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.     

Differential in vitro metabolism of beta-endorphin in schizophrenia

Biologically active peptide fragments derived from the proteolytic cleavage of beta-endorphin (beta E) have been shown to be present in the brain. Based on clinical results using some of these fragments in neuropsychiatric disease studies we investigated the in vitro metabolism of beta E by twice-washed membrane homogenates of postmortem putamen from sex and age matched controls versus subjects with a diagnosis of schizophrenia. The present study demonstrates that frozen (-80 degrees C) postmortem human tissues are viable for these studies and that metabolism in control tissue proceeds similarly to fresh tissues. Furthermore, a significant increase in the formation of the putative neuroleptic-like peptide fragment des-enkephalin-gamma-endorphin in postmortem schizophrenic putamen versus controls was shown. A significant decrease in the formation of beta E was also reported. These data suggest that an approach using postmortem human brain is possible in studying beta-endorphin catabolism and is therefore applicable to other neuropeptide systems.     

Modulation of spontaneous seizures in the mongolian gerbil: effects of beta-endorphin

Intraventricular injection of beta-endorphin (0.1-3 micrograms) into gerbils from the UCLA seizure sensitive strain reduced the incidence and severity of spontaneous epileptiform seizures, both the motor manifestations and the preceding high voltage focal spiking and accompanying seizure activity in the cortical EEG. This "'anticonvulsant" effect of beta-endorphin was prevented by prior administration of naloxone (1 mg . kg-1 IP). These findings suggest that the endogenous opioid peptide may be involved in the normal suppression of the epileptic diathesis in these animals during the interictal periods.     

Characterization of in vitro proteolytic processing of beta-endorphin by reversed-phase HPLC

In vitro, central and peripheral proteolytic processing of beta-endorphin by membrane-bound enzymes results in the formation of specific active fragments that have been recently shown to function in behavior, intestinal motility and in the central control of urinary bladder activity. A high resolution, reversed phase high performance liquid chromatography system capable of separating 28 beta-endorphin related fragments simultaneously was used to study the time-course processing of beta-endorphin by membrane associated peptidases in the brain and regions of the small intestine. The hypothesis we tested was that a homeostatic balance between alpha- and gamma-type endorphins exists in these tissues. The results of the study show that the rate and quantity of fragments produced between the mucosa and nerve-muscle regions of the small intestine are significantly different. Metabolic rates, pattern, and the ratio of alpha/gamma-type endorphins in the brain were very similar to the nerve-muscle region of the small intestine. This suggests that beta-endorphin processing to active fragments is occurring at the nerves of the small intestine and that a specific and similar balance of alpha/gamma-type endorphin exists in the brain and gastrointestinal system at neutral pH.     

Acetylation of alpha MSH and beta-endorphin by rat neurointermediate pituitary secretory granule-associated acetyltransferase

ACTH(1-8) and ACTH(9-13)NH2 were used as potential enzyme inhibitors to begin examining the relationship between the acetylation of ACTH- and beta-endorphin-related peptides. ACTH(1-8) was a potent inhibitor of the acetylation of both ACTH- and beta-endorphin-related peptides, whereas ACTH(9-13)NH2 was an effective inhibitor only of the acetylation of ACTH-related substrates. This inhibition pattern indicated that there may be an unusual interaction between some ACTH- and beta-endorphin-related peptides as substrates for the acetyltransferase. Utilizing HPLC to separate ACTH- and beta-endorphin-related peptides present in the same reaction mixture, ACTH(1-14) and beta-endorphin(1-27) at Km and saturating concentrations were used as substrates to examine the ability of one peptide substrate to affect the acetylation of the other. It was observed that the acetylation of ACTH(1-14), even at Km concentration, was relatively unaffected by the presence of beta-endorphin(1-27). However, the acetylation of beta-endorphin(1-27) was significantly reduced by the presence of ACTH(1-14). This preferential acetylation of ACTH-related peptides over the acetylation of beta-endorphin-related peptides might have physiological importance under some conditions.     

Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Cerebral Cortex: Effect of Quinolinic Acid Lesions of the Basal Forebrain

The effects of neurotensin (NT) on endogenous acetylcholine (ACh) release from basal forebrain, frontal cortex, and parietal cortex slices were tested. The results show that NT differentially regulates evoked ACh release from frontal and parietal cortex slices without altering either spontaneous or evoked ACh release from basal forebrain slices. In the frontal cortex, NT significantly inhibited evoked ACh release by a tetrodotoxin (TTX)-insensitive mechanism, suggesting an action directly on cholinergic terminals. In the parietal cortex, NT enhanced evoked ACh release by a TTX-sensitive mechanism, suggesting an action of NT on the cholinergic neuron or in close proximity to the cholinergic neuron. The effects of NT on ACh release were confined to evoked ACh release; that is, spontaneous ACh release was not affected. NT did not affect spontaneous or potassium-evoked ACh release from occipital cortex slices. The second set of experiments tested the effects of quinolinic acid (QUIN) lesions of the basal forebrain cell bodies on the NT-induced regulation of evoked ACh release in the cerebral cortex. QUIN lesions of basal forebrain cell bodies caused decreases in choline acetyltransferase activity (27 and 28%), spontaneous ACh release (14 and 21%), and evoked ACh release (38 and 44%) in frontal and parietal cortex, respectively. In addition, 11 days following QUIN lesions of basal forebrain cell bodies, the action of NT to regulate evoked ACh release in frontal cortex or parietal cortex was no longer observed. The results suggest that in the rat frontal and parietal cortex, NT differentially regulates the activity of cholinergic neurons by decreasing and increasing evoked ACh release, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced ACTH, while normal beta-endorphin CSF levels in early epileptic encephalopathies

Since ACTH and the opioids display opposite effects on experimentally-induced seizures, cerebrospinal fluid (CSF) levels of ACTH and beta-endorphin (beta-EP) were measured in 6 children (4-8 months) affected by infantile spasms with hypsarhythmia, an idiopathic early onset encephalopathy, and in 8 age-matched controls. beta-EP levels in the patients (76.3 +/- 14.7 fmol/ml, M +/- SD) did not differ from those in controls (109.8 +/- 42.7) while babies with epileptic encephalopathy showed reduced ACTH levels in the CSF (3.8 +/- 1.5) as compared to controls (9.0 +/- 3.7, p less than 0.01). This resulted in an increased beta-EP/ACTH ratio. Another patient previously treated with ACTH showed a normal CSF level of ACTH (9.0) with a normal beta-EP/ACTH ratio while in clinical and EEG remission. These results are consistent with the hypothesis that some infantile seizures unrelated to brain injuries could originate from an ACTH deficiency at central level and/or an imbalance of neuropeptidergic pathways.     

Beta-endorphin-like peptide SLTCLVKGFY reduces the production of 11-oxycorticosteroids by rat adrenal cortex through nonopioid beta-endorphin receptors

β-Endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (K_d=31.6±0.2 nM, B_max=37.4±2.2 pmol/mg protein). Immunorphin at concentrations of 10⁻⁹ to 10⁻⁶ M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10–100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.     

Regional distributions of somatostatin and cholecystokinin-like immunoreactivities in rat and bovine brain

The distribution of somatostatin- and cholecystokinin octapeptide (CCK-8)-like immunoreactivities in the central nervous system of bovine and rat has been studied using a sensitive and specific radioimmunoassay. The most interesting information is the high concentration of CCK-8 in the various cortical areas in rat and bovine. The nuclei of amygdala contain the highest amount of octapeptide in rat while in bovine brain, this structure contains the second highest concentration after polus frontalis.     

Action of selected serotonin antagonists on hyperthermia evoked by intracerebrally injected beta-endorphin

Methergoline, an antagonist of cerebral serotonin receptors, has been shown to significantly reduce the rise in rectal temperature (Tre) produced by the intracerebral microinjection of beta-endorphin. In this study the role of serotonin in the increase in Tre elicited by beta-endorphin was further examined using three additional serotonin antagonists. beta-Endorphin was administered twice to rats using a crossover design in which half of the animals were first pretreated with the vehicle solution and half with the antagonist. Serotonin antagonists used were: methergoline, methysergide, cinanserin and cyproheptadine. Although methergoline did cause a marked reduction in the beta-endorphin-induced rise in Tre, neither methysergide, nor cinanserin, nor cyproheptadine produced a marked reduction in the hyperthermia. Since methergoline also interacts with the dopamine receptor, the effect of a dopamine antagonist, haloperidol, on the endorphin-evoked response was also examined. Haloperidol failed to attenuate the rise in Tre. The reason for the apparent discrepancy in the action of these serotonin antagonists is unclear. Further research may reveal distinct subpopulations of serotonin receptors at which these antagonists exert differential effects.     

Neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity in the cat hypothalamus

Immunohistochemical studies were conducted on sections of cat hypothalamus in order to determine the distribution of neurons containing alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity. A large number of neurons in the arcuate nucleus were stained after incubation of sections with antisera to either substance. Analysis of serial sections suggested that each neuron revealed with one antiserum was also revealed with the other antiserum, indicating the co-existence of alpha-melanocyte stimulating hormone and beta-endorphin immunoreactivity within these arcuate neurons. In contrast, a more diffuse group of lateral hypothalamic neurons which extended from the retrochiasmatic level to the posterior hypothalamus were stained only with the antiserum directed against alpha-melanocyte stimulating hormone. The present results largely confirm findings in the rat hypothalamus, although the lateral hypothalamic group of alpha-melanocyte stimulating hormone immunoreactive neurons appears to be more extensive in the cat.     

