Suppressive effect on food intake of a potato extract (Potein®) involving cholecystokinin release in rats.

We have recently reported that oral gavage of a potato extract (Potein®) suppressed the food intake in rats. The satiating effect of the potato extract was compared in the present study to other protein sources, and the involvement of endogenous cholecystokinin (CCK) secretion was examined. Food consumption was measured in 18-h fasted rats after oral gavage of the potato extract or other protein sources. The CCK-releasing activity of the potato extract was then examined in anesthetized rats with a portal cannula. Oral gavage of the potato extract reduced the food intake in the rats, the effect being greater than with casein and a soybean ß-conglycinin hydrolysate. The suppressive effect on appetite of the potato extract was attenuated by treating with a CCK-receptor antagonist (devazepide). The portal CCK concentration was increased after a duodenal administration of the potato extract to anesthetized rats. These results indicate that the potato extract suppressed the food intake in rats through CCK secretion.

Effect of heat exposure on gene expression of feed intake regulatory peptides in laying hens.

The aim of this paper was to investigate the effect of heat stress on the regulation of appetite-associated genes in laying hens. Forty eight laying hens were randomly divided into two circumstances: high (31 ± 1.5°C; relative humidity, 82.0 ± 2.2%) or normal (20 ± 2°C, control; relative humidity, 60.1 ± 4.5%) ambient environment. Heat stress decreased body weight gain (P < 0.01), feed intake (P < 0.01), laying rate (P < 0.05), average egg mass (P < 0.01), egg production (P < 0.01), shell thickness (P < 0.01), and feed efficiency (P < 0.05). High ambient temperature decreased plasma uric acid (P < 0.05). Heat stress significantly increased mRNA levels of ghrelin and cocaine- and amphetamine-regulated transcript (P < 0.05) and decreased mRNA levels of cholecystokinin (P < 0.05) in the hypothalamus. Heat stress significantly increased (P < 0.05) mRNA levels of ghrelin in the glandular stomach and jejunum but significantly decreased (P < 0.05) mRNA levels of cholecystokinin in the duodenum and jejunum. In conclusion, heat stress plays a unique role in some special neuropeptides (e.g., ghrelin, cocaine- and amphetamine-regulated transcript, and cholecystokinin), which might participate in the regulation of feed intake in laying hens under high ambient temperature.

Duodenal lipid-induced symptom generation in gastroesophageal reflux disease: role of apolipoprotein A-IV and cholecystokinin.

BACKGROUND: Duodenal lipid intensifies the perception of esophageal acid perfusion. Recently, we showed that genes implicated in lipid absorption were upregulated in the duodenum of fasting gastro-esophageal reflux disease (GERD) patients. This suggests that chylomicron production and secretion may be enhanced and, consequently, the release of apolipoprotein A-IV (apoA-IV), a chylomicron-derived signaling protein. ApoA-IV may stimulate release of cholecystokinin (CCK), an activator of vagal afferents. This study evaluated putative involvement of abnormal apoA-IV and CCK responses to lipid in GERD. METHODS: Ten GERD patients and 10 healthy volunteers (HV) underwent duodenal perfusion with Intralipid 20%, 2 kcal min(-1) , for 60 min. Symptoms were scored, blood samples collected every 15 min during lipid perfusion and 15 min after discontinuation when duodenal biopsies were taken. Plasma and mucosal concentrations of apoA-IV and CCK and transcript levels of 21 genes implicated in lipid absorption, differentially expressed under fasting conditions, were quantified. KEY RESULTS: Heartburn (P = 0.003), abdominal discomfort (P = 0.037) and nausea (P = 0.008) only increased significantly during lipid infusion in GERD patients. Following lipid infusion mean mucosal apoA-IV concentration was lower in GERD patients compared with HV (P = 0.023), whereas plasma concentration tended to be elevated (P = 0.068). Mean mucosal CCK concentration was also lower in GERD patients (P = 0.009). Two genes, HIBADH and JTB, were upregulated in GERD patients (P = 0.008 and P = 0.038, respectively). CONCLUSIONS & INFERENCES: Our results suggest excessive duodenal lipid-induced release of apoA-IV and CCK in GERD. We postulate that the resulting heightened activation of duodenal vagal afferents may underlie central sensitization, thereby increasing the perception of reflux events.

Effect of Salvia miltiorrhiza pretreatment on the CCK and VIP expression in hepatic ischemia-reperfusion-induced digestive tract congestion.

The inhibitory effect of different reperfusion periods 45 min following hepatic ischemia on the expression of cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) in the jejunum and the effect of salvia miltiorrhiza pretreatment were investigated, and the possible mechanism and implications were explored. Eighty rats were randomly divided into four groups: normal control group (CO group), sham-operated group (SO group), ischemia/reperfusion (I/R) injury group (IR group) and salvia miltiorrhiza pretreatment group (SM group). The rat model of I/R was established by using a non-invasive artery clamp to clip (45 min) or relax the hepatic pedicle. In the SM group, saline (40 mL/kg) and salvia miltiorrhiza injection (6 g/kg) were injected via the tail vein 30 min before clipping the hepatic pedicle. In the SO group only the porta hepatis was dissected after laparotomy without clamping the hepatic pedicle. At 0, 3, 12, 24 and 72 h post-reperfusion, respectively, upper jejunum samples were taken for immunohistochemistry of CCK and VIP. It was found that 0 h after I/R, the expression of CCK and VIP in the upper jejunum was upregulated. With prolongation of the reperfusion period, the expression of CCK and VIP was also increased, reached the peak at the 24th h, and gradually returned to the normal level at the 72nd h after reperfusion. The levels of both CCK and VIP in the SM group were lower than those in the IR group. It is suggested that the digestive tract congestion injury caused by liver ischemia can upregulate the expression of CCK and VIP in the jejunum following reperfusion. Salviae pretreatment can partly reduce the increased expression of CCK and VIP in the jejunum in the same period, which might contribute to the early recovery of gastrointestinal motility.

[Overdose of iodine on expression of CCK gene in rat brains].

OBJECTIVE: To observe the effect of overdose iodine on the expression of CCK gene in brains of rats and identify the possible mechanisms. METHODS: One-month weaning Wistar rats were randomly divided into five groups which were fed with normal feedstuff and water supplemented with different concentrations of potassium iodide, named A group (iodine ration was about 6.15 microg per day), B group (iodine ration was about 30.75 microg per day), C group (iodine ration was about 61.5 microg per day), D group (iodine ration was about 307.5 microg per day) and E group (iodine ration was about 615 microg per day). Rats were sacrificed after being fed for three or six months. Then serum thyroid hormones were measured by radioimmunoassay and the mRNA level of CCK gene was studied by using RT-PCR technique. RESULTS: At the end of three months, the values of thyroid hormones in E group [TT4 (45.2 +/- 13.7) nmol/L, TI'3 (0.65 +/- 0.20) nmol/L, FT3 (0.93 +/- 0.45) pmol/L, FT4 (7.07 +/- 2.43) pmol/L, rT3 (0.15 +/- 0.04) nmol/L] were all lower than those in A group [TT4 (76.0 +/- 18.8) nmol/L, TT3 (1.34 +/- 0.41) nmol/L, FT3 (2.45 +/- 0.62) pmol/L, FT4 (15.12 +/- 3.40) pmol/L, rT3 (0.24 +/- 0.04) nmol/L]. There were significant differences between E group and A group on the levels of serum TH (F values are 14.68, 16.03, 21.16, 20.25, 13.52 respectively, P < 0.01); FT3 levels in C and D groups were significantly decreased as compared to A and B groups (F = 21.16, P < 0.05). rT3 level in D group was significantly decreased compared with A,B and C groups (F = 13.52, P < 0.05). At the end of six months, the levels of serum TH in E group (TT4 (51.84 +/- 15.83) nmol/L, TT3 (0.77 +/- 0.22) nmol/L, FT4 (6.88 +/- 2.23) pmol/L, FT3 (0.74 +/- 0.28) pmol/L, rT3 (0.14 +/- 0.03) nmol/L) were lower than those in any other groups (F values were 6.05, 12.22, 11.25, 13.42, 5.89 respectively, P < 0.05). At the end of both three and six months, the mRNA levels of CCK gene in E group were lower than any other groups (F values were 4.04, 3.95 respectively, P < 0.01). The results of correlation analysis showed that serum FT4 had linear correlation with levels of CCK mRNA (r values were 0.990, 0.948 respectively; P < 0.05); However serum FT3 had no linear correlation with the levels of CCK mRNA (r values are 0.970, 0.932 respectively). CONCLUSIONS: Exposure to overdose of iodine (iodine ration was 100-fold higher than that of A group) could decrease the mRNA level of CCK gene. Compared with FT3, FT4 might have more important role on the regulation of CCK mRNA induced by excess of iodine.

Central expressions of ghrelin and cholecystokinin in rats with gastric electrical stimulation.

BACKGROUND: Gastric electrical stimulation (GES) has recently been proposed for the treatment of obesity. The aim of this study was to explore the possible central mechanisms involved in GES by investigating the expression of orexigenic and anorexigenic peptides in the rodent hypothalamus and hippocampus. METHODS: The experiment was designed in two parts: an acute experiment with 2 h GES and a chronic experiment with 14-day continuous GES. After stimulation, the expressions of an orexigenic hormone, ghrelin, in the hypothalamus and an anorexigenic hormone, cholecystokinin (CCK), in the hippocampus were detected by the immunohistochemical method. GES was performed using parameters similar to those used in clinical studies for treating obesity. RESULTS: Compared with the control group, 2 h GES resulted in a decrease in the number of ghrelin-immunoreactive (ghrelin-IR) neurons in the hypothalamic paraventricular nucleus (PVN, 34.8 +/- 1.86 vs 57.2 +/- 2.95, P = 0.02) and the supraoptic nucleus (SON, 51.2 +/- 3.21 vs 82.8 +/- 3.08, P = 0.01); the CCK-immunoreactive (CCK-IR) neurons in the hippocampus were of no changes (7.4 +/- 0.87 vs 6.2 +/- 0.58, P = 0.29). After the 14-day GES, the number of CCK-IR neurons in the hippocampus was increased compared with that of the control group (4.0 +/- 0.32 vs 2.4 +/- 0.51, P = 0.03). However, there were no changes in the number of ghrelin-IR neurons either in the PVN or in the SON. CONCLUSIONS: These results indicate that the expression of ghrelin and CCK can be altered by GES. GES may be able to alter energy homeostasis by modulating the expressions of food intake-related hormones in the central nervous system: reducing the level of orexigenic ghrelin acutely and increasing the level of anorexigenic CCK chronically.

Regional brain cholecystokinin changes as a function of rough-and-tumble play behavior in adolescent rats.

Brain cholecystokinin (CCK) levels have been shown to be elevated in animals defeated during adult social aggression. The present experiment evaluated whether similar effects are evident in prolonged bouts of juvenile social-play fighting, which tend to switch from largely positive to some negative affect after approximately 15 min into a half-hour play session, as indexed by a gradual shift from positively valenced 50 kHz ultrasonic vocalizations (USVs) to negatively valenced 20 kHz USVs. Given the role of CCK in both positive and negative emotional events, we examined levels of CCK-8 in tissue homogenates from 14 brain areas in animals 6h after a 30 min play bout compared to no-play control animals tested similarly in isolation for 30 min. As with patterns observed following adult defeat, significantly higher CCK levels were evident after play in the posterior neo-cortex compared to no-play control animals (+26%). Levels of CCK were also elevated in the midbrain (+35%). However, unlike in adult aggression, CCK levels were reduced in the hypothalamus (-40%) and basal forebrain (-24%) as compared to no-play animals. Posterior cortex CCK levels were positively correlated to the duration that each animal was pinned (r = +.50) which suggests that elevated CCK in the posterior cortex may be related to the negative aspects of play. Hypothalamic CCK levels were negatively related to dorsal contacts and pins (r's = -.57), and suggest that the lower CCK levels may reflect the more positive valenced aspects of play. The data indicate that CCK utilization in the brain is dynamically responsive to rough-and-tumble play.

Complementary roles of cholecystokinin- and parvalbumin-expressing GABAergic neurons in hippocampal network oscillations.

In the hippocampal CA1 area, a relatively homogenous population of pyramidal cells is accompanied by a diversity of GABAergic interneurons. Previously, we found that parvalbumin-expressing basket, axo-axonic, bistratified, and oriens-lacunosum moleculare cells, innervating different domains of pyramidal cells, have distinct firing patterns during network oscillations in vivo. A second family of interneurons, expressing cholecystokinin but not parvalbumin, is known to target the same domains of pyramidal cells as do the parvalbumin cells. To test the temporal activity of these independent and parallel GABAergic inputs, we recorded the precise spike timing of identified cholecystokinin interneurons during hippocampal network oscillations in anesthetized rats and determined their molecular expression profiles and synaptic targets. The cells were cannabinoid receptor type 1 immunopositive. Contrary to the stereotyped firing of parvalbumin interneurons, cholecystokinin-expressing basket and dendrite-innervating cells discharge, on average, with 1.7 +/- 2.0 Hz during high-frequency ripple oscillations in an episode-dependent manner. During theta oscillations, cholecystokinin-expressing interneurons fire with 8.8 +/- 3.3 Hz at a characteristic time on the ascending phase of theta waves (155 +/- 81 degrees), when place cells start firing in freely moving animals. The firing patterns of some interneurons recorded in drug-free behaving rats were similar to cholecystokinin cells in anesthetized animals. Our results demonstrate that cholecystokinin- and parvalbumin-expressing interneurons make different contributions to network oscillations and play distinct roles in different brain states. We suggest that the specific spike timing of cholecystokinin interneurons and their sensitivity to endocannabinoids might contribute to differentiate subgroups of pyramidal cells forming neuronal assemblies, whereas parvalbumin interneurons contribute to synchronizing the entire network.

Distribution of glutamic acid decarboxylase, neurotensin, enkephalin, neuropeptide Y, and cholecystokinin neurons in the septo-preoptic region of male rats.

Neurons in the lateral septum (LS) and preoptic area (POA) are known to play an inhibitory role in feminine sexual behavior regulation in male rats. In this study, the distribution of neurons containing glutamic acid decarboxylase (GAD) and of the peptidergic neurotransmitters neurotensin (NT), enkephalin (ENK), neuropeptide Y (NPY), and cholecystokinin (CCK), was examined immunohistochemically in the LS and POA of castrated male rats subcutaneously implanted with estrogen-containing Silastic tubes. Colchicine was injected into the lateral ventricle of the animals. The forebrain sections were immunostained for each substance. A large number of GAD-immunoreactive (ir) cells were found in the LS. Many NT-ir cells were seen in the intermediate and ventral parts of the LS at the rostral and middle levels. A considerable number of ENK-ir cells were scattered over the LS at the rostral and middle levels and were observed in the ventral part of the caudal LS. There were only a few NPY-ir cells in the LS. No CCK-ir cells were observed in the LS. In the POA, GAD-ir cells were observed in abundance. Many NT-ir cells were seen, especially in the medial preoptic nucleus. Some ENK-ir cells and a few NPY-ir cells were found in the medial POA. CCK-ir cells of the POA were restricted to the periventricular and paraventricular hypothalamic nuclei.

A transient increase in CCK mRNA levels in hippocampus following audiogenic convulsions in audiogenic seizure-prone rats.

