Sulfated Cholecystokinin Octapeptide Research Articles

Cholecystokinin potentiates morphine anticonvulsant action through both CCK-A and CCK-B receptors

Recent studies have suggested that cholecystokinin may have a role in modulating the effects of the endogenous opioid system in physiological functions such as thermoregulation and pain control. However, the possible interaction of cholecystokinin and morphine in epileptogenesis is unknown. We studied the effect of subcutaneous morphine and intracerebroventricularly administered cholecystokinin octapeptide sulphate ester and receptor antagonists CCK-A (MK 329) and CCK-B (L 365,260) on seizures provoked by maximal electroshock in male Sprague-Dawley rats. Seizures were induced through electrode-gel-coated ear clip electrodes by a high voltage, high internal resistance constant current generator, 30 minutes after morphine administration and 10 minutes after cholecystokinin-8-SE, CCK-A and CCK-B infusion. Morphine decreased the length of the tonic component of the seizure and cholecystokinin potentiated this decrease. Cholecystokinin antagonists blocked the effects of both cholecystokinin and morphine. The results suggest that cholecystokinin acts as an endogenous agonist with opioids in the regulation of seizure susceptibility through both CCK-A and B receptors and may be responsible for part of the anticonvulsant action of morphine.

Inhibition of murine peritoneal macrophage functions by sulfated cholecystokinin octapeptide

The effect in vitro of the sulfated octapeptide form of cholecystokinin, CCK-8, at concentrations from 10 −12 M to 10 −6 M on several functions of resting peritoneal macrophages from BALB/c mice: adherence to substrate, mobility (spontaneous and directed by chemical gradient or chemotaxis), ingestion of inert particles (latex beads) or cells ( Candida albicans), and production of superoxide anion measured by nitroblue tetrazolium reduction was studied. CCK-8, at concentrations from 10 −10 M to 10 −8 M, inhibited significantly all functions studied with the exception of adherence to substrate, which was increased. A dose-response relationship was observed, with a maximum inhibition of macrophage functions found at 10 −8 M. This neuropeptide induced in murine macrophages a significant, but transient, increase of cAMP levels at 60 sec. On the contrary, CCK-8 produced a slight but significant decrease of protein kinase C (PKC) activity at 5 min of incubation. These results suggest that CCK-8 is a negative modulator of several macrophage functions, and that the inhibition of these activities is carried out through an increase of intracellular cAMP levels and a decrease in PKC activity.

Involvement of different subtypes of cholecystokinin receptors in opioid antinociception in the mouse.

Various doses of sulfated cholecystokinin octapeptide (CCK-8s) injected intracerebroventricularly (ICV) alone did not show any antinociceptive effect. CCK-8s (0.01-1 ng) pretreated ICV for 10 min dose-dependently attenuated the inhibition of the tail flick response induced by ICV-administered morphine (2 micrograms). beta-endorphin (1 microgram), and U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl]benzeocetamide), 60 micrograms). However, ICV pretreatment with CCK-8s was not effective in reducing the inhibition of the tail flick response induced by [D-Pen(2)-D-Pen5]enkephalin (DPDPE; 10 micrograms) administered ICV. To determine what subtype(s) of CCK receptors are involved in antagonizing the antinociception induced by these opioids, effect of lorglumide sodium salt (a CCKA receptor antagonist) or PD135,158 N-methyl-D-glucamine salt (a CCKB receptor antagonist) on opioid-induced inhibition of the tail flick response was examined. Various doses of lorglumide sodium salt (lorglumide) or PD135,158 N-methyl-D-glucamine salt (PD135,158) injected ICV alone did not affect the basal tail flick response. The antagonistic effect of CCK-8s on morphine-, beta-endorphin-, and U50,488H-induced inhibition of the tail flick response was blocked in a dose-dependent manner by the co-ICV injection of PD135,158 (0.001-0.1 ng). The co-ICV injection of lorglumide (0.001-0.1 ng) dose-dependently blocked the antagonistic effect of CCK-8s on beta-endorphin- and U50,488H-induced, but not morphine-induced, inhibition of the tail flick response. Our results suggest that both CCKA and CCKB receptors are involved in antagonizing antinociception induced by beta-endorphin and U50,488H administered supraspinally. However, only CCKB (but not CCKA) receptors are involved in antagonizing antinociception induced by morphine administered supraspinally. CCK receptors are not involved in antagonizing the supraspinally administered DPDPE-induced antinociception.

