A new simple mouse model for the in vivo evaluation of cholecystokinin (CCK) antagonists: comparative potencies and durations of action of nonpeptide antagonists

A new simple mouse assay for the in vivo evaluation of CCK antagonists which is based upon visual determination of the gastric emptying of a charcoal meal is described. CCK-8 (24 micrograms/kg s.c.) but not various other peptide and nonpeptide agents effectively inhibited gastric emptying in this test system. The effect of CCK-8 was antagonized by established peripheral CCK antagonists but not representative agents of various other pharmacological classes. The rank order of potency of the CCK antagonists were: L-364,718 (ED50 = 0.01 mg/kg, i.v.; 0.04 mg/kg, p.o.) greater than Compound 16 (ED50 = 1.5 mg/kg, i.v.; 2.0 mg/kg p.o.) greater than asperlicin (ED50 = 14.8 mg/kg i.v.) greater than proglumide (ED50 = 184 mg/kg i.v.; 890 mg/kg, p.o.). Duration of action studies based upon ED50 values determined at various time intervals after oral administration showed that L-364,718 and proglumide are considerably longer acting than Compound 16. Asperlicin (ED50 greater than 300 mg/kg, p.o.) was ineffective as a CCK antagonist when administered orally. These data provide the first direct comparisons of the in vivo potencies of current CCK antagonists and demonstrate the utility of a new simple mouse assay for the in vivo characterization of peripheral CCK antagonists.     

Antisense oligodeoxynucleotide to δ opioid receptors attenuates morphine dependence in mice

The effect of intracerebroventricular (i.c.v.) treatment with antisense oligodeoxynucleotide (A-oligo) to δ opioid receptor mRNA on the morphine-induced place preference and naloxone-precipitated jumping was examined in morphine-dependent mice. Morphine (5 mg/kg, s.c.) produced a significant place preference. I.c.v. pretreatment with A-oligo (0.01–1 μg/mouse) dose-dependently attenuated this morphine (5 mg/kg, s.c.)-induced place preference, while mismatched oligodeoxynucleotide (M-oligo; 1 μg/mouse, i.c.v.) was ineffective. Naloxone (3 mg/kg, s.c.) precipitated jumping in morphine-dependent mice. I.c.v. pretreatment with A-oligo (1 μg/mouse) attenuated this naloxone (3 mg/kg, s.c.)-precipitated jumping in morphine-dependent mice, while M-oligo (1 μg/mouse, i.c.v.) was ineffective. These data demonstrate that the selective reduction in supraspinal δ opioid receptor function caused by pretreatment with A-oligo attenuated the morphine-induced place preference and naloxone-precipitated jumping in morphine-dependent mice, suggesting that the rewarding effect of and physical dependence on morphine may be modulated by central δ opioid receptors.     

Increased circulating cholecystokinin contributes to anorexia and anxiety behavior in mice overexpressing pancreatic polypeptide

We have previously reported that pancreatic polypeptide (PP) overexpressing mice display thin phenotype with delayed gastric emptying and decreased food intake. In the present study, we further examined if CCK contributes to anorexia and anxiety behavior in PP overexpressing mice. Plasma CCK levels in PP overexpressing mice and their littermates were determined by radioimmunoassay using antisera specific to sulfated CCK-8 and CCK-33. To elucidate the role of CCK in PP overexpressing mice, CCK-1 receptor antagonist (L-364,718) or saline was administered intraperitoneally and food intake was measured for 2 h. CCK-2 antagonist (L-365,260) or saline was injected intraperitoneally and the elevated plus-maze test was performed to assess anxiety. Plasma CCK levels were significantly increased in PP overexpressing mice. Administration of L-364,718 increased food intake in PP overexpressing mice compared to the saline-injected PP overexpressing group, while L-364,718 did not increase food intake in non-transgenic littermates. PP overexpressing mice exhibited anxiety in the plus-maze test. Administration of CCK-2 receptor antagonist (L-365,260) reversed the decreased percentage of entry into the open arms in PP overexpressing mice. These results indicated that elevated CCK may contribute to anorexic and anxious phenotype of PP overexpressing mice.     

