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.     

Transfer of immunity by transfer of bone marrow cells: T-cell dependency.

Thymectomized, lethally irradiated mice reconstituted with normal bone marrow cells succumbed when challenged ip with rat Yoshida ascites sarcoma (YAS) cells 40 days after irradiation and reconstitution. In contrast, thymectomized irradiated mice reconstituted with bone marrow cells from YAS-immune donors rejected the subsequent tumor challenge. Pretreatment of the bone marrow cells from immune donors with anti-Thy 1.2 antiserum and complement completely abolished the transfer of anti-YAS resistance.Bone marrow cells from donors thymectomized 2 months before immunization enabled almost all recipients to reject YAS, but bone marrow cells from donors thymectomized 8 months before immunization protected only 50% of the recipients. Further analysis showed that mice thymectomized 8 months before immunization failed to generate anti-YAS antibody response, whereas the antibody response of mice thymectomized 2 months before immunization did not differ from that of non-thymectomized age-matched control mice. The data suggest that the immune reaction of mice against xenogeneic YAS requires long-lived T₂ lymphocytes.     

Passive avoidance deficit following intracerebroventricular administration of cholecystokinin tetrapeptide amide in rats

The effect of cholecystokinin tetrapeptide amide (CCK-4) injected into the lateral cerebral ventricle on memory processes was examined by a one-trial passive avoidance test in the rat. CCK-4 injection 30 and 60 min before the first retention test caused a shortened latency to response, and its chronic infusion into the lateral ventricle at a rate of 2 micrograms/day shortened the latency of the response to the level of almost complete amnesia. CCK-4 also reduced arginine-vasopressin effect on memory processes when administered simultaneously 30 min before the first retention test, but its amnestic action is short-lasting and antagonized by relatively small amounts of cholecystokinin octapeptide (CCK-8). In addition, the shortened latency to response was admitted to be not always associated with the motility effect of CCK-4.     

Cholecystokinin tetrapeptide,proglumide and open-field behavior in rats

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

八肽胆囊收缩素(CCK_8)的人工合成

本文报道了用液相法合成八肽胆囊收缩素(H-Asp-Tyr(SO_3H)-Met-Gly-Trp-Met-Asp-Phe-NH_2,ccK_8)。先分别合成C-端四肽和N-端四肽,然后缩合成八肽,并用温和的乙酰硫酸吡啶盐(PAS)法使其酪氨酸的酚羟基硫酯化。根据其氨基酸组成,层析行为和生物活性等证实产物为纯品。     

Adoptive immunotherapy of a BALB/c lymphoma by syngeneic anti-DBA/2 immune lymphoid cells: Characterization of the effector population and evidence for the role of the host's non-T cells

Summary It has been previously shown that the BALB/c lymphoma YC8 is susceptible to lysis by syngeneic anti-DBA/2 lymphocytes and that YC8-bearing BALB/c mice can be cured by adoptive transfer of such immune effectors. In this study in vivo and in vitro functions of the curative immune lymphocytes have been evaluated together with the role of the host immune system in the mechanism of tumor eradication. It was found that the curative anti-DBA/2 lymphocytes were not directly cytotoxic to YC8 cells although they developed into YC8-lytic cells after in vitro restimulation with YC8. In vivo, the immune lymphocytes were able to mediate a tumor-specific delayed type hypersensitivity reaction against YC8 but had a low tumor-neutralizing activity in the Winn assay. Proliferation of infused BALB/c anti-DBA/2 lymphocytes was necessary for the in vivo therapeutic effect, since irradiation of effector cells or treatment of the donor immune lymphocytes with vinblastine abolished their curative capacity. Immunodepression of the T cell compartment of the prospective tumor-bearing animals by thymectomy plus irradiation or its abrogation in B mice (thymectomized, lethally irradiated, and reconstituted with fetal liver cells) did not interfere with the therapeutic effect of the transferred anti-DBA/2 lymphocytes. Blocking the macrophage functions of the host by carrageenan, however, abolished the therapeutic effect of immune lymphocytes. These data indicate that a radiation-resistant, non-T cell is involved in the tumor eradication induced by anti-DBA/2 lymphocytes. It was also shown that cured mice, tested 90 days after therapy, become resistant to 5×10³ LD₈₀ YC8 cells and that this resistance was due to the presence of memory cells derived from the transferred and not from the host lymphocyte population.     

Cortical tissue from syngenous brain as a stimulator of immunogenesis in thymectomized mice]

Experiments on CBA mice showed that the gray matter homogenate of the syngeneic brain contained components reconstituting the T-cell population in the spleen of thymectomized animals and possessing a stimulating effect on the immune response to sheep red blood cells. The white matter homogenate was much less active, it is not excluded that the trace activity is due to the gray matter admixture in the preparation. Syngeneic muscle tissue homogenate possessed no biological activity.     

Immunocorrective action of tuftsin during exposure to ionizing radiation

The effect of the tetrapeptide tuftcin (Thr-Lys-Pro-Arg) on the humoral immune response in CBA mice exposed to sublethal irradiation in a dose of 450 sGy was studied. The drug was injected intraperitoneally for 5 days before (series I) or after (series II) radiation exposure. It has been shown that taftcin has no protective action but decreases considerably the immune response lowering due to radiation.     

The immunotropism of sunflower oil and starch in peroral administration to mice]

It has been shown that per os introduction of sunflower oil and starch to adult thymectomized mice resulted in reconstitution of the number of Thy-I+ spleen cells similar of the glutamic acid effect. Glutamic acid restored the immune response to sheep red blood cells (SRBC), however, sunflower oil and starch were not effective. The influence of these compounds on the immune response to SRBC in normal and sham-thymectomized mice was different: glutamic acid stimulated it, starch had no influence on the immune response, sunflower oil suppressed it.     