Cholinergic nerve-mediated excitation in the guinea pig choledochoduodenal junction activated by distension.

The electrical basis of propulsive contractions in the guinea pig choledochoduodenal junction (CDJ), which are triggered by distension, was investigated using intracellular microelectrode recording techniques. The isolated CDJ was placed in a continuously perfused tissue chamber at 37 degrees C. Membrane potential was recorded from smooth muscle cells in either the ampulla or in the upper CDJ (upper junction) regions, which were immobilized by pinning. Distension of the upper junction (20-30 s) by increasing intraductal hydrostatic pressure (mean elevation: 2.0 +/- 0.3 kPa, n = 13) triggered "transient depolarizations" (TDs: < 5 mV in amplitude and 2-5 s in duration) and action potentials in the circular muscle layer of the ampulla. The frequency of TDs in the ampulla was increased from 2.2 +/- 0.2 to 15.9 +/- 2.2 min-1 (n = 13) during distension. Simultaneous impalements of cells in the longitudinal and circular muscle layers in the ampulla revealed that subthreshold TDs in the circular layer were associated with an increased rate of action potential discharge in the longitudinal layer. Atropine (Atr; 1.4 x 10(-6) M) and tetrodotoxin (TTX; 3.1 x 10(-6) M blocked the distension-evoked increase in TD frequency, without affecting the frequency of ongoing TDs. The sulfated octapeptide of cholecystokinin (1-5 x 10(-8) M) increased the amplitude of TDs recorded in the circular muscle layer of the ampulla and increased action potential discharge rate. In separate recordings, radial stretch of the ampulla region increased the rate of discharge of action potentials in the smooth muscle of the upper junction.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term perfusion of the cerebroventricular system of dogs without leakage to the peripheral circulation

Methods developed previously for studying the effect of cerebroventricular injection or ventriculocisternal perfusion of test substances are unsatisfactory because the test substance is not confined to the central compartment. Most likely the test substance enters the peripheral circulation via the arachnoid villi. The purpose of this paper is to describe a method for perfusing the cerebroventricular system of conscious dogs without passage of test substances to the peripheral circulation. With the method described, the mean (+/- SE) cerebroventricular pressure in conscious dogs was 7.4 +/- 0.8 cmH2O (n = 16), and the mean (+/- SE) production of cerebrospinal fluid (CSF) was 25 +/- 0.3 microliter/min (n = 16). Endogenously occurring migrating myoelectric complexes (MMCs) of the small intestine were recorded in dogs before catheters were implanted in the left and right lateral ventricles and the fourth ventricle and after catheter implantation during cerebroventricular perfusion with artificial CSF alone or with CSF containing sulfated (S-CCK-OP) or nonsulfated cholecystokinin octapeptide (NS-CCK-OP). Only cerebroventricular perfusion with S-CCK-OP (1.2 pmol.kg-1.min-1; n = 20) replaced spontaneously occurring MMCs with a fed-like pattern of myoelectric activity. The results suggest that replacement of the fasting pattern of myoelectric activity with a fed-like pattern in the fasted dog was mediated by CCK-A receptors located in one or more brain nuclei surrounding the third ventricle.

CCK B receptor signaling in rat pituitary GH3 cells. CCK-8S-induced intracellular calcium mobilization by Ca 2+ release and Ca 2+ influx

We describe the effect of sulphated cholecystokinin octapeptide (CCK-8S) on [Ca 2+] i in rat pituitary GH3 cells. Investigations were performed on fluo-3 loaded cells by using a confocal imaging system MRC-600 (Bio-Rad). Because CCK-8S mobilized intracellular calcium in cells bathed in Ca 2+-free buffer it must be able to release calcium from internal stores. Furthermore, influx of Ca 2+ from outside the cells seems to contribute to CCK-8S induced increase in [Ca 2+] i as demonstrated by calcium mobilization in GH3 cells preincubated with thapsigargin in Ca 2+ containing buffer.