CCK antagonists interact with CCK-B receptors on human small cell lung cancer cells

The ability of cholecystokinin (CCK) receptor antagonists to interact with CCK receptors in small cell lung cancer (SCLC) cells was investigated. L-365,260, CCK-8, L-364,718, CBZ-CCK(27-32)-NH2 and proglumide analogue 10 inhibited specific 125I-CCK-8 binding to SCLC cells with IC50 values of 0.2, 2, 500, 100,000 and 500,000 nM, respectively. Gastrin-I and CCK-8 elevated the cytosolic Ca2+ when SCLC cells were loaded with Fura 2-AM. L-365,260 inhibited the cytosolic Ca2+ increase caused by 10 nM CCK-8 in a dose-dependent manner. The effects of 10 nM L-365,260 were reversed by high concentrations of CCK-8. These data indicate that L-365,260 functions as a reversible CCK-8 antagonist using SCLC cells.     

Evidence for a nucleus accumbens CCK2 receptor regulation of rat ventral pallidal GABA levels: a dual probe microdialysis study

We employed dual probe microdialysis in the nucleus accumbens and ipsilateral ventral pallidum of the halothane anaesthetized rat to investigate the effect of intra-accumbens perfusion with the sulphated octapeptide cholecystokinin (CCK-8S, 10-1000 nM, 60 min) alone and in the presence of the selective CCK1 and CCK2 receptor antagonists L-364,718 (10 and 100 nM) and PD134308 (10 nM), tetrodotoxin (TTX, 1000 nM) and the GABA(A) receptor antagonist bicuculline (1000 nM), on dialysate GABA levels in the ventral pallidum. Intra-accumbens perfusion with the 100 and 1000 nM concentration of CCK-8S was associated with a significant decrease (-16+/-3% and -23+/-3% vs basal, respectively) in ventral pallidum GABA levels. The CCK-8S (1000 nM) induced decrease in ventral pallidal dialysate GABA levels was abolished when PD134308, TTX and bicuculline, but not L-364,718, were included into the perfusion medium of the accumbens probe. The data indicate that nucleus accumbens CCK-8S exerts a CCK2 receptor mediated inhibition of ventral pallidal GABA levels. Furthermore, the TTX and bicuculline sensitivity of this effect suggests that this is possibly mediated via CCK2 receptors probably located on local GABA interneurons.     

Effects of Cholecystokinin Peptides and GV 150013, a Selective CholecystokininB Receptor Antagonist, on Electrically Evoked Endogenous GABA Release from Rat Cortical Slices

The effects of cholecystokinin (CCK) agonists and antagonists on spontaneous and electrically evoked endogenous GABA release from rat cerebral cortex slices were evaluated. Neither the nonselective and CCK(B)-selective receptor agonists CCK-8S (3-1,000 nM) and CCK-4 (3-1,000 nM), respectively, nor the selective CCK(B) and CCK(A) receptor antagonists GV 150013 (3-30 nM) and L-364,718 (10-100 nM), respectively, significantly affected spontaneous GABA release. CCK-8S (1-1,000 nM) and CCK-4 (1-1,000 nM) increased the electrically (5 and 10 Hz)-evoked GABA release. On the contrary, GV 150013 (10 and 30 nM) significantly decreased the electrically evoked GABA release only when the slices were stimulated at the higher 10 Hz frequency. The CCK-8S- and CCK-4-induced increases in electrically evoked GABA release were counteracted by GV 150013, but not by L-364,718. Furthermore, GV 150013 at 3 nM shifted to the right the CCK-4 concentration-response curve, whereas at the higher 10 nM concentration it dramatically flattened the curve. Finally, in cortical slices obtained from rats chronically treated with GV 150013, the concentration-response curve of CCK-4 was shifted to the left and the peak effect of the peptide was significantly higher than that observed in naive animals. These results suggest that CCK increases electrically evoked, but not spontaneous, endogenous GABA release from rat cortical slices, possibly by activating local CCK(B) receptors. In addition, chronic treatment with the novel CCK(B) receptor antagonist GV 150013 leads to an enhanced responsiveness of cortical slices to CCK-4 application.     