Unsulfated CCK-8 not blocked by proglumide or CR 1409 in the mouse abdominal irritant-induced stretching assay: possible central site of action

Previous studies have shown that unsulfated cholecystokinin octapeptide (CCK-8-U) shares with the sulfated octapeptide (CCK-8-S) and the carboxyl-terminal tetrapeptide (CCK-4) the ability to block abdominal irritant-induced stretching when administered intracerebroventricularly. The effects of CCK-8-U, however, are not naloxone-reversible and do not appear upon systemic administration. To assess the hypothesis that the antistretching effects of CCK-8-U are mediated by central-type (CCK-B), rather than peripheral-type (CCK-A) receptors, the present experiments examined the reversal of these effects by CR 1409, a CCK receptor antagonist with in vitro selectivity for CCK-A receptors, and by proglumide. Both antagonists, when administered ICV, blocked the antistretching effects of CCK-8-S and CCK-4 (ICV), but not those of CCK-8-U. CR 1409 was approximately 40 times more potent against CCK-8-S by the ICV route than subcutaneously, indicating a likely action on CCK-A receptors in the brain. The effects of morphine, bombesin and neurotensin (ICV) were not blocked by CR 1409, indicating specificity for CCK receptor-mediated effects. The antistretching effects of CCK-8-U do not appear to be mediated by CCK-A receptors, and the possibility of a CCK-B receptor site of action must be considered.     

Characteristics of the reaction of immunologically tolerant mice to the polyclonal stimulant salmozan

The nonspecific stimulant of the immune response salmozan (Sal) increases the number of PFC against SRBC in intact mice, B mice (thymectomized, irradiated and reconstituted with embryonic liver cells), and in mice pretreated with cyclophosphamide (CY). This effect was decreased in mice pretreated with SRBC and CY. The subsequent injections of SRBC and Sal into tolerant mice did not increase the response under study. It is concluded that the effect observed is due to partial alteration of antigen-specific B cells of tolerant mice and this alteration cannot be explained by the lack of the regeneration of membrane immunoglobulins.     

Neonatal thymectomy affects follicle populations before the onset of autoimmune oophoritis in B6A mice

Using a variation on a standard follicle classification technique, 5 classes of follicles were quantified in serial sections of ovaries from intact mice and mice thymectomized on Day 3 at 5-day intervals from 5 to 40 days of age. Sera from these animals and from animals 60 days of age were analysed for the presence of anti-oocyte antibodies. Ovaries from intact animals 10 to 40 days of age were examined for the presence of antigen(s) using anti-oocyte antibody-positive sera from all ages of mice. There was a dramatic decrease in the primordial follicle population at 10 days of age in thymectomized mice and that population remained significantly lower until 40 days of age. The growing follicle population was also significantly lower at 20 days of age in thymectomized mice and remained lower through 40 days of age. Anti-oocyte antibodies were not detectable until 30 days of age and at that age reacted with oocytes from all follicle types including primordial. Ovarian antigens were present in similar patterns in ovaries from mice at all ages tested. We conclude that thymectomy has an earlier influence on the ovary than previously thought and this influence does not appear to involve the immune activity associated with autoimmune ovarian dysgenesis. This suggests that the effect of thymectomy on the ovary may be biphasic: (1) an early effect, possibly involving a disruption in the hypothalamic-pituitary-ovarian-thymic axis, that influences the primordial and growing follicle populations before 20 days of age; and (2) a later effect involving an immune imbalance first evident by 25-30 days of age that ultimately results in the destruction of the ovary.     

Role of the thymus in regulation of the macrophage migration inhibitory factor production in mice of different genotypes]

The influence of the thymus on the production of the macrophage migration inhibitory factor (MIF) was studied in C57BL and CBA mice thymectomized at 4--6 weeks of age. On the 1--21st day after the operation they were immunized intraperitoneally with complete Freund's adjuvant. MIF production stimulated by tuberculin was determined on the maximum of the immune response. MIF production was abolished in mice of both lines already during the first days. To elucidate a relationship between MIF production and the presence of the thymus the former was investigated in the thymectomized "nude" mice. The mice showed no MIF production. It was found as well that thymectomy can interrupt the immune response in early stages of its development and completely eliminates MIF production the first days after immunization. Moreover, thymectomy in adult mice also changes spontaneous migration of macrophages both in immunized and non-immunized mice. These changes were more pronounced in C57BL mice.     

Effects of CCK antagonists on CCK-induced suppression of locomotor activity in mice.

Sulfated cholecystokinin octapeptide (CCK-8) was administered either intraperitoneally or into the cerebral ventricle of fully conscious mice, and locomotor activity was quantified. CCK-8 administered by either route suppressed locomotor activity. Subcutaneously administered selective CCK-A receptor antagonist, L-364,718 (1 mg/kg), reversed the inhibitory effect of centrally as well as peripherally administered CCK-8, but the selective CCK-B receptor antagonist, L-365,260 (1 mg/kg), did not. These results demonstrate that centrally as well as peripherally administered CCK-8 suppresses locomotor activity in mice through an interaction with CCK-A, but not CCK-B, receptors.     

Double-negative T regulatory cells can develop outside the thymus and do not mature from CD8+ T cell precursors

Recent studies have demonstrated that activated peripheral alphabeta TCR+ CD3+ CD4- CD8- NK1.1- (double-negative, DN) regulatory T cells (Tregs) from both mice and humans are able to down-regulate immune responses in vitro and in vivo. However, the origin and developmental requirements of functional DN Tregs remain unclear. In this study, we investigated the requirement for CD8 expression as well as the presence of a thymus for the development of functional DN Tregs. We demonstrate that DN Tregs exist in CