Potentiation of beta-endorphin effects by cholecystokinin antiserum in rats

Intracerebroventricular administration of a C-terminal cholecystokinin (CCK) antiserum potentiated the analgesic and cataleptic effects of beta-endorphin. The results are opposite to those observed after injection of CCK-8. It was suggested that CCK-8 may play a physiological role antagonizing the action of beta-endorphin.     

Dual action of beta-endorphin on insulin release in genetically obese and lean mice

Intraperitoneal administration of beta-endorphin (1 mg/kg) to ob/ob mice doubled fasting plasma insulin concentrations within 30 min, while plasma glucose concentrations were unaltered. In lean mice, beta-endorphin failed to alter plasma insulin or glucose responses. In glucose-loaded ob/ob mice, beta-endorphin (1 mg/kg) reduced insulin levels at 40 min, and delayed glucose disposal. A lower dose of beta-endorphin (0.1 mg/kg) decreased plasma insulin at 90 min, with no effect on plasma glucose disposal. In lean mice, only the higher dose of beta-endorphin suppressed the glucose-stimulated rise in plasma insulin concentrations, without affecting plasma glucose. Beta-endorphin's actions were blocked by naltrexone and could not be mimicked by N-acetyl-beta-endorphin. Beta-endorphin (10(-8)M) enhanced insulin release from isolated ob/ob and lean mouse islets incubated in medium containing 6 mM glucose, but inhibited release when 20 mM glucose was present. These effects were naloxone reversible. The results indicate that 1) ob/ob mice display a greater magnitude of response in vivo to beta-endorphin's actions on insulin release compared with lean mice, 2) high concentrations of beta-endorphin exacerbate glucose disposal in ob/ob mice. 3) the prevailing glucose concentration is an important determinant of whether beta-endorphin's effects on insulin release will be stimulatory or inhibitory and 4) these actions are mediated via opiate receptors.     

Specificity of nucleus accumbens to activities related to cholecystokinins in rats

Cholecystokinin octapeptide (CCK-8) or cholecystokinin tetrapeptide (CCK-4) were bilaterally injected into the areas where dopamine (DA) terminals and receptors have been detected; nucleus accumbens (NA), nucleus caudatus (NC), medial profrontal cortex (MPC), or prefrontal cortex (PC). The amount injected to each animal varied from 0 (control), 1 to 500 ng of CCK-8 and 0 (saline control), 0.5 to 2.5 micrograms of CCK-4 in NA in a volume of 1 microliter. The other areas received 500 ng CCK-8, 2.5 micrograms CCK-4 and proper control injections. The effects were observed in an open-field apparatus by measuring locomotor and rearing responses, the latency to move out of a specified area where the animal was first placed, and the amount of excretory bolus during a 5 min period following injections. When injected into NA, CCK-8 decreased locomotion and rearing at doses of 2.5 ng or more in a dose-related manner whereas CCK-4 increased locomotion and rearing at 1 microgram or more. The effects on latency and defecation were not detected. When the peptides were injected into NC, MPC or PC no effects were detectable. It appears that the effects of CCK-8 and CCK-4 on the exploratory responses are site-specific at NA where CCK-8 and DA are found to coexist in same neurons. CCK-4, a metabolite of CCK-8, could exert a negative feedback to moderate the effect of CCK-8.     

Characterization of cholecystokinin binding sites in rat cerebral cortex using a 125I-CCK-8 probe resistant to degradation

Specific binding sites for cholecystokinin (CCK) have been characterized in a particulate membrane fraction of rat cerebral cortex using a biologically active 125I-labeled derivative of the C-terminal octapeptide of CCK (CCK-8) prepared by reaction with the iodinated form of the imidoester (125IIE), methyl-p-hydroxybenzimidate. The time course of binding to cortical membranes was rapid, temperature dependent, and saturable. Half-maximal binding at 24 degrees C was reached in 30 min and full binding at 120 min. At 37 degrees C there was only a slight increase in 125IIE-CCK-8 bound after 15 min. The addition of a large excess of CCK-8 after 30 min of binding at 24 degrees 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 membranes with increasing concentrations of CCK-8 and other structurally related peptides. CCK-8 displaced 50% of the radioligand at 4 nM, CCK-33 at 10 nM, and gastrin (desulfated CCK-8) at 60 nM. Secretin, a structurally unrelated peptide, was unable to displace the radioligand from cortical membranes at 1.0 microM. Finally, 125IIE-CCK-8 exposed to cortical membranes or to buffers that had previously contained such membranes for 60 min at 24 degrees C bound equally as well to fresh cortical membranes as control radioligand that had not been exposed to the same conditions. Thus the 125I-CCK-8 radioligand used in this study was highly resistant to degradative processes in rat brain tissue.     

Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.     

Effects of CCK-8 in the nucleus accumbens

The electrophysiological effects of CCK-8 were studied in the rabbit nucleus accumbens. CCK-8 was found to influence neurotransmitter (modulator) systems so as to enhance their action. For example, CCK-8 enhanced the effects of stimulation of the glutaminergic pathways, the fimbria. In addition, when CCK-8 was co-administered with dopamine and acetylcholine, their suppressive effect on the fimbria evoked response was enhanced. Therefore, CCK-8 appears to be capable of enhancing the influence of multiple neurotransmitter (modulator) systems.     

Receptor binding of cholecystokinin analogues in isolated rat pancreatic acini

The receptor binding of CCK analogues was determined in terms of the inhibition of [125I]CCK binding in isolated rat pancreatic acini. The inhibition curve produced by CCK-8 showed the same feature as that produced by synthetic human CCK-33. The relative potency values of CCK analogues to half-maximally inhibit specific CCK binding were calculated; CCK-8 was equal to human CCK-33, 3-fold stronger than natural porcine CCK-33 and 39, and 700-fold stronger than the unsulphated form of synthetic human CCK-33. Our data suggest that CCK-33, one of the longer molecular forms of CCK, is as important as CCK-8 in the mechanism of physiological actions of CCK.     

CCK-8 inhibits feeding-specific neurons in Navanax, an opisthobranch mollusc

Cholecystokinin has been implicated as a satiety factor in mammals because it inhibits feeding through peripheral and central mechanisms. The cellular mechanisms of the central actions of CCK have been difficult to study because of the complex circuitry of the mammalian brain. Navanax is an opisthobranch mollusc with a defined neural network for feeding behavior in which the central effects of CCK can be studied at the cellular level. Here we report the localization of CCK-immunoreactivity in neuronal cell bodies and varicose fibers in the buccal ganglion of Navanax and that CCK-8 inhibits buccal ganglion neurons selectively and at tenth picomolar concentrations: expansion motoneurons responsible for prey capture are strongly inhibited by CCK-8; circumferential motoneurons responsible for swallowing are weakly inhibited by CCK-8. A large cell, cell X, is described which is sensitive to very low doses of CCK-8. These data imply the existence of a CCK-like peptide with transmitter-like actions in the buccal ganglion of Navanax.     

Differential bile-pancreatic secretory effects of CCK-58 and CCK-8

In this work, we 1) synthesized rat CCK-58, 2) determined the amounts and forms of rat CCK in whole blood after stimulation of its release by casein, 3) determined the potency of CCK-8 and CCK-58 peptides to displace labeled CCK-8 from CCK(A) and CCK(B) receptors transfected into Chinese hamster ovary (CHO) cells, and 4) examined the biological actions of CCK-8 and rat CCK-58 in an anesthetized rat model. CCK-58 was the only detected endocrine form of CCK in rat blood. Synthetic rat CCK-58 was less potent than CCK-8 for displacing the label from CCK(A) and CCK(B) receptors in transfected CHO cells. However, rat CCK-58 was more potent than CCK-8 for stimulation of pancreatic protein secretion in the anesthetized rat. In addition, CCK-58 but not CCK-8 stimulated fluid secretion in this anesthetized rat model. These data suggest that regions outside the COOH terminus of rat CCK-58 influence the expression of CCK biological activity. The presence of only CCK-58 in the circulation and the fact that its biological activity differs from CCK-8 suggests that CCK-58 deserves scrutiny in other physiological models of CCK activity.     

