Decreased activity of hepatic alkaline protease in rats with carbon tetrachloride-induced liver cirrhosis

To investigate the cause of accumulation of oxidised proteins in the livers of rats with carbon tetrachloride (CCl4) induced liver cirrhosis, the activity of alkaline protease (a high molecular weight, multisubunit cysteine proteinase) was determined in the cirrhotic livers. A significant decrease (P < 0.05) in the activity of hepatic alkaline protease was observed in the cirrhotic rats. Decreased activity of alkaline protease in the liver of cirrhotic rats may contribute to the accumulation of the oxidised proteins in the liver.     

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.     

Nitric Oxide Production in Striatum and Pallidum of Cirrhotic Rats

Ammonium and manganese are neurotoxic agents related to brain metabolic disturbances observed after prolonged liver damage. The aim of this study was to assess the production of nitric oxide (NO) in the brain of cirrhotic rats exposed to manganese. We induced cirrhosis by bile duct ligation for 4 weeks in rats. From brain, striatum and globus pallidus were dissected out, and NO synthase activity and the content of nitrites plus nitrates (NOx) were determined. In pallidum we found a diminished constitutive NO synthase activity from cirrhotic rats, independently of manganese exposure. This result was confirmed by low levels of NOx in the same brain area (P<0.05, two-way ANOVA). This finding was not related to protein expression of NO synthase since no differences were observed in immunoblot signals between cirrhotic and sham-operated animals. Results from present study suggest that the production of NO is reduced in basal ganglia during cirrhosis.     

Plasma cholecystokinin-octapeptide like immunoreactivity in patients with hepatic cirrhosis

Molecular forms of cholecystokinin (CCK) in the peripheral circulation were studied in normal subjects and cirrhotic patients. Fractionation of plasma extract collected 20 min after intraduodenal infusion of fat revealed four major peaks by Sephadex G-50 column chromatography in normal subjects. Peak I eluted at a position similar to CCK-33, peaks II and III eluted between CCK-33 and CCK-14, and peak IV eluted between CCK-14 and CCK-8. In cirrhotic patients, there was a prominent peak (peak V) eluted at a position similar to CCK-8, in addition to those four peaks. These findings are consistent with the previous observations of hepatic elimination of CCK-8, and suggest that smaller forms of CCK similar in size to CCK-8 are not major forms of CCK in plasma in normal subjects but circulate substantially in cirrhotic patients.     

Role of cholecystokinin-A and cholecystokinin-B receptors in anxiety

Evidence from several laboratories indicates that the anxiogenic effects of cholecystokinin (CCK) are mediated by CCKB receptors. However, it has been reported that CCKA receptors have been found in brain and CCKA antagonists have anxiolytic properties. The aim of this work was to study whether CCKA receptors are also involved in the modulation of anxiety. Anxiogenic effects were observed in the elevated plus maze in rats when pure CCKB receptor agonists (CCK-4 and CCK-8 non-sulfated) or CCK-8S, a CCKB/CCKA agonist, were injected into the lateral ventricle. In contrast, CCK-33, a CCKA agonist or CCK-(1-21) and CCK-(26-29) were ineffective. Furthermore, the anxiogenic effects of CCK-8S were prevented by blocking CCKB but not CCKA receptors. Finally, CCK-33 injected into the postero-medial nucleus accumbens failed to affect the anxiety level of the rats. These results indicate that CCKA receptors are not involved in anxiety, as measured by the paradigms used in this work.     

Unimpaired induction of drug-metabolizing enzymes in hepatocytes isolated from rats with micronodular cirrhosis

To test further the competence of the cirrhotic liver to metabolize xenobiotics, hepatocytes were isolated from control and CCl4-induced cirrhotic male or female rats. Histologically micronodular cirrhosis was present in all CCl4-treated rats, while control rats had normal livers. Portal perfusion pressure and intrahepatic collagen content were also significantly increased by CCl4 administration. In male rats, no significant differences in levels of circulating transaminases nor in alkaline phosphatase was observed between cirrhotic and control rats, while CCl4-treated females had slightly higher than normal serum transaminase levels at the time of the studies. Hepatocytic cytochrome P-450 and basal xenobiotic biotransformation were unaffected by micronodular cirrhosis in both genders; calculation of the aminopyrine and 7-ethoxycoumarin intrinsic clearances (Cli) revealed, however, a slightly decreased transformation potential in hepatocytes obtained from cirrhotic females, a phenomenon not observed in cirrhotic male rats. It is speculated that the observed reduction in Cli may have been independent of cirrhosis per se, owing to the perduring cytotoxic effect of CCl4 as evidenced by the higher than normal level of transaminases in female rats. Finally, male rats were subjected to in vivo administration of phenobarbital or 3-methylcholanthrene; both compounds led to significant induction of the mixed-function oxidase system, which was similar in magnitude and in selectivity in control and cirrhotic rats as illustrated by calculation of the Michaelis-Menten kinetic parameters for aniline p-hydroxylation, aminopyrine-N-demethylation, 7-ethoxycoumarin-O-deethylation, and p-nitrophenol UDP-glucuronyl transferase. We conclude that in well-established but compensated and hepatolysis-free micronodular cirrhosis, hepatocytes are fully able to transform xenobiotics and to respond normally and selectively to inducers of drug metabolism.     