Cholecystokinin (CCK-8S) prolongs ‘unsaturated’ θ-pulse induced long-term potentiation in rat hippocampal CA1 in vitro

The effects of bath-applied sulphated cholecystokinin octapeptide (CCK-8S; 500 nM) and the selective CCK B receptor antagonist PD 135158 (1μM) on the induction and maintenance of an ‘unsaturated’ long-term potentiation (LTP) were examined using recordings of field excitatory postsynaptic potentials (fEPSP) and population spikes (PS) in the hippocampal CA1 region of the rat. LTP was induced by a single θ-pulse stimulation (10 pulses at 7 Hz) of the Schaffer collateral-commissural pathway. CCK-8S had minor effects on the induction of LTP but substantially prolonged LTP of both fEPSP and PS from about 1 to at least 3 h. The antagonist PD 135158 had no influence on the induction of potentiation of fEPSP slope, but decreased the potentiation of PS amplitude. The results support the view that CCK-8S may facilitate long-term changes in glutamatergic synaptic transmission of the hippocampus, such as LTP.

Evidence that cholecystokinin induces immediate early gene expression in the brainstem, hypothalamus and amygdala of the rat by a CCK A receptor mechanism

The effect of sulphated cholecystokinin octapeptide (CCK-8S) on immediate early gene expression in the rat CNS was investigated using the technique of in situ hybridization. A rapid and transient induction of c- fos, NGFI-A and NGFI-B (nerve growth factor-induced genes A and B) mRNA was demonstrated in the nucleus tractus solitarius (NTS), area postrema (AP), hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, and central nucleus of the amygdala, following peripheral administration of CCK-8S (1–100 μg/kg i.p.). In contrast, levels of c-jun, jun B and jun D mRNA were unaffected by the peptide. The closely related decapeptide, caerulein (50 mg/kg i.p.), induced the same pattern of IEG expression as CCK-8S, whereas the desulphated octapeptide, CCK-8DS (50 mg/kg i.p.), had no effect on levels of mRNA for any IEG studied. Expression of IEG mRNA in these areas was suppressed by bilateral subdiaphragmatic vagotomy, and by pretreatment with the selective CCK A receptor antagonist, devazepide (0.1 and 1 mg/kg i.p.). In contrast, CCK-8S induction of IEG mRNA was not blocked by pretreatment with the selective CCK B receptor antagonist, CI-988 (1 or 10 mg/kg i.p.). In addition, the selective CCK B receptor agonists, CCK-4 (50 μg/kg i.p.) or pentagastrin (2 mg/kg i.p.), failed to induce IEG expression in any of the areas studied. These results suggest that systemic CCK-8S primarily acts via CCK A receptors on vagal afferents to stimulate IEG mRNA expression in the rat CNS.

Effects of cholecystokinin tetrapeptide and sulfated cholecystokinin octapeptide in rat models of anxiety

The effects of the acutely administered cholecystokinin (CCK) agonists CCK tetrapeptide (BOC-CCK-4) and sulfated CCK octapeptide (CCK-8S) were examined in four animal models of anxiety in rats. In the elevated plus maze, BOC-CCK-4 reduced the time spent in the open arms and the number of entries into the open arms. BOC-CCK-4 but not the anorectic acting CCK-8S increased the suppression of feeding in a conflict paradigm based on novelty suppressed feeding in hungry rats. In the two-compartment black-and-white box, BOC-CCK-4 decreased the time spent and locomotor activity in the white compartment. In the ultrasound vocalization test, using rat pups separated from the mother, BOC-CCK-4 increased the number of distress calls. No evidence was found for inducing anxiety-like behaviour by CCK-8S.