AIM: To examine the effects of a single convulsion and multiple convulsions on cholecystokinin (CCK) mRNA expression in hippocampus of audiogenic seizure-prone rats (P77PMC). METHODS: Ringing (electric bell, 100 dB, 60 s) was used to induce convulsions, hippocampal CCK mRNA expression was exhibited by in situ hybridization. RESULTS: 1) The number of CCK mRNA-positive neurons in principal hippocampus of normal rats was 34 +/- 5, which elevated markedly after a single (155 +/- 7, P < 0.01) or multiple convulsions (95 +/- 8, P < 0.01). 2) CCK mRNA levels in multiple consecutive convulsion rats were lower than that in a single convulsion rats (P < 0.01). CONCLUSION: The increased number of CCK mRNA-positive neurons in hippocampus may have important functional consequences in convulsion-associated processes.

Identification and expression of gastrin and cholecystokinin mRNAs from the turtle, Pseudemys scripta: evidence of tissue-specific tyrosyl sulfation(1).

Gastrin and cholecystokinin (CCK) are related peptide hormones expressed in the brain and gut of vertebrates. In this study, complementary DNAs have been characterised from the red-eared slider turtle, Pseudemys scripta. The encoded preproCCK contains mono and dibasic endoproteolytic processing sites for formation of the previously identified CCK-70, CCK-40 and CCK-8 products, whereas preprogastrin contains two dibasic processing sites for the generation of gastrin-52. Alignment of the predicted preprohormone structures with those of other species, showed that preproCCK has been well conserved among all vertebrates, whereas progastrin is less conserved. Both gastrin and CCK mRNA display expression patterns similar to their mammalian counterparts, with CCK being expressed in the brain, duodenum and small intestine, and gastrin in the antrum. Heterologous expression of turtle preprogastrin in a mammalian endocrine cell line led to production of carboxyamidated gastrin-52 as observed in turtle antrum. However, in contrast to the non-sulfated endogenous peptide, the heterologously expressed gastrin was completely Tyr sulfated. Consequently, it appears that either gastrin producing cells in the turtle gut do not express tyrosylprotein sulfotransferases or the enzyme(s) present in turtle antrum is unable to sulfate turtle gastrin.

Peripubertal ontogeny and estrogen stimulation of cholecystokinin and preproenkephalin mRNA in the rat hypothalamus and limbic system.

The neuropeptide cholecystokinin (CCK) is expressed in limbic system and hypothalamic nuclei that form a circuit that regulates the display of the female rodent reproductive behavior, lordosis. CCK mRNA and peptide levels fluctuate across the estrous cycle and have been shown to be modulated by estrogen exposure. The objective of these experiments was to examine the expression of CCK mRNA during postnatal development of this limbic-hypothalamic, lordosis regulating circuit, and to determine the age at which CCK mRNA expression becomes responsive to estrogen stimulation, by using quantitative in situ hybridization histochemistry. CCK mRNA levels were below the level of detectability within the circuit during the postnatal period, but increased during the peripubertal period. Rats were injected with either estradiol benzoate (EB), EB and progesterone, progesterone, or oil, and were killed 48 hours later on postnatal day (PND) 15, 20, and 25. Alternate brain sections were processed for CCK and preproenkephalin (PPE) mRNA in situ hybridization histochemistry. EB treatment induced CCK mRNA expression in the central portion of the medial preoptic nucleus and posterodorsal medial amygdala at PND 20 and 25, respectively. However, EB treatment increased PPE mRNA levels within the nuclei of the circuit at all ages examined. Progesterone had neither an independent nor additive effect on the EB induction of these neuropeptide messages. The estrogenic induction of CCK mRNA appears to be dependent on estrogen sensitive pathways of neurotransmission, or components of second messenger pathways which regulate CCK mRNA expression in the adult limbic-hypothalamic lordosis regulating circuit, which are not functional before PND 18-25.

[Changes in the cholecystokinin-synthetizing hypothalamic system during experimental diabetes mellitus in rats]. / Izmeneniia kholetsistokinin-sinteziruiushchei sistemy gipotalamusa pri éksperimental'nom sakharnom diabete u krys.

The investigation was performed in 96 Wistar rats. Diabetes mellitus was induced by single injection of 50 mg/kg of streptozotocin. Cholecystokinin (CCK) synthesizing neurons were identified in hypothalamic structures using indirect immunofluorescence. In latent period of diabetes (2 wks) number of CCK--immunopositive neurons increases, especially in paraventricular and suprachiasmatic nuclei, while in ventrolateral subnucleus of arcuate nucleus and parvicellular subnucleus of paraventricular nucleus areas occupied by immunoreactive material in neurons and their CCK content are reduced. By the end of wk 5 of the disease increase in number of CCK immunopositive neurons was registered only in medial parvicellular subnucleus of paraventricular nucleus whereas in other structures their number was reduced. The administration of CCK to intact animals causes increase of insulin content in endocrinocytes of pancreatic islets, but does not affect the level of hypoglycemia. The administration of the peptide to animals with diabetes leads to destruction of pancreatic islets, decline in endocrinocyte number and insulin content and marked hypoglycemia. Thus, the data obtained indicate the significant role of hypothalamic peptidergic system and CCK in regulation of beta-endocrinocyte function.

Opiate receptors modulate estrogen-induced cholecystokinin and tachykinin but not enkephalin messenger RNA levels in the limbic system and hypothalamus.

Cholecystokinin, substance P and methionine enkephalin all regulate the display of reproductive behaviour. Their expression is exquisitely regulated by estrogen in the limbic-hypothalamic circuit, a circuit that regulates the display of estrogen-sensitive female reproductive behavior. Relatively little is known, however, about the interaction of endogenous opioid peptides with cholecystokinin and substance P in the limbic-hypothalamic circuit. Opiates antagonize the release of cholecystokinin and substance P in the hypothalamus and periaqueductal gray and stimulate cholecystokinin messenger RNA levels in the amygdala. To determine the effect of endogenous opioid input on estrogen-induced cholecystokinin, enkephalin and substance P expression, in situ hybridization histochemistry was used to examine estrogen-induced messenger RNA levels of these neuropeptides in specific nuclei of the limbic system and hypothalamus in the presence of opiate receptor antagonists. Estrogen treatment of ovariectomized rats significantly elevated cholecystokinin messenger RNA levels in the central portion of the medial preoptic nucleus, the encapsulated portion of the bed nucleus of the stria terminalis and the posterodorsal medial amygdala, as well as increased preproenkephalin and preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus and the posterodorsal medial amygdala. The universal opiate receptor antagonist naltrexone and the delta-opiate receptor antagonist naltrindole each potentiated the estrogen-induced increase and elevated cholecystokinin messenger RNA levels an additional 1.9- to 2.8-fold depending on the nucleus examined, but had no effect on the estrogen-induced expression of either preproenkephalin or preprotachykinin messenger RNA. beta-Funaltrexamine, a mu-opiate receptor antagonist, had no effect on the medial preoptic or medial amygdaloid cholecystokinin messenger RNA levels or on the estrogen-induced expression of preproenkephalin messenger RNA but did cause a decrease in estrogen-induced cholecystokinin messenger RNA levels in the bed nucleus of the stria terminalis and a decrease in the preprotachykinin messenger RNA levels in the ventromedial hypothalamic nucleus. These results indicate that endogenous opioids, acting on the delta-opiate receptor within nuclei of the limbic-hypothalamic circuit, restrain the estrogen-induced increase of cholecystokinin messenger RNA expression. Activation of the mu-opiate receptor, however, may facilitate cholecystokinin messenger RNA expression in the bed nucleus of the stria terminalis and preprotachykinin messenger RNA expression in the ventromedial hypothalamic nucleus. Thus, endogenous opioid peptides may act in a site- and receptor-specific manner to modulate estrogen-induced neuropeptide levels in the limbic system and hypothalamus.