兔侧脑室注射八肽胆囊收缩素抗血清对血浆自由脂肪酸浓度的影响

本工作根据抗原抗体间具有高度特异性的中和作用的原理,将微量 CCK-8抗血清注射入制备有埋藏套管的慢性实验兔的侧脑室内,观察在中和脑内外源性或内源性的 CCK-8后,血浆 FFA 浓度的变化,结果如下:1.侧脑室内注射正常兔血清,对血浆 FFA 浓度无明显影响;在注射 CCK-8同时注射兔正常血清,也不影响 CCK-8降低血浆 FFA 浓度的作用。2.侧脑室内注射 CCK-8抗血清能有效地阻断外源性脑室注射 CCK-8降低血浆 FFA 的作用,此阻断作用随 CCK-8抗血清剂量的增加而增强。3.侧脑室内单独注射 CCK-8抗血清,血浆 FFA 浓度明显升高,这一作用可能是由于中和了脑内内源性 CCK-8的作用所致。以上结果表明,CCK-8脑室注射引起的血浆 FFA 降低的作用具有一定的特异性;而在正常生理情况下,脑内释放的内源性 CCK-8可能参与血浆 FFA 代谢的调节。     

Behavioral effect of cholecystokinin octapeptide in vagotomized rats

Cholecystokinin octapeptide (CCK-8) was administered intracerebroventricularly (icv) or subcutaneously (sc) into subdiaphragmatically vagotomized and sham-operated rats, and the behavioral effects were quantified by an open-field test. Intracerebroventricularly injection of CCK-8 decreased locomotion and rearing to the same extent in both vagotomized and sham-operated rats, while sc injection produced behavioral changes only in sham-operated rats but not in vagotomized ones. The results indicate that CCK-8 affects both central and peripheral receptors, and the vagal nerve may be the major pathway causing behavioral effects from the visceral organs to the brain.     

Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.     

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.     

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.     

Fronto-cortical regulation of beta-endorphin actions in the rat

Ablation of the frontal neocortex markedly enhanced the antinociceptive and cataleptic actions of beta-endorphin injected into the lateral ventricle of rat brain. This enhanced response was not affected by simultaneous administration of cholecystokinin octapeptide (CCK-8). In sham-operated rats, however, CCK-8 suppressed the effects of beta-endorphin in a dose-related manner. Moreover, ablation of a similar amount of occipital neocortex did neither affect beta-endorphin actions nor the interactions of CCK-8.     

Effects of forebrain microinjection of cholecystokinin on dopamine cell firing rate.

In anesthetized rats, midbrain dopamine (DA) neuronal firing rate was differentially sensitive to focal brain microinjection of cholecystokinin peptides (CCK-4 and CCK-8) and N-methyl-D-aspartate (NMDA) into nucleus accumbens, amygdala and prefrontal cortex. Whereas changes in DA neuronal firing rate were frequently observed in response to intra-amygdalar microinjection of CCK peptides, NMDA was most effective in eliciting changes in DA neuronal activity following intra-accumbal microinjection. Thus, stimulation of amygdalar CCK receptors and accumbal excitatory amino acid receptors may participate in the afferent regulation of midbrain DA neuronal function.     

Degradation of cholecystokinin-like peptides by a crude rat brain synaptosomal fraction: a study by high pressure liquid chromatography

Degradation of CCK-8, CCK-4, and related peptides by a crude synaptosomal fraction of rat brain was investigated by monitoring the tryptophan fluorescence of reaction products after HPLC fractionation. At 20 degrees C, the half disappearance time was 52 min for CCK-8, 35 min for unsulphated CCK-8, 20 min for unsulphated CCK-7, 6 min for Tyr(SO3H)-Trp-Met-Asp-Phe-NH2, and 3 min only for CCK-4. Caerulein was much more resistant than CCK-8, and Boc-CCK-4 and Aoc-CCK-4 remained stable for at least 3 h. The apparent Km for CCK-8 and CCK-4 was 40 microM and maximal activity on CCK-8 was observed at pH 7.0. Zn2+ was strongly inhibitory. The protease inhibitors puromycin and bacitracin, the metal chelator 1,10-phenanthroline, and the sulphydryl blocking agents N-ethylmaleimide and p-chloromercuribenzoate greatly reduced the release of tryptophan from CCK-8. Puromycin inhibition of CCK-8 degradation provoked the accumulation of a CCK-7-like peptide, and that of CCK-4 degradation was of a competitive type (Ki = 2 microM). The CCK-8 degrading activity of brain synaptosomes was present in the cytosol as well as in synaptic membranes.     