CCK-8 excites substantia nigra dopaminergic neurons by increasing a cationic conductance

Using the whole-cell patch-clamp technique, we investigated electrophysiological effects of cholecystokinin on acutely isolated dopaminergic (DA) neurons of rat substantia nigra (SN). During voltage-clamp recordings, sulfated cholecystokinin octapeptide (CCK-8) dose-dependently induced an inward current at the holding potential of −70 mV. Under current-clamp recordings, CCK-8 depolarized DA neurons and triggered action potentials. CCK-8-evoked inward current reversed its direction at 1.0 ± 1.9 mV (n = 9), and the amplitude of inward current induced by CCK-8 was reduced in an external solution with low sodium concentration. Cholecystokinin tetrapeptide (CCK-4), a selective CCK-B receptor agonist, failed to induce an inward current. CCK-8-evoked cationic current was antagonized by lorglumide, a selective CCK-A receptor antagonist. PD135, 158, a highly selective and potent CCK-B receptor antagonist, failed to attenuate CCK-8-induced cationic currents. These results suggest that by activating CCK-A receptors, CCK-8 excites SN DA neurons via increasing a non-selective cationic conductance.

MK-329 blocks the inhibition of alcohol intake by CCK-8

Peripheral administration of sulfated cholecystokinin octapeptide (CCK-8) potently reduces alcohol intake, preference, and blood levels in rats. MK-329 (L-364,718 or Devazepide) acts at peripheral cholecystokinin (CCK A) receptors to antagonize CCK-8's physiological and behavioral effects, such as pancreatic stimulation and inhibition of feeding. We determined whether CCK A receptor blockade would also prevent CCK-8's alcohol satiety effect. Water-deprived female and male rats ( n = 7 for each) received randomized combinations of intraperitoneal injections of MK-329 (0, 100, 200, or 400 μg/kg) followed by CCK-8 (0 or 4 μg/kg). Rats were then given access to 5% w/v ethanol for 30 min, followed by 30-min access to water, with food ad lib. MK-329 at all doses significantly ( p < 0.05) reduced the suppression of alcohol intake and food intake by CCK-8. MK-329 alone increased alcohol intake at 400 μg/kg, and increased food intake, in females and males at 100 and 200 μg/kg, respectively. We concluded that CCK-8's alcohol and food satiation effects depend on specific, peripheral CCK A receptors, and satiation of alcohol consumption and drinking-associated feeding reflect an endogenous functional interaction of CCK-8 with CCK A receptors.

Abdominal vagotomy dissociates the anorectic mechanisms for peripheral serotonin and cholecystokinin

These studies compared the effects of total abdominal vagotomy (VGX) on ingestive actions produced by peripheral serotonergic and cholecystokinergic (CCKergic) stimulation in rats. Subcutaneous injection of 0.01-0.16 mumol/kg of the serotonin (5-HT) analogue 5-carboxamidotryptamine (5-CT) dose-dependently reduced mash intake equally in VGX rats and their laparotomized (LAP) controls but concurrently stimulated drinking only in the controls. The sulfated octapeptide of cholecystokinin (CCK-8, 4.0 nmol/kg ip) also reduced food intake only in the controls. In a second set of rats, vagotomy did not alter anorexia after intraperitoneal administration of either 2.0 or 8.0 mumol/kg of 5-HT or of 0.03 mumol/kg of 5-CT but abolished anorexia after a large dose of CCK-8 (8.0 nmol/kg). The completeness of vagotomy was verified histologically by immunohistochemical staining of the vagal bundles for the high molecular weight form of neurofilament-H protein. We report for the first time that 5-CT produces anorexia by a vagally independent mechanism. In contrast, 5-CT stimulates drinking by a pathway that does involve vagal function. Finally, we confirm the prediction that vagotomy dissociates the neural mechanisms for the anorectic action of peripheral 5-HTergic and CCKergic stimulation.

Role of perinatal estrogens in sexual differentiation of the inhibition of lordosis by exogenous cholecystokinin

Microinjections of sulphated cholecystokinin octapeptide (sCCK-8) into the ventromedial nucleus of the hypothalamus inhibit lordosis behavior in receptive female rats. This effect of sCCK-8 seems to differentiate under the control of gonadal steroids shortly after birth. Neonatally castrated males and normal females show similar responses, while androgenized females are less sensitive to sCCK-8. The current study investigated estrogen's role on the differentiation of the response to sCCK-8. On the day of birth male rat pups were castrated, given sham surgeries, or implanted with the antiestrogen tamoxifen or the aromatase inhibitor androst-1, 4, 6-triene-3, 17-dione (ATD). Females were implanted with testosterone propionate or tamoxifen, or given sham surgeries. Implants were removed 10 days later. As adults, rats were tested for female sexual behavior after microinjections of sCCK-8 into the ventromedial nucleus of the hypothalamus. Neonatally castrated males, ATD-treated males, and control females showed profound inhibition of lordosis behavior after sCCK-8. These results suggest that elimination of estrogen postnatally prevents defeminization of the reproductive circuitry that responds to sCCK-8.