Thermoregulatory (core, surface and metabolic) responses of unrestrained rats to repeated POAH injections of beta-endorphin or adrenocorticotropin

Repeated preoptic-anterior hypothalamic (POAH) injections of saline and 10 or 25 micrograms/microliters of beta-endorphin or ACTH were given to groups of male Sprague-Dawley rats. One hr after the fifth injection of beta-endorphin or ACTH, each rat received a POAH injection of naloxone HCl (10 micrograms/microliters). Core (Tre-rectal) and surface (Tt-tail) temperatures, metabolic (VO2) and behavioral responses were recorded 30 min before and 60 min after each drug injection. The initial POAH injection of either dose of beta-endorphin produced a hyperthermia. Peak hyperthermia was reduced in the group given 10 micrograms/microliters of beta-endorphin repeatedly. TtS rose after each beta-endorphin injection but temporally lagged Tre increases. Metabolic rate (VO2) was increased with repeated POAH injections of beta-endorphin. Naloxone reduced the elevated Tre seen with beta-endorphin by increasing Tt's further and reducing VO2. POAH administration of ACTH evoked only a slight hyperthermic Tre response, but elevated TtS and VO2S, due to enhanced grooming and explorative behavior. With repeated ACTH injections, TreS did not change from those on the first day as TtS and VO2 remained enhanced. Naloxone reduced VO2 and TtS of the ACTH-treated rats but TreS still were unchanged. Results suggest that the hyperthermia of unrestrained rats given an acute as opposed to repeated POAH beta-endorphin injections is mediated by different effector mechanisms. With the doses used, the slight and unchanging TreS seen with ACTH occurred because this peptide increased heat production due to locomotor activation yet also exaggerated heat loss by vasodilating the peripheral vasculature.     

Pineal mediation of the thermoregulatory and behavioral activating effects of beta-endorphin

Intraventricular administration of the opioid peptide, beta-endorphin to goldfish altered their body temperatures and activity levels. Low doses (0.5-5.0 pg g-1 body weight) of beta-endorphin significantly increased behaviorally selected body temperatures while higher doses (15 pg g-1) decreased the preferred temperatures selected in horizontal thermal gradients. There was a significant day-night rhythm in the extent of these effects. These thermoregulatory effects could be blocked and reversed by systemic administration of the opiate antagonist, naloxone, supporting mediation of the thermoregulatory effects at opioid receptors. In addition, administration of naloxone by itself significantly decreased preferred temperature. Removal of the pineal gland significantly increased the preferred temperatures selected by goldfish and eliminated the thermoregulatory effects of beta-endorphin administration in both the day and the night. The behavioral activity effects of beta-endorphin were dependent on the thermal conditions. In fish held at a constant temperature (20 degrees C) beta-endorphin caused a dose-dependent increase in activity, while in individuals held in thermal gradients administration of beta-endorphin had no effects on activity. In both situations naloxone caused a decrease in activity levels. Pinealectomy also eliminated the behavioral activating effects of beta-endorphin, though it had no apparent effects on the actions of naloxone. These results indicate that the pineal gland is involved in the mediation of the thermoregulatory and behavioral activating effects of beta-endorphin. Speculations are made as to the possible mechanisms of action of the pineal gland in mediating the effects of opioid neuropeptides.     

Postmortem Stability of Monoamines, Their Metabolites, and Receptor Binding in Rat Brain Regibns

Abstract: The effects of postmortem delay, time of storage, and freezing, thawing, and refreezing tissue samples were studied in postmortem rat brain using conditions that reflect the handling of postmortem human brain before neurochemical analysis. The levels of monoamines and metabolites in the striatum and cingulate and occipital cortex were measured using alumina extraction and HPLC methods. Binding of raclopride to dopamine D₂, SCH- 23390 to dopamine D₁, ketanserin to serotonin 5-HT₂, 8- hydroxy-2-(di- n -propylamino)tetralin to serotonin 5-HT_1A, and cholecystokinin (CCK)-8 to CCK-B sites was measured in tissue homogenates from the striatum or frontoparietal cortex. An 18-h postmortem delay before dissection and storage resulted in region-specific changes in monoamine and metabolite levels. Binding to striatal D₁and frontoparietal cortex CCK-B sites was reduced over the course of a 27-h postmortem delay. Binding to D₂ and 5-HT sites was relatively stable. Storage of tissue for up to 8 months also resulted in region-specific changes in monoamine and metabolite levels. No changes in receptor binding were seen after long-term storage. Freezing, thawing, and refreezing tissue samples resulted in increased levels of striatal 3, 4-dihydroxyphenylacetic acid and decreased binding to striatal D₂ sites. These results demonstrate time-, temperature-, and storage-dependent regional differences in.the stability of monoamines and their metabolites and in binding to various receptor sites. These differences in stability and binding should be accounted for to interpret accurately the effects of neurological disorders on neurotransmitter dynamics in postmortem human brain tissue.     