Role of PGE2 on gallbladder muscle cytoprotection of guinea pigs

H2O2 and taurochenodeoxycholic acid (TCDC) impair the contraction induced by CCK-8, ACh, and KCl without affecting the actions of PGE2 and damage functions of membrane proteins except for PGE2 receptors. The aim of this study was to examine whether the preserved PGE2 actions contribute to cytoprotective mechanisms against reactive oxygen species. Muscle cells from guinea pig gallbladder were obtained by enzymatic digestion. Levels of lipid peroxidation and activities of SOD and catalase were determined by spectrophotometry. Pretreatment with PGE2 prevented the inhibition of H2O2 or TCDC on agonist (CCK-8, ACh, and KCl)-induced contraction and reduced the expected increase in lipid peroxidation and activities of catalase and SOD caused by H2O2 and TCDC. Incubation with CCK-8 for 60 min desensitized CCK-1 receptors up to 30 min, whereas no receptor desensitization was observed after PGE2 pretreatment. Cholesterol-rich liposome treatment enhanced the inhibition of H2O2 and TCDC on agonists-induced contraction, including that of PGE2. Pretreatment with PGE2 before H2O2 and TCDC did not completely block their inhibition on agonist-induced contraction. Cholesterol-rich liposome treatment impaired the expected increase in catalase activities in response to PGE2. We conclude that pretreatment with PGE2 prevents the muscle cell damage caused by H2O2 and TCDC due to the resistance of PGE2 receptors to agonist-induced desensitization. The preservation of PGE2 receptors may be designed to conserve these cytoprotective functions that are, however, impaired by the presence of excess cholesterol in the plasma membrane.     

Plasma glucose-lowering effect of beta-endorphin in streptozotocin-induced diabetic rats.

The effect of beta-endorphin on plasma glucose levels was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). A dose-dependent lowering of plasma glucose was observed in the fasting STZ-diabetic rat fifteen minutes after intravenous injection of beta-endorphin. The plasma glucose-lowering effect of beta-endorphin was abolished by pretreatment with naloxone or naloxonazine at doses sufficient to block opioid mu-receptors. Also, unlike wild-type diabetic mice, beta-endorphin failed to induce its plasma glucose-lowering effect in the opioid mu-receptor knock-out diabetic mice. In isolated soleus muscle, beta-endorphin enhanced the uptake of radioactive glucose in a concentration-dependent manner. Stimulatory effects of beta-endorphin on glycogen synthesis were also seen in hepatocytes isolated from STZ-diabetic rats. The blockade of these actions by naloxone and naloxonazine indicated the mediation of opioid mu-receptors. In the presence of U73312, the specific inhibitor of phospholipase C (PLC), the uptake of radioactive glucose into isolated soleus muscle induced by beta-endorphin was reduced in a concentration-dependent manner, but it was not affected by U73343, the negative control of U73312. Moreover, chelerythrine and GF 109203X diminished the stimulatory action of beta-endorphin on the uptake of radioactive glucose at a concentration sufficient to inhibit protein kinase C (PKC). The data obtained suggest that activating opioid mu-receptors by beta-endorphin may increase glucose utilization in peripheral tissues via the PLC-PKC pathway to lower plasma glucose in diabetic rats lacking insulin.     

Pharmacological examination of cholecystokinin (CCK-8)-induced contractile activity in the rat isolated pylorus

The actions of cholecystokinin (CCK) in the production of a satiety-like state have been suggested to be mediated via receptors for CCK which are located in the pylorus. We investigated the actions of CCK and other pharmacological agents upon the isolated rat pylorus in vitro. We used the change in isometric tension of the tissue preparation (contraction amplitude) as the measure of the effects of the pharmacological agents. Cholecystokinin COOH-terminal octapeptide (CCK-8) was observed to elicit contraction in a dose-dependent manner, with the half-maximal dose (ED50) in the vicinity of 1 nM. Rapid desensitization to CCK was observed. The contraction amplitude was atropine-independent, and was not significantly antagonized by a wide variety of other pharmacological agents. The Na+-channel blocker tetrodotoxin was without effect upon contractile amplitude, as was the K+-channel blocker 4-aminopyridine, except at very high concentrations. Neurotensin, bombesin, and the substance P and bombesin antagonist spantide all elicited contraction in the isolated tissue; neurotensin had a similar potency to CCK-8 and bombesin was 10-15-fold less potent than CCK-8. Unsulfated CCK-8 was at least 170-fold less potent than sulfated CCK-8 and tetragastrin was at least 500-fold less potent than CCK-8. These results suggest that pyloric CCK receptors, which appear to have a pharmacological profile typical of peripheral CCK receptors, may have a physiological role in the peptidergic control of gastric emptying in the rat.     