Modulation of paired-pulse activation in the hippocampal dentate gyrus by cholecystokinin, baclofen and bicuculline

Paired-pulse stimulation was used to evaluate the effects of the sulfated octapeptide of cholecystokinin (CCK8-S), the γ-aminobutyric acid B (GABA B) agonist (−) baclofen, and the GABA A antagonist (−) bicuculline on hippocampal dentate gyrus (DG) granule cell excitability. Evoked action potentials (EAPs) and excitatory postsynaptic potentials (EPSPs) were recorded in response to orthodromic stimulation of the medial (MPP) or lateral (LPP) perforant pathway. Paired-pulse indices were determined using interpulse intervals (IPIs) across the range of 5–1000 ms. As reported by others, three phases of paired-pulse effects were revealed under control (drug-free ACSF) conditions: early paired-pulse inhibition (PPI), intermediate paired-pulse facilitation (PPF) and late PPI. With EAPs, CCK8-S enhanced only the intermediate PPF on both pathways, with no effect on the early or late PPIs. The effects of (−) baclofen were similar to CCK8-S. (−) Bicuculline attenuated the early and late PPI as well as the PPF. No differences were measured on the MPP- or LPP-evoked EPSPs in any of the drug conditions. These results indicate a similarity of CCK8-S- with GABA B-mediated modulation on neuronal activation in the DG. CCK8-S disinhibition of DG granule cells may play a role in the induction of long-lasting synaptic modifications.

2‐Naphthalenesulphonyl l‐aspartyl‐(2‐phenethyl)amide (2‐NAP)‐a selective cholecystokinin CCKA‐receptor antagonist

1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.

Opposite effects of mesencephalic microinjections of cholecystokinin octapeptide and neurotensin-(1–13) on brain stimulation reward

Changes in operant responding for brain stimulation reward were studied before and after a microinjection of 1 nmol of sulphated cholecystokinin octapeptide, neurotensin or saline into the ventral tegmental area. Neurotensin produced a significant and long lasting decrease in the stimulation frequency required to produce a half-maximal rate of responding; cholecystokinin had the opposite effect, attenuating the rewarding efficacy the stimulation during the first 30 min post-injection. It is suggested that the opposite effects of the peptides on reward are due to their differential modulatory effects on a subpopulation of mesencephalic dopamine neurones.

Cholecystokinin-induced ventral root depolarization of neonate rat hemicord in vitro

1. 1. Sulphated cholecystokinin octapeptide (CCK8S, 0.03–1.00 μM), pentapeptide (CCK5) and tetrapeptide (CCK4) elicited concentration dependent depolarizations of neonate rat ventral roots in vitro. 2. 2. CCK5 was equipotent with CCK8S although CCK4 was weaker (equipotent molar ratio 17.5). 3. 3. CCK8S-induced depolarizations were depressed by tetrodotoxin (0.1 μM), Mg 2+ ions (0.75 mM) and the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP5, 10 μM). These results suggest that CCK8S-induced depolarizations were predominantly mediated through the release of an excitatory amino acid from interneuronal sites. 4. 4. The selective CCK A and CCK B receptor antagonists, L-364,718 and L-365,260 both depressed CCK8S-induced depolarizations. CCK8S dose ratios in the presence of 1 μM L-364,718 or L-365,260 were 4.5 and 11.2 respectively, suggesting the response was mediated predominantly through stimulation of CCK B receptors. 5. 5. These results suggest that the neonate rat hemicord preparation is a suitable tissue for functional CCK receptor assays.

Cholecystokinin and somatostatin modulate the glucose-induced insulin secretion by different mechanisms in pancreatic islets. A study on phospholipase C activity and calcium requirement.