Pharmacological characterization of cholecystokinin receptors mediating contraction of human gallbladder and ascending colon

Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK(1) and CCK(2) receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration-effect (E/[A]) curves obtained by cumulative dosing with sulphated CCK. The CCK(1) antagonist L-364,718 produced a rightward shift of the CCK-8S [E/[A] curve in the human gallbladder (pA(2)=9.15 +/- 0.26) and ascending colon (pA(2)=9.20 +/- .33). In both tissues, the CCK(2) receptor antagonist, JB93182, had no effect on the CCK E/[A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK(1) receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.     

家兔中脑导水管周围灰质中注射八肽胆囊收缩素（CCK—8）拮抗吗啡镇痛和...

We have reported that intracerebroventricular (i. c. v.) injection of 1-4 ng of CCK-8 to the rat produced a remarkable antagonistic effect on morphine analgesia. In order to study the species specificity and the site of action, CCK-8 was microinjected into the PAG of the rabbit, and its influence on morphine analgesia and electroacupuncture analgesia was observed. The latency of the escape response (ERL) to radiant heat focused on the snout was measured as an index of the pain threshold. Microinjections were made via cannulae chronically implanted into the PAG. The drug solutions were delivered in a volume of 1 microliter, at a speed of 0.125 microliter/min. The ERL was measured for a period of 60 or 70 minutes at 10 min intervals. 1. CCK-8 administered unilaterally to the PAG of the rabbit at a dose of 3 ng antagonized the analgesia induced by morphine (4 mg/kg, i. v.) by 73% (P less than 0.001), and reduced the analgesic effect of electroacupuncture by 67% (P less than 0.001). These effects were dose-dependent within the range from 1.5 ng to 6.0 ng. The effect of CCK-8 was reversed by CCK receptor blocker proglumide (4 microliters, intra-PAG injection). Unsulfated CCK-8 (CCK-us) had no effect in this regard. These results indicate that in the PAG of the rabbit, exogenously administered CCK-8 was capable of antagonizing opioid analgesia by the activation of CCK receptors. 2. Two groups of rabbits were given with morphine (2 mg/kg, i. v.) and simultaneous injection of CCK-8 antiserum (CCK-AS, 1 microliter) or normal rabbit serum (NRS) into the PAG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of cell proliferation by the cholecystokinin antagonist L-364,718

Specific cholecystokinin (CCK) receptor and gastrin receptor antagonists were used to assess what role, if any, these receptors have in autocrine cell growth. Although the cholecystokinin receptor antagonist, L-364,718, inhibited cell proliferation in a broad spectrum of cell lines, the gastrin antagonist, L-365,260, had no effect on cell proliferation. In addition neither added gastrin17, nor sulfated cholecystokinin8, could reverse the inhibitory action of L-364,718. It is proposed that L-364,718 inhibits cell proliferation independently of classical gastrin/CCK receptors.     

Functional CCK-A and Y2 receptors in guinea pig esophagus

Effects of cholecystokinin octapeptide (CCK-8), peptide YY (PPY), neuropeptide Y (NPY) and their analogs on muscle contractions of esophageal strips were investigated. CCK-8 induced a tetrodotoxin and atropine-sensitive contraction. The relative potencies for CCK related peptides to induce contractions were CCK-8 > desulfated CCK-8 > gastrin-17-I. The CCK-A receptor antagonist L-364,718 was 300-fold more potent than the CCK-B receptor antagonist L-365,260 at inhibiting CCK-8-induced contraction. These indicate that neural CCK-A receptors mediate this contraction. PYY or NPY did not cause muscle contraction or inhibit muscle contraction induced by carbachol, endothelin-1 or KCl. However, both PYY and NPY concentration-dependently inhibited contraction induced by CCK-8. This inhibition was not affected by nitric oxide (NO) synthase inhibitors L-NMMA or L-NAME. The relative potencies of PYY related peptides to inhibit CCK-8 induced contraction were PYY > NPY > NPY13-36 > [Leu(31), Pro(34)]NPY > pancreatic polypeptide (PP). We conclude that CCK interacts with neural CCK-A receptors to cause esophageal muscle contraction. PYY and NPY interact with Y2 receptors to inhibit this CCK-induced muscle contraction by an effect not related to NO.     