Feeding and drinking responses in the golden hamster following treatment with cholecystokinin and angiotensin II

Drinking and feeding behaviours of female golden hamsters were examined following intracerebroventricular injections of angiotensin II or systemic and intracerebroventricular injections of cholecystokinin octapeptide. Injections of angiotensin II into the brain produced a dose-dependent drinking response in water repleted animals. Systemically, a low dose (0.5 microgram/kg body wt) of cholecystokinin was ineffective at reducing food intake of fasted animals during a 1 hr test. Larger peripheral doses (1.0 to 4.0 microgram/kg body wt), however, were effective at decreasing food intake. Injected in the lateral cerebral ventricle, nanogram doses of cholecystokinin decreased food consumption in a dose dependent manner. These results are discussed in relation to how these peptides regulate feeding and drinking behaviours in other species.     

Cholecystokinin octapeptide: effects on the excitability of cultured spinal neurons

The actions of cholecystokinin octapeptide (CCK) on the membrane properties of mouse spinal neurons grown in monolayer culture were examined using intracellular recording techniques. In a subpopulation of cells, application of CCK (0.2-100 micron) by pressure ejection from micropipettes produced a small (approximately 2 mV) membrane depolarization that was accompanied by a decrease in membrane conductance (approximately 11 percent). These effects were associated with an enhanced tendency of the cells to generate action potentials when stimulated with intracellular depolarizing current. The unsulfated analog of CCK, which possesses weak biological activity in the gut, had little or no effect on cultured spinal neurons. A number of differences were noted between the responses to CCK and the excitatory amino acid glutamate. First, the effects of CCK were more delayed in onset (approximately 17 sec) and prolonged in duration (approximately 124 sec). Second, the depolarizations produced by glutamate were of larger magnitude and associated with variable effects on membrane conductance. Third, the response to CCK showed tachyphylaxis with repeated applications whereas glutamate remained effective as often as it was applied. It is concluded that CCK facilitates the excitability of spinal neurons in a manner distinct from that of the conventional excitant glutamate.     

Effects of beta-endorphin on body temperature in mice at different ambient temperatures

The effect of intracerebroventricular injection of beta-endorphin (beta-END) on body temperature of mice was studied at ambient temperatures (Ta) of 10 degrees, 20 degrees and 31 degrees C. Doses between 0.1 and 10.0 microgram/mouse were studied. The lower (less than 1 microgram) doses of beta-END produced a hyperthermia at all Ta's studied. The higher doses of beta-END produced hyper- or hypothermia depending on the Ta. The subcutaneous injection of naloxone (1 mg/kg) antagonized the high dose hypothermic effects, but not the hyperthermic effect of beta-END. These data suggest that there may be different receptors and/or sites of action for high and low doses of beta-END.     

Neuropeptides in the gut: quantification and characterization of cholecystokinin octapeptide-, bombesin- and vasoactive intestinal polypeptide-like immunoreactivities in the myenteric plexus of the guinea-pig small institute

The localization of CCK8-, bombesin- and VIP-like immunoreactivities in the myenteric plexus of the guinea pig small intestine has been studied by radioimmunoassay of extracts of longitudinal muscle strips obtained with and without adherent myenteric plexus; concentrations were compared with those in other regions of the gut. In innervated strips of longitudinal muscle of ileum there was approximately 14 pmol/g CCK8-, 32 pmol/g bombesin- and 135 pmol/g VIP-like immunoreactivity; concentrations were reduced by over 70% in denervated strips. Gel filtration and ion exchange chromatography indicated that over 80% of CCK immunoreactivity was due to CCK8; no evidence was found of significant amounts of smaller COOH-terminal fragments. Bombesin immunoreactivity occurred in two forms, the major one resembling the amphibian tetradecapeptide in its elution from gel filtration columns. Immunoreactive VIP differed markedly from porcine VIP in immunochemical and chromatographic properties; the data suggest that guinea pig VIP is less basic than porcine VIP and that the two peptides differ in structure in their NH2-terminal regions. Some functional implications of these findings are discussed.