Cholecystokinin tetrapeptide,proglumide and open-field behavior in rats

The effect of cholecystokinin tetrapeptide (CCK-4) was studied in an open field situation. CCK-4 increased locomotion and rearing and the effect was enhanced by proglumide, a selective antagonist of CCK-8. This is in sharp contrast to our earlier findings that CCK-8 decreased the open-field behavior and that proglumide completely blocked the effect. Thus, the effects of CCK-4 and CCK-8 appear to be opposite to each other in that one is excitatory and the other inhibitory to open-field responses.     

Modulation of passive avoidance behaviour of rats by intracerebroventricular administration of cholecystokinin octapeptide sulfate ester and nonsulfated cholecystokinin octapeptide

The effects of intracerebroventricular administration of different doses of cholecystokinin octapeptide sulfate ester (CCK-8-SE) and nonsulfated cholecystokinin octapeptide (CCK-8-NS) were tested on the latency of passive avoidance behaviour in rats. Treatments were carried out prior to learning trial, immediately after electroshock and prior to testing 24 h retention. Both CCK-8-NS and CCK-8-SE enhanced the latency of passive avoidance after all forms of treatment while showing different dose-response patterns depending on time of administration. These data indicate that CCK-8-SE and CCK-8-NS might play a role in the regulation of memory consolidation and retrieval.     

Reversed behavioral effect of cholecystokinin after frontal decortication in rats

Cholecystokinin tetrapeptide (CCK-4) is likely to posses opposite central action against cholecystokinin octapeptide (CCK-8). In the present study, the behavioral effects of CCK-4 and CCK-8 were compared in frontal decorticated rats. In sham-operated rats, CCK-4 produced marked excitatory responses, while CCK-8 had no stimulatory effects. In frontal decorticated rats, CCK-4 did not cause any excitatory behaviors, while CCK-8 produced markedly enhanced responses. We speculate that an appropriate balance of these CCK peptides might be involved in the maintenance of normal mental states.     

CCK-8 stimulation of ventromedial hypothalamic neurons in vitro: A feeding-relevant event?

Bath application of sulfated or non-sulfated cholecystokinin octapeptide (CCK-8s or CCK-8ns, respectively) at concentrations of 25 to 250 nM stimulated the firing activity of 40 to 80% of neurons recorded from the ventromedial nucleus (VMN) in hypothalamic slices maintained in vitro. On the basis of molarity or the percentages of neurons affected, CCK-8s was about 2 to 10 times more potent than CCK-8ns. However, qualitatively, the two forms of CCK-8 were virtually identical: both had a stimulatory action on VMN neurons; both affected VMN neurons in a dose-dependent fashion; both could desensitize their own stimulatory action; and both could cross-desensitize the stimulatory action of the other. These results indicate that not only CCK-8s but also CCK-8ns, which is biologically inactive peripherally, can serve as excitatory neurotransmitters in the VMN, and that both peptides stimulated neurons through the same or a similar neuronal mechanism. It was also found that in the VMN, the stimulatory action of CCK-8 correlated with the actions of norepinephrine, and affected all of the VMN neurons responsive to glucose. Since the actions of glucose and norepinephrine on the activity of VMN neurons are feeding-relevant, our data support the notion that, in addition to acting as a peripheral satiety agent, CCK-8 can also act as a neurotransmitter centrally to mediate satiety.     