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.     

Stimulation of either cholecystokinin receptor subtype reduces while antagonists potentiate or sensitize a morphine-induced excitatory response.

Cholecystokinin peptides (CCK) have been shown to antagonize many opioid-mediated effects. The present study was undertaken to determine whether peripheral injections of cholecystokinin sulphated octapeptide (CCK8), cholecystokinin tetrapeptide (CCK4), the CCK(1) (lorglumide) and the CCK(2) (PD-135,158 and LY-225910) receptor antagonists can influence a classic morphine excitatory effect, i.e. the display of Straub tail reaction in mice (STR). A total of 570 female Balb/C mice were tested. Experiment 1 was undertaken to determine whether i.p. injections of CCK8 or CCK4 can influence STR. Each animal was treated with i.p. injections of saline or CCK8 (10 and 20 nmol/kg) or CCK4 (20 and 40 nmol/kg). After 30 min all animals received an i.p. injection of morphine hydrochloride (10.0 mg/kg). The highest doses of both CCK8 (35% STR) and CCK4 (40% STR) significantly reduced STR as compared to saline (85% STR) treated mice (Fisher test; P < 0.01). In experiment 2 each animal was treated with ip injections of saline or 1.0 mg/kg lorglumide or PD-135,158 fifteen minutes before an injection of morphine at doses ranging from 1.0 to 50.0 mg/kg. In experiment 3 animals were treated with injections of saline, 0.1 or 10.0 mg/kg lorglumide or LY-225910 before an injection of a fixed MC dose (2.0 mg/kg). Both lorglumide and PD-135,158 induced a significant shift to the left in the morphine dose-response curves as well as a significant decrease in ED50 of the STR. ED50 for lorglumide was significantly lower than ED50 for PD-135,158. Both doses of lorglumide and the highest dose of LY-225910 significantly increased the percent of animals displaying STR. Experiment 4 was undertaken to determine whether repeated peripheral injections of morphine or the morphine-potentiating agents CCK(1) (lorglumide) and the CCK(2) (LY-225910) receptor antagonists can induce morphine sensitization. Each animal was treated with 5 daily i.p. injections of saline (control group), 1.5 mg/Kg morphine hydrochloride (group morphine), and 1.0 mg/Kg lorglumide (group LOR) or LY-225910 (group LY). One, two, three and four weeks after the last treatment day, all animals were challenged with one i.p. injection of morphine (1.5 mg/Kg). The morphine, LOR groups and group LY showed a significant increase in percentage of animals displaying STR. These data demonstrate that the blockade of endogenous CCK actions leads to morphine sensitization probably through both CCK receptors. The present data are consistent with the antagonistic effects of CCK and opioids in the control of morphine-induced STR. In addition, these results suggest that both CCK receptors are involved in the modulatory effects of CCK on this morphine effect.     

Characterization of interactions between CCK-33 and CCK receptors in isolated dispersed pancreatic acini.