The role of cholecystokinin octapeptide (CCK-8) in regulating thyrotropin secretion in rats. In vivo studies

The effect of cholecystokinin octapeptide (CCK-8) on basal and TRH-stimulated secretion of TSH was investigated in 67 male Sprague-Dawley rats. Blood for TSH determinations was sampled by cannulation of right heart auricle in urethane narcosis before and four times during 60 minutes following CCK-8 administration. It was found that CCK-8 administered to the lateral brain ventricle at a dose of 0.5 microgram per animal caused a decrease in blood serum TSH concentration but did not change the response of TSH to TRH. Intravenous administration of CCK-8 at doses of 2 and 20 micrograms/kg had no effect on blood serum concentration of TSH.     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.     

Chronotropic actions of cholecystokinin octapeptide on the rat heart

Cholecystokinin octapeptide (CCK-8) administered i.v. to urethane-anaesthetized rats or added to the perfusion stream of isolated rat hearts produced an immediate bradycardia. The size of this response was dose-related. Studies in vivo and in vitro using atropine and propranolol indicated that the response to CCK-8 was largely due to a direct action of the peptide on the heart. N-carbobenzoxy-tryptophan (CBZ-Trp), a cholecystokinin receptor antagonist, abolished the response of the isolated heart to CCK-8. Gastrin I did not produce bradycardia. The receptors on rat heart were similar to the classes of cholecystokinin receptors found in brain and exocrine pancreas in that CCK-8 rather than cholecystokinin tetrapeptide (CCK-4) was the preferred agonist.     

The depolarization-induced release of cholecystokinin C-terminal octapeptide (CCK-8) from rat synaptosomes and brain slices

Studies on the subcellular distribution of immunoreactive cholecystokinin (CCK) in homogenates of rat cerebral cortex showed that approximately 95% was associated with particulate fractions, including presynaptic terminals (synaptosomes). Chromatography of extracts of tissue and medium from incubated synaptosomes revealed that this material was almost exclusively in the form of COOH-terminal octapeptide (CCK-8), very little CCK-33 being present. There was a wide range of CCK-8 concentrations in synaptosomes from different brain regions (cortex > striatum ? hypothalamus > brain stem). Cerebral cortex synaptosomes were incubated in vitro and showed a complex pattern of CCK-8 release with varying concentrations of tissue: amounts in the medium rose rapidly with increasing synaptosome concentrations, then fell to a plateau at higher tissue values. A mechanism for the rapid disposal of extracellular CCK-8 was associated with synaptosomal fractions. Depolarization-induced (high K⁺) release of CCK-8 was observed with cortex and corpus striatum synaptosomes. A rapid and reversible enhancement of CCK-8 release from cortex slices was observed in response to elevated K⁺. Veratrine also released CCK-8 from cortex slices, although this was not reversible. Stimulus-induced release of CCK-8 from synaptosomes and slices required extracellular Ca²⁺. The storage, release and degradation of CCK-8 by nerve-endings suggest a synaptic function for this peptide.     

Carotid ligation alters cholecystokinin-8 (CCK-8) immunoreactivity in gerbil brains

The unilateral or bilateral carotid arteries were ligated in gerbils used as a model of cerebral ischemia. The effect of different times of bilateral ischemia on the content of CCK-8 in fore regions of gerbil brain and the effect of 30 min of unilateral ischemia on the content of CCK-8 of the same regions in gerbils with or without neurological signs were observed. Our results show that the content of CCK-8 of cortex, basal ganglia, thalamus and hypothalamus decreased significantly. But, in brain stem it remained basically unchanged no matter whether the ischemia was unilateral or bilateral. This suggests that there is a close relationship between CCK-8 and cerebral ischemia, and raises the possibility that CCK-8 may be involved in cerebral ischemia through a yet unclear mechanism.     

Cholecystokinin octapeptide, proglumide, and passive avoidance in rats

Rats were conditioned to avoid a darkened chamber using electric footshock (0.25 mA for 2 sec). Cholecystokinin octapeptide (CCK-8), a CCK-8 antagonist proglumide, or 0.9% NaCl solution was injected immediately following the footshock to study the effect upon passive avoidance behavior. The passive avoidance behavior was observed one day following the conditioning footshock and treatment. CCK-8 produced a reduction of the passive avoidance latency of rats at doses ranging from 30 micrograms/kg to 500 micrograms/kg. Proglumide (5 mg/kg) was able to block the CCK-8 effect on rat passive avoidance conditioning. Proglumide by itself at a dose of 2 mg/kg decreased the latency to enter the darkened chamber. Endogenous CCK-8 activity may be involved in passive avoidance conditioning in rats.