To study the interaction between the phospholipase C activation and the insulin secretion, isolated pancreatic islets were stimulated with glucose and the sulfated cholecystokinin octapeptide (CCK). To discriminate between intracellular mechanisms, experiments with agents inhibiting adenylyl cyclase and calcium-channels like somatostatin and verapamil, were performed. The phospholipase C activity, i.e. the accumulation of inositol phosphates, was increased by CCK (100 nmol l-1) at 3.3 mmol l-1 glucose. This effect of CCK did not require extracellular Ca2+, was not inhibited by somatostatin (100 nmol l-1), and no concomitant increase in the insulin secretion was observed. Both the phospholipase C activity and the insulin secretion increased in response to 12 mmol l-1 glucose. Somatostatin was able in some extent to inhibit these effects of glucose. At 12 mmol l-1 glucose, the phospholipase C activity and the insulin secretion were potentiated by CCK. CCK also counteracted the effect of somatostatin on the phospholipase C activity and the insulin secretion. Verapamil (2.5 umol l-1) more or less completely inhibited both the glucose-induced phospholipase C activity and the insulin secretion. Moreover, whereas the CCK-induced increase in the phospholipase C activity was unaffected, verapamil blocked the CCK-induced increase in the insulin secretion. We conclude that CCK directly activates phospholipase C, whereas glucose and somatostatin modulates phospholipase C via a Ca(2+)-dependent mechanism. CCK potentiates the insulin secretion by increased phospholipase C activity, but with a requirement of glucose at an apparent threshold level of Ca(2+)-influx.

Effects of various neuroactive substances on single-unit activities of hypothalamic arcuate neurons in brain slices

Extracellular single-unit activities of 288 dorsomedial/ventrolateral hypothalamic arcuate (ARC) neurons were studied electrophysiologically in brain slices in vitro. We tested the effects of several neuroactive substances plus some of their analogs in this study. Among them, baclofen, a GABA b-receptor agonist, inhibited 95.6% of ARC neurons tested, although GABA itself was much less effective (23.8%). About half of baclofen's effect was blocked by phaclofen, a GABA b antagonist. Serotonin and dopamine also exhibited mostly inhibitory effects on responsive ARC neurons (80 and 78.4%, respectively), although more than half ARC neurons tested (53.3% of 169) were not responsive to dopamine. Neither ketanserin, a 5-HT 2, nor domperidone, a D 2 receptor antagonist, had much effect on blocking the actions of 5-HT or DA. Histamine and carbachol excited 67.4% and 52.2% of ARC neurons tested, respectively. Moreover, their effects were completely blocked by pyrilamine, a H 1 and atropine, a muscarinic receptor antagonist, respectively. Ranitidine, a H 2 receptor antagonist, however, was less effective. Norepinephrine had about equal number of excitation (33.3%) and inhibition (38.5%) on ARC neurons. Cholecystokinin-octapeptide sulphate (CCK-8S), a neuropeptide tested exerted potent excitatory effects on ARC neurons (62.8% of 137). In summary, ARC neurons in a more localized region aiming at the tuberoinfundibular dopaminergic neurons were selected in this study. Their responses to many agents and the implications on the regulation of prolactin secretion were discussed.

Role of Endogenous Corticotropin Releasing Factor in Mediation of Neuroendocrine and Behavioral Responses to Cholecystokinin Octapeptide Sulfate Ester in Rats

The possible involvement of endogenous corticotropin-releasing factor (CRF) in the anxiogenic and pituitary-adrenal-axis-activating effects of cholecystokinin octapeptide sulfate ester (CCK 8) was investigated in rats. Intracerebroventricularly (i.c.v.) administered CCK 8 induced an anxiogenic response in an elevated plus-maze test, and enhanced the plasma corticosterone level. Pretreatment with different dilutions (1:10, 1:20 and 1:100, i.c.v.) of CRF antiserum and different doses of a CRF receptor antagonist, alpha-helical CRF (ahCRF, 0.001-1.0 microgram, i.c.v.) prevented the anxiogenic response to CCK 8 in a dose-dependent manner. None of the doses of CRF antiserum or ahCRF alone produced any alteration in either the elevated plus-maze paradigm or corticosterone level in saline-treated control rats. The results strongly suggest that the anxiogenic and hypothalamo-pituitary-adrenal-activating effects of CCK 8 are mediated via CRF.