In isolated dispersed pancreatic acini, we have characterized the interactions between cholecystokinin (CCK) and CCK receptors by simultaneously measuring CCK-33 immunoreactivity and CCK bioactivity. Incubation of acinar cells with CCK-33 at cell density of 0.2-0.3 mg acinar protein per ml resulted in stimulation of amylase release concomitant with significant and time-dependent decrease of the immunoreactive CCK. With L-364,718 (0.1 microM), a specific CCK receptor antagonist, immunoreactive CCK levels in the media were not significantly altered during incubation; however, CCK-stimulated amylase release was almost completely abolished (94% inhibition). Vasoactive intestinal peptide (1 nM) significantly potentiated CCK stimulated amylase release without affecting immunoreactive CCK in the media. Insulin (167 nM) did not affect the CCK stimulated amylase release or immunoreactive CCK in the media. Incubation of acinar cells with CCK-33 at 4 degrees C did not affect the levels of immunoreactive CCK; however, a significant change in levels of immunoreactive CCK were found at 37 degrees C at 90 min. Incubation of cell free medium with CCK-33 in the presence or absence of secreted enzymes revealed no changes in CCK immunoreactivity in the medium at 90 min. Addition of bacitracin in the incubation media did not affect the CCK immunoreactivity or bioactivity. These findings indicate that in isolated rat pancreatic acini, CCK-33 stimulates amylase release through a receptor that is specifically blocked by L-364,718. Specificity of the interactions of CCK-33 with acinar cells in the media appears to be receptor-mediated and time- and temperature-dependent.     

Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibers

Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake, and afford similar gastroprotective activity. The present study was designed to investigate the putative protective effects of CCK and leptin on acute colonic inflammation. Leptin or CCK-8s was injected to rats intraperitoneally immediately before and 6 h after the induction of colitis with acetic acid. CCK-A receptor antagonist (L-364,718) or CCK-B receptor antagonist (L-365,260) was injected intraperitoneally 15 min before leptin or CCK treatments. In a group of rats, vagal afferent fibers were denervated by topical application of capsaicin on the cervical vagi. Rats were decapitated at 24 h, and the distal 8 cm of the colon were removed for macroscopic scoring, determination of tissue wet weight index (WWI), histologic assessment and tissue myeloperoxidase (MPO) activity. All inflammation parameters were increased by acetic acid-induced colitis compared to control group. Leptin or CCK-8s treatment reduced these parameters in a similar manner, while co-administration of leptin and CCK was found to be more effective in reducing the macroscopic score and WWI. CCK-8s-induced reduction in the score and WWI was prevented by CCK-A, but not by CCK-B receptor antagonist, whereas neither antagonist altered the inhibitory effect of leptin on colitis-induced injury. On the other hand, perivagal capsaicin prevented the protective effects of both CCK-8s and leptin on colitis. Our results indicate that leptin and CCK have anti-inflammatory effects on acetic acid-induced colitis in rats, which appear to be mediated by capsaicin-sensitive vagal afferent fibers involving the reduction in colonic neutrophil infiltration.     

Effects of amylin on feeding of goldfish: interactions with CCK

In mammals, amylin (AMY) is a peptide that is secreted from the pancreas in response to a meal. AMY inhibits food intake and may also contribute to the anorectic effects of the brain-gut peptide cholecystokinin (CCK). In this study, we assessed the role of AMY in the regulation of food intake in goldfish (Carassius auratus) and its interactions with CCK. Fish were injected intraperitoneally (i.p.) with mammalian AMY and intracerebroventricularly (i.c.v.) with mammalian AMY, alone or in combination with the sulfated octapeptide CCK-8S. We also assessed the effects of i.c.v. injections of AC187, an amylin receptor antagonist on the central actions of both AMY and CCK-8S, as well as the effects of i.c.v. injections of proglumide, a CCK receptor antagonist, on the central effects of AMY. AMY injected i.p. at 100 ng/g but not 25 or 50 ng/g or i.c.v. at 10 ng/g but not 1 ng/g significantly decreased food intake as compared to saline-treated fish. Fish co-treated i.c.v. with AMY at 1 ng/g and CCK-8S at 1 ng/g had a food intake lower than that of control fish and fish treated with either 1 ng/g CCK-8S or 1 ng/g AMY, suggesting a synergy between the two systems. Whereas low i.c.v. doses of AC187 (30 ng/g) had no effect, moderate doses (50 ng/g) induced an increase in food intake, indicating a role of endogenous AMY in satiety in goldfish. Blocking central amylin receptors with i.c.v. AC187 (30 ng/g) resulted in an inhibition of both i.c.v. AMY- and CCK-induced reduction in feeding. Blocking central CCK receptors with i.c.v. proglumide (25 ng/g) resulted in an inhibition of both i.c.v. CCK-induced and AMY-induced decrease in food intake. Our results show for the first time in fish that AMY is a potent anorexigenic factor and that its actions are interdependent with those of CCK.     