Cholecystokinin antisense RNA increases the analgesic effect induced by electroacupuncture or low dose morphine: conversion of low responder rats into high responders.

The analgesic effects of the rat in response to electroacupuncture (EA) or low-dose morphine (3 mg/kg) show marked individual variations. In the midbrain periaqueductal gray (PAG) of the rat, the content of the neuropeptide cholecystokinin octapeptide (CCK-8) was found to be significantly higher in the low responder (LR) rats as compared to that in the high responders (HR). Since PAG has been shown to be a strategic site for CCK-8 to exert an anti-opioid action, a high CCK content in PAG may account for the low analgesic responsiveness to EA and morphine. In order to block the expression of the gene encoding preproCCK in the brain, antisense CCK expression vector pSV2-CCKAS was microinjected into the lateral cerebral ventricle of the rat, leading to a decrease of the CCK-mRNA as well as the CCK-8 content in rat brain. This effect started 4 days after the intracerebroventricular (i.c.v.) injection of the antisense expression vector, and lasted no more than 1 week. This procedure was shown to be very effective in converting LR rats into HR for EA analgesia and morphine analgesia, and also delayed the development of tolerance elicited by prolonged EA stimulation or repeated morphine administration. The time course of the augmentation of opioid analgesia (4-6 days after the i.c.v. injection of the expression vector) paralleled the decrease of the brain CCK-8 content. The results argue that blocking the CCK gene expression in the brain may tilt the balance between opioid and anti-opioid peptides in favor of the former, thus strengthening the EA analgesia and morphine analgesia, and delaying the development of opioid tolerance.

Estrogen modulation of opioid and cholecystokinin systems in the limbic-hypothalamic circuit.

The display of lordosis behavior has been correlated with the estrogen-induced expression of cholecystokinin (CCK) and enkephalin within the limbic-hypothalamic circuit. These neuropeptides have opposing effects on lordosis; for example, in the medial preoptic nucleus, CCK facilitates and opiates inhibit lordosis. Antisense oligodeoxynucleotide blockade of receptor expression indicated that CCK modulates lordosis in the medial preoptic nucleus through the CCK(A)-receptor. Sequence-specific antibodies directed against delta- and mu-opiate receptor proteins labeled fibers in the medial preoptic nucleus. Estrogen treatment of ovariectomized rats or etorphine (a nonselective opiate agonist) treatment altered the appearance of the immunoreactivity from a diffuse pattern to one of distinctly stained mu-opiate receptor immunoreactive cells and varicose fibers in the medial preoptic nucleus. Such a pattern of staining reflects an internalization of mu-opiate receptors following agonist stimulation. This type of internalization has been used as an indication of synaptic activity. The distribution of receptor internalization surrounds the distribution of CCK cells in the medial preoptic nucleus, suggesting that endogenous opioid peptides may modulate estrogen-induced CCK mRNA expression. Interestingly, nonselective and delta-opiate receptor selective antagonists potentiated the estrogen-induced CCK mRNA expression in the medial preoptic nucleus. Together, these results suggest that endogenous opioid peptides may modulate the estrogenic upregulation of CCK mRNA expression and demonstrate an important level of regulation of gene expression in which synaptic activity modifies hormonal input.

Expression of cholecystokinin mRNA in corticothalamic projecting neurons: a combined fluorescence in situ hybridization and retrograde tracing study in the ventrolateral thalamus of the rat.

Cholecystokinin (CCK), a well-known neuroactive peptide, has been observed in the axon endings within the thalamic reticular nucleus and the adjacent ventrolateral nucleus of the thalamus. The origin of this CCK innervation remains undefined. In this study, a fluorescence in situ hybridization (FISH) technique was used in conjunction with latex microsphere retrograde tracing to investigate whether cortical neurons may provide a source of CCK afferents to the ventrolateral thalamic nucleus. Rhodamine latex beads were injected into the ventrolateral thalamic nucleus of adult male rats to retrogradely label corticothalamic cells. After 7 days, tissues were processed for FISH using a 24-base oligonucleotide probe complementary to the 3' coding region of rat preprocholecystokinin mRNA. It was found that CCK transcripts are expressed in about 80% of identified corticothalamic projecting neurons. We therefore conclude that the descending cortical projections to the ventrolateral thalamus may provide an important source of CCK innervation to this region of the brain.