Heterogeneous effects of cholecystokinin on neuronal response properties in deep layers of rat barrel cortex

Abstract

Cholecystokinin (CCK) is one of the most studied neuropeptides in the brain. In this study, we investigated the effects of CCK-8s and LY225910 (CCK₂ receptor antagonist) on properties of neuronal response to natural stimuli (whisker deflection) in deep layers of rat barrel cortex. This study was done on 20 male Wistar rats, weighing 230–260?g. CCK-8s (300?nmol/rat) and LY225910 (1?µmol/rat) were administered intracerebroventricularly (ICV). Neuronal responses to deflection of principal (PW) and adjacent (AW) whiskers were recorded in the barrel cortex using tungsten microelectrodes. Computer controlled mechanical displacement was used to deflect whiskers individually or in combination at 30?ms inter-stimulus intervals. ON and OFF responses for PW and AW deflections were measured. A condition-test ratio (CTR) was computed to quantify neuronal responses to whisker interaction. ICV administration of CCK-8s and LY225910 had heterogeneous effects on neuronal spontaneous activity, ON and OFF responses to PW and/or AW deflections, and CTR for both ON and OFF responses. The results of this study demonstrated that CCK-8s can modulate neuronal response properties in deep layers of rat barrel cortex probably via CCK₂ receptors.     

Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.     

Effects of cholecystokinin-8s in the nucleus tractus solitarius of vagally deafferented rats

We have shown recently that cholecystokinin octapeptide (CCK-8s) increases glutamate release from nerve terminals onto neurons of the nucleus tractus solitarius pars centralis (cNTS). The effects of CCK on gastrointestinal-related functions have, however, been attributed almost exclusively to its paracrine action on vagal afferent fibers. Because it has been reported that systemic or perivagal capsaicin pretreatment abolishes the effects of CCK, the aim of the present work was to investigate the response of cNTS neurons to CCK-8s in vagally deafferented rats. In surgically deafferented rats, intraperitoneal administration of 1 or 3 mug/kg CCK-8s increased c-Fos expression in cNTS neurons (139 and 251% of control, respectively), suggesting that CCK-8s' effects are partially independent of vagal afferent fibers. Using whole cell patch-clamp techniques in thin brain stem slices, we observed that CCK-8s increased the frequency of spontaneous and miniature excitatory postsynaptic currents in 43% of the cNTS neurons via a presynaptic mechanism. In slices from deafferented rats, the percentage of cNTS neurons receiving glutamatergic inputs responding to CCK-8s decreased by approximately 50%, further suggesting that central terminals of vagal afferent fibers are not the sole site for the action of CCK-8s in the brain stem. Taken together, our data suggest that the sites of action of CCK-8s include the brain stem, and in cNTS, the actions of CCK-8s are not restricted to vagal central terminals but that nonvagal synapses are also involved.     

Cholecystokinin-58 and cholecystokinin-8 produce similar but not identical activations of myenteric plexus and dorsal vagal complex

The enteric nervous system (ENS: myenteric and submucosal plexuses) of the gastrointestinal tract may have a role in the reduction of food intake by cholecystokinin (CCK). Exogenous cholecystokinin-8 (CCK-8) activates the myenteric plexus and the feeding control areas of the dorsal vagal complex (DVC) of the brainstem. An increasing number of reports, however, have shown that CCK-58 is the sole or the major circulating form of CCK in rat, human and dog, and that it is qualitatively different from CCK-8 in evoking various gastrointestinal physiological responses (e.g., contraction of the gallbladder and exocrine pancreatic secretion). In the current report, we compared the abilities of exogenous CCK-58 to activate the myenteric plexus and the dorsal vagal complex with those of exogenous CCK-8 by quantifying Fos-like immunoreactivity (Fos-LI; a marker for neuronal activation). We report that CCK-58 (1, 3, and 5 nmol/kg) increased Fos-LI in the myenteric plexus (p<0.001) and in the DVC (p<0.001) compared to the saline vehicle. The highest dose of CCK-58 increased Fos-LI more than an equimolar dose of CCK-8 in the myenteric plexus and the area postrema. Thus, CCK-8 and CCK-58 produce the same qualitative pattern of activation of central and peripheral neurons, but do not provoke identical quantitative patterns at higher doses. The different patterns produced by the two peptides at higher doses, in areas open to the circulation (myenteric plexus and area postrema) may reflect endocrine actions not observed at lower doses.