Intracerebroventricular injections of cholecystokinin octapeptide suppress feeding in rats--pharmacological characterization of this action

Cholecystokinin octapeptide (CCK-8), administered intracerebroventricularly (i.c.v.), will suppress feeding. The aim of the present study was to determine the pharmacological characteristics of this satiety inducing effect in rats. For this purpose, we employed a feeding bioassay model in 24 h fasted rats and examined the effects of CCK-8 and a variety of structurally related analogs on latency to feed after i.c.v. injection and on the amount of food and water consumed as measured after the initiation of feeding in sequential 20-min epochs for 1 h. CCK-8, given in doses of 0.1, 1 and 10 nmol, produced a dose-dependent increase in feeding latency and a reduction of food intake during the first 20 min after initiation of feeding. Food intake during the next 40 min and water consumption were not altered. Plasma levels of CCK-like immunoreactivity after an i.c.v. injection of a dose of CCK-8 which blocked feeding (10 nmol) rose insignificantly from 117 to 125 pg/ml. In contrast, at the minimally effective dose of CCK-8 after i.v. administration (10 nmol), which also produced an inhibition of feeding, the plasma level was 1430 pg/ml. This difference indicates that plasma levels of CCK after i.c.v. CCK-8 are not adequate to produce the observed feeding suppression and suggests that the effects of i.c.v. CCK-8 are not mediated by a peripheral redistribution. Systematic dose response studies revealed the following rank order of potencies: CCK-8 greater than or equal to G-17 II much greater than CCK-8 NS = G-17 I greater than or equal to CCK-4 = CCK 26-29 = 0. Only gastrin-17 II (sulfated) produced an effect comparably significant to CCK-8. I.c.v. proglumide at 2500 nmol failed to modify the effects of CCK-8 at 10 nmol after i.c.v. injection. These data demonstrate that the structural requirements for feeding suppressive activity in rat brain are the carboxyterminus with a sulfated tyrosine residue, located 6 to 7 residues from the carboxyterminus, as present in CCK-8 and gastrin-17 II.     

CCK-8 Inhibits Acute Morphine-induced Spatial Reference Memory Impairment in Mice

Administration of morphine may impair learning and memory processes. Cholecystokinin has been reported to be involved in various types of memory, and our previous study found that Cholecystokinin octapeptide attenuates spatial memory impairment in chronic morphine-treated mice. However, the effect of CCK-8 on acute morphine-induced memory impairment is not clear. In this study, effect of acute CCK-8 and morphine on spatial reference memory was evaluated using Morris water maze in KM mice. Acetylcholine (Ach) content was measured using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS). Pre-training with morphine (5, 10 mg/kg, i.p.) significantly impaired spatial reference memory acquisition without disturbing the performance in the visible platform task, while pre-test morphine has no effect on memory retrieval. Pre-training (0.01, 0.1 and 1 μg, i.c.v.) or pre-test (0.1 and 1 μg, i.c.v.) of CCK-8 facilitated spatial reference memory acquisition and retrieval, respectively. CCK-8 (0.1 and 1 μg) significantly attenuated memory loss by pre-training morphine. Furthermore, CCK-8 (1 μg, i.c.v) increased acetylcholine contents of hippocampus in saline or morphine-treated mice. Our study identifies CCK-8 reversed spatial reference memory loss induced by acute morphine, and the mnemonic effect could be related to the facilitation of CCK-8 on memory acquisition and retrieval through accelerating acetylcholine release in hippocampus.     