Cholecystokinin effect on human lymphocyte ionized calcium and mitogenesis.

Cholecystokinin (CCK) is a peptide present in large amounts in gut, brain, and neurons innervating lymphatic tissues. Plasma CCK levels increase in enterally alimented patients. Enteral alimentation is also associated with enhanced immune function. The effects of CCK and a CCK antagonist were studied on human peripheral blood mononuclear cells (H-PBMC), lymphocyte intracellular ionized calcium ([Ca2+]i), and lymphocyte mitogenesis. CCK receptors transduce their signal via the release of [Ca2+]i. CCK octapeptide caused a specific increase in [Ca2+]i measured by Fura-2 fluorometry in H-PBMC and human T helper lymphocytes. Neither gastrin-17 nor pentagastrin produced a signal. While the highly specific CCK antagonist MK329 blocked the CCK [Ca2+]i signal, it had no effect on the PHA-mediated signal. At high dosages (10(-7)-10(-8) M), CCK was a comitogen with "complete" lymphocyte mitogens such as anti-CD3 monoclonal antibody (mAb) or low-dose PHA, but not for "partial" mitogens such as phorbol esters. CCK comitogenic effect occurred even in the presence of cyclosporine. CCK radioimmunoassay demonstrated that H-PBMC contained CCK and that anti-CD3 mAb- or PHA-mediated H-PBMC mitogenesis caused release of CCK. MK329 blocked PHA and anti-CD3 mAb mitogenesis and CCK comitogenic effects. We conclude that CCK octapeptide may be a coregulator of lymphocyte Ca2+ activation signals. The immunologically beneficial effect of enteral nutrition may, in part, be mediated by increased levels of CCK.

Gonadal steroid control of preprocholecystokinin mRNA expression in the limbic-hypothalamic circuit: comparison of adult with neonatal steroid treatments.

The neuropeptide cholecystokinin (CCK) is involved in the regulation of female, but not male, reproductive behavior. In both sexes, estrogen regulates the expression of CCK in adulthood within the bed nucleus of the stria terminalis and medial amygdaloid nucleus. These areas are parts of an interconnected limbic system-hypothalamic circuit, the development of which is influenced by estrogen during the early postnatal period. This is the same period during which central nervous system (CNS) expression of CCK is dramatically increased, suggesting that the male and female patterns of CCK expression may be the result of early postnatal exposure to estrogen. In the present experiment, the expression of preprocholecystokinin (pCCK) mRNA was determined by in situ hybridization with an isotopically labeled pCCK complementary RNA and emulsion autoradiography in animals whose neonatal and adult gonadal steroid levels had been manipulated. The number of pCCK-expressing cells in animals that were gonadectomized as adults was determined by neonatal estrogen, but stimulation with steroids in adulthood induced a similar number of pCCK-expressing cells in both sexes in the medial amygdala and bed nucleus of the stria terminalis. Neonatal treatment of females with estrogen or testosterone, followed by ovariectomy in adulthood, eliminated the sex difference in pCCK mRNA expression. Males treated neonatally with the aromatase inhibitor androstenedione (to block metabolism of testosterone to estrogen) and orchidectomized in adulthood had a level of pCCK mRNA expression that was similar to that of ovariectomized females. These data suggest that, during neonatal development, estrogen determines the constitutive expression of pCCK mRNA in the medial amygdala and bed nucleus of the stria terminalis, resulting in higher levels of pCCK mRNA expression in males than in females. However, exogenous gonadal steroids induce the same levels of pCCK mRNA expression in adult females, indicating that the levels of gonadal steroids and the patterns of their secretion are the predominant influences on the sexually dimorphic adult levels of pCCK mRNA expression.