretory states.

The histamine (H2)-receptor antagonist famotidine was compared with ranitidine and cimetidine in its ability to control gastric acid hypersecretion in 33 patients with gastric hypersecretory states (32 patients with Zollinger-Ellison syndrome and one patient with idiopathic hypersecretion). Equipotent doses of each drug were determined in nine patients and used to determine relative onset of action, duration of action, and potency. Each drug had a similar time course of onset with a maximal effect at three to four hours after oral ingestion. The duration of action of famotidine was 30 percent longer than that of either cimetidine or ranitidine. In terms of relative potency, famotidine was nine times more potent than ranitidine and 32 times more potent than cimetidine. Thirty-two patients underwent long-term famotidine treatment for up to 34 months (mean, 10 months) with a duration in 21 patients of at least six months, in nine patients of at least 12 months, and in six patients of at least 24 months. The mean daily maintenance dose with famotidine was 0.33 g per day (range, 0.05 to 0.8 g). Prior to famotidine therapy, 27 patients were taking ranitidine and the mean daily dose required was 2.3 g per day (range, 0.6 to 5.4 g), whereas six patients were taking cimetidine and the mean daily dose was 4.6 g per day (range, 1.2 to 9.0 g). Fourteen of the 32 patients required an anticholinergic agent in addition to ranitidine or cimetidine to maintain control, whereas only five patients required an anticholinergic agent with famotidine. Gastric acid hypersecretion was controlled in seven patients with less frequent dosing with famotidine than with cimetidine or ranitidine. Long-term treatment with famotidine was not associated with any hematologic or biochemical toxicity or clinical side effects. These results demonstrate that famotidine has a similar onset of action to other H2-receptor antagonists but has a 30 percent longer duration of action and is nine times more potent than ranitidine and 32 times more potent than cimetidine. Famotidine is safe and highly effective in the long-term treatment of gastric hypersecretory states.

Molecular cloning, functional expression, and chromosomal localization of the human cholecystokinin type A receptor.

The results presented here describe for the first time the molecular cloning of the human CCKA-R. Expression of the recombinant receptor shows the expected subtype pharmacology and coupling to phosphoinositide hydrolysis reported for the native human CCKA-R. This knowledge will enhance our understanding of its distribution, pharmacology, and structure and will improve our understanding of its physiological role in the gastrointestinal and nervous systems in humans. Ultimately, this should hasten the understanding and therapy of gastrointestinal and neuropsychiatric disorders.

Cholecystokinin receptor family. Molecular cloning, structure, and functional expression in rat, guinea pig, and human.

A review of the literature encompassing numerous pharmacological, physiological, and biochemical studies indicates the presence of at least four CCK receptor types, CCKA, CCKB, gastrin, and CG-4 receptors. Multiple subtypes of the CCKAR have been postulated to account for the differences in pharmacology or affinity cross-linking of CCKARs between pancreas and gallbladder and the presence of high and low affinity CCKARs on pancreatic acini. Multiple subtypes of the CCKBR have been postulated to explain the differences in pharmacology and physiology between gastric and gallbladder smooth muscle CCKBRs. We recently cloned and functionally expressed both the CCKAR and the CCKBR from rat, guinea pig, and human. The CCKAR and CCKBR are 48% homologous and constitute a family of receptors within the guanine nucleotide-binding regulatory protein-coupled superfamily of receptors. Each receptor is highly conserved between species. A single cDNA encoding a single protein is present in both pancreas and gallbladder and can account for both high and low affinity CCKARs found on pancreatic acini when transfected into COS-7 cells. A single cDNA encoding a single CCKBR protein is present in both the central nervous system and the periphery including the gastrointestinal system. Therefore, the gastrin receptor is actually a CCKBR present on parietal cells. Genomic and cDNA library hybridization as well as Northern and Southern hybridization studies among rat, guinea pig, and human species identifies only two members of the CCK receptor family, CCKAR and CCKBR. Although these studies do not identify other closely related members of the CCK receptor family, they do not rule out the existence of other less closely related members. Furthermore, differences in tissue and species-specific posttranslational processing, receptor coupling, and associated membrane protein and lipid heterogeneity may be among some of the other factors that may account for the phenotypic expression of more receptor subtypes than molecular studies would predict.

Differential signaling and immediate-early gene activation by four splice variants of the human pituitary adenylate cyclase-activating polypeptide receptor (hPACAP-R).