八肽胆囊收缩素对家兔内毒素休克时肺动脉张力的影响

为探讨八肽胆囊收缩素(CCK-8)缓解内毒素休克(ES)时肺动脉血压(PAP)增高的机制,观察了CCK-8对脂多糖(LPS)引起家兔ES时PAP变化以及离体肺动脉环(PARs)张力改变的影响。实验用新西兰大耳白雄性家兔40只,分为颈静脉注入LPS(8mg／kg i．v.)复制的家兔ES模型、LPS注入前15min给CCK-8(15μg/kg,i.v.)、LPS注入前15min给CCK受体拮抗剂丙谷胺(Pro 1mg／kg,i.v.)、单独注入CCK-8(15μg/kg,i.v.)和注射生理盐水(对照)共5组。用生理记录仪监测平均动脉压(MAP)和PAP的变化;5h后制备PARs,应用血管张力测定技术,检测各组PARs张力。结果为：(1)ES时MAP降低、PAP升高,CCK-8可完全翻转ES时PAP的增高,而Pro加剧ES时PAP的增高;(2)LPS组的PARs对苯肾上腺素(PE)的收缩反应增强,对ACh内皮依赖性舒张反应降低,而CCK-8可逆转LP5的上述作用。上述结果提示CCK—8可缓解ES时的PAP升高,这可能与其调节肺动脉张力改变有关。     

Extinction of conditioned opiate withdrawal in rats in a two-chambered place conditioning apparatus

Conditioned opiate withdrawal contributes to relapse in addicts and can be studied in rats by using the opiate withdrawal-induced conditioned place aversion (OW-CPA) paradigm. Attenuation of conditioned withdrawal through extinction may be beneficial in the treatment of addiction. Here we describe a protocol for studying OW-CPA extinction using a two-chambered place conditioning apparatus. Rats are made dependent on morphine through subcutaneous implantation of morphine pellets and then are trained to acquire OW-CPA through pairings of one chamber with naloxone-precipitated withdrawal and the other chamber with saline. Extinction training consists of re-exposures to both chambers in the absence of precipitated withdrawal. Rats tested after the completion of training show a decline in avoidance of the formerly naloxone-paired chamber with increasing numbers of extinction training sessions. The protocol takes a minimum of 7 d; the exact duration varies with the amount of extinction training, which is determined by the goals of the experiment.     

慢性吗啡依赖大鼠条件性位置厌恶模型杏仁中央核蛋白激酶A表达变化

目的:分析慢性吗啡依赖大鼠纳洛酮催瘾戒断条件性位置厌恶(CPA)建立前后、消退后及重建后,与成瘾密切相关的脑区杏仁中央核(CeA)内蛋白激酶A(PKA)蛋白表达的适应性变化,探讨阿片依赖戒断后厌恶动机形成的生物学基础。方法:①将雄性SD大鼠分为实验组(慢性吗啡注射+纳洛酮催瘾组,MN),对照组(慢性吗啡注射+生理盐水催瘾组,MS),慢性生理盐水注射+纳洛酮催瘾组,SN),每组24只。采用慢性吗啡注射(10 mg/kg,BID,ip)后给予一次纳洛酮(0.3 mg/kg)催瘾注射(同时与条件性位置训练箱搭配)建立大鼠CPA模型。②在CPA建立前后、消退后及重建后,采用免疫组织化学方法检测CeA内PKA蛋白表达情况。结果:MN组在CPA建立前后、消退后及重建后CeA内PKA的蛋白表达出现适应性变化(P<0.05),建立后(Day7,134.43±4.481,P<0.05),消退后(Day13,141.01±3.360,P<0.01)及重建后(Day14,137.18±40.330,P<0.05)PKA蛋白表达水平均低于建立前(Day5,124.48±6.722)。而MS组(P>0.05)和SN组(P>0.05)在CPA的各个时间点PKA的蛋白表达变化差异均无显著性。结论:①CeA内PKA蛋白的低表达导致的厌恶的中枢状态,可能是CPA建立的关键的神经机制。②CeA内PKA的适应性变化可能是物质依赖戒断后CPA相关神经可塑性变化的重要分子基础。