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the VIP/secretin/glucagon family, is present in the hypothalamus, anterior pituitary, and adrenal gland where it regulates hormone release, in the GI tract where it modulates motility, and in human tumoral cell lines where it shows a growth-promoting effect. It is now appreciated that alternative splicing of two exons of the rat PACAP-R gene generate four major rPACAP-R splice variants that are differentially expressed in tissues and variably coupled to intracellular second messengers. Because of the potential implications of these findings in human physiology, we cloned the hPACAP-R gene. Similar to the rat, two exons (SV-1 and SV-2) are alternatively spliced to account for four major hPACAP-R receptor splice variants. These splice variants (hPACAP-R-null, hPACAP-R-SV1, hPACAP-R-SV2, hPACAP-R-SV-3) were cloned from a human frontal cortex cDNA library, stably transfected in NIH/ 3T3 cells and each characterized for ligand affinity, stimulation of adenylate cyclase (AC) and phospholipase C (PLC), and ligand-induced expression of the proto-oncogenes, c-fos, and c-myc. Stably transfected NIH/3T3 cells expressing similar numbers of receptors of the four splice variants showed nearly identical responses for ligand affinity and potency for P-38- and P-27-stimulated increases in cAMP and total inositol phosphates. However, each receptor splice variant differed in their ligand-stimulated efficacy for total inositol phosphate stimulation. The hPACAP-R-SV2 showed the greatest efficacy for stimulating phospholipase C that was approximately seven-fold greater than the hPACAP-R-SV1, twofold greater than the hPACAP-R-Null, and 1.5-fold greater than the hPACAP-R-SV-3 splice variants. To determine whether the splice variants also differ in their ability to stimulate immediate early gene expression, c-fos and c-myc transcripts were assayed by Northern blot and quantified by densitometry. PACAP-38 increased c-fos and c-myc expression for all four of the receptor splice variants that paralleled the efficacy for PLC stimulation, with the the SV-2 splice variant showing the greatest stimulation. These results show that the hPACAP-R-SV2 exhibits enhanced efficacy for coupling to both PLC and activation of the protooncogenes, c-fos and c-myc suggesting a novel and potentially important mechanism for differentially activating signal transduction pathways that influence cellular growth and differentiation.

Characterization of the bombesin receptor on mouse pancreatic acini by chemical cross-linking.

Bombesin (BN), gastrin-releasing peptide (GRP) and GRP(18-27) (neuromedin C) were equipotent and 30-fold more potent than neuromedin B (NMB) in inhibiting binding of 125I-GRP to and in stimulating amylase release from mouse pancreatic acini. In the present study we used 125I-GRP and chemical cross-linking techniques to characterize the mouse pancreatic BN receptor. After binding of 125I-GRP to membranes, and incubation with various chemical cross-linking agents, cross-linked radioactivity was analyzed by SDS-PAG electrophoresis and autoradiography. With each of 4 different chemical cross-linking agents, there was a single broad polypeptide band of Mr 80,000. Cross-linking did not occur in the absence of the cross-linking agent. Cross-linking was inhibited only by peptides that interact with the BN receptor such as GRP, NMB, GRP(18-27) or BN. Dose-inhibition curves for the ability of BN or NMB to inhibit binding of 125I-GRP to membranes or cross-linking to the 80,000 polypeptide demonstrated for both that BN was 15-fold more potent than NMB. The apparent molecular weight of the cross-linked polypeptide was unchanged by adding dithiothreitol. N-Glycanase treatment reduced the molecular weight of the cross-linked peptide to 40,000. The present results indicate that the BN receptor on mouse pancreatic acinar cell membranes resembles that recently described on various tumor cells in being a single glycoprotein with a molecular weight of 76,000. Because dithiothreitol had no effect, this glycoprotein is not a subunit of a larger disulfide-linked structure.

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.

A new method for determining gastric acid output using a wireless pH-sensing capsule.

BACKGROUND: Gastro-oesophageal reflux disease (GERD) and gastric acid hypersecretion respond well to suppression of gastric acid secretion. However, clinical management and research in diseases of acid secretion have been hindered by the lack of a non-invasive, accurate and reproducible tool to measure gastric acid output (GAO). Thus, symptoms or, in refractory cases, invasive testing may guide acid suppression therapy. AIM: To present and validate a novel, non-invasive method of GAO analysis in healthy subjects using a wireless pH sensor, SmartPill (SP) (SmartPill Corporation, Buffalo, NY, USA). METHODS: Twenty healthy subjects underwent conventional GAO studies with a nasogastric tube. Variables impacting liquid meal-stimulated GAO analysis were assessed by modelling and in vitro verification. Buffering capacity of Ensure Plus was empirically determined. SP GAO was calculated using the rate of acidification of the Ensure Plus meal. Gastric emptying scintigraphy and GAO studies with radiolabelled Ensure Plus and SP assessed emptying time, acidification rate and mixing. Twelve subjects had a second SP GAO study to assess reproducibility. RESULTS: Meal-stimulated SP GAO analysis was dependent on acid secretion rate and meal-buffering capacity, but not on gastric emptying time. On repeated studies, SP GAO strongly correlated with conventional basal acid output (BAO) (r = 0.51, P = 0.02), maximal acid output (MAO) (r = 0.72, P = 0.0004) and peak acid output (PAO) (r = 0.60, P = 0.006). The SP sampled the stomach well during meal acidification. CONCLUSIONS: SP GAO analysis is a non-invasive, accurate and reproducible method for the quantitative measurement of GAO in healthy subjects. SP GAO analysis could facilitate research and clinical management of GERD and other disorders of gastric acid secretion.

Cholecystokinin receptors.

The cholecystokinin (CCK) and gastrin families of peptides act as hormones and neuropeptides on central and peripheral CCK receptors to mediate secretion and motility in the gastrointestinal (GI) tract in the physiological response to a normal meal. CCK and its receptors are also widely distributed in the central nervous system (CNS) and contribute to the regulation of satiety, anxiety, analgesia, and dopamine-mediated behavior. Although the wide distribution, myriad number of functions, and reported pharmacological heterogeneity of CCK receptors would suggest a large number of receptor subtypes, the application of modern molecular biological techniques has identified two CCK receptors, CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR), that mediate the actions of CCK and gastrin; gastrin receptors have been found to be identical to CCK-BR. CCK-AR, found predominantly in the GI system and select areas of the CNS, have high affinity for CCK and the nonpeptide antagonist L-364,718, whereas CCK-BR, found predominantly in the CNS and select areas of the GI system, have high affinity for CCK and gastrin and the nonpeptide antagonist L-365,260. Both CCK-AR and CCK-BR are highly conserved between species, although there is some tissue-specific variation in expression. Recombinant receptor expression faithfully reproduces the native receptor pharmacology and signal transduction pathways, allowing direct comparisons of receptor function between species as well as serving as a convenient source of receptor. Our present knowledge of the chromosomal localization, receptor gene structure, and primary sequence will allow further studies in disease association, receptor regulation, and structure-function analysis.

G protein-coupled receptors in gastrointestinal physiology. I. CCK receptors: an exemplary family.

The CCK and gastrin families of peptides act as hormones and neuropeptides on central and peripheral receptors to mediate secretion and motility in the gastrointestinal tract in the physiological response to a normal meal. Thus far, two CCK receptors have been molecularly identified to mediate the actions of CCK and gastrin, CCK-A and CCK-B receptors (CCK-AR and CCK-BR, respectively). The regulation of CCK-AR and CCK-BR affinity by guanine nucleotides and the receptor activation of G protein-dependent stimulation of phospholipase C and adenylyl cyclase suggested that they were guanine nucleotide-binding protein-coupled receptors [G protein-coupled receptors (GPCRs)]; however, the eventual cloning of their cDNAs revealed their heptahelical structure and confirmed their membership in the GPCR superfamily. The gastrointestinal system is a rich source of neuroendocrine hormones that interact with a large number of GPCRs to regulate the complex tasks of digestion, absorption, and excretion of a meal. This article focuses on the CCK family of GPCRs, and its activities in the gastrointestinal system.

A segment of five amino acids in the second extracellular loop of the cholecystokinin-B receptor is essential for selectivity of the peptide agonist gastrin.

The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors having approximately 50% amino acid homology. Both the CCKAR and CCKBR have high affinity for sulfated CCK peptides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for gastrin, we first constructed a series of CCKB/AR chimeras in which restriction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expressed in COS-1 cells were screened for the selective loss of gastrin affinity according to the displacement of 125I-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100 of the expected 300-fold lower affinity of gastrin-17-I observed for the control CCKAR compared to the control CCKBR. Using site-directed mutagenesis in this segment of the CCKBR, we identified a sequence of 5 amino acids in the second extracellular loop responsible for this 100-fold selective loss in gastrin affinity. 125I-labeled Bolton-Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective antagonist) was unaffected by the changes in these 5 amino acids. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.

Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C.

Alternative splicing of two exons of the rat pituitary adenylate cyclase activating polypeptide (PACAP) receptor gene generates four major splice variants that are differentially expressed in specific tissues and variably coupled to intracellular second messengers. To evaluate the potential implications of these findings in human physiology, the human PACAP receptor gene was cloned. Alternative splicing about two exons of the gene allowed for four major splice variants that were subsequently identified on cDNA cloning. Each of the four splice variant cDNAs (null, SV-1, SV-2, and SV-3) was stably expressed in NIH/3T3 cells at similar receptor densities. For each splice variant, PACAP (both PACAP-38 and PACAP-27) had similar affinity and potency for stimulating either adenylate cyclase or phospholipase C. However, each receptor splice variant differed in their ligand-stimulated maximal response (efficacy) for total inositol phosphate accumulation with the SV-2 showing the greatest efficacy, followed by the null, SV-1, and SV-3 splice variants. Therefore, unlike the rat, PACAP binds and stimulates signal transduction with nearly equal affinity and potency for each of the receptor splice variants although with varying efficacy for the stimulation of phospholipase C. These results suggest a novel and potentially important mechanism for a single hormone to not only couple to dual signal transduction cascades but also elicit tissue-specific differential activation of phospholipase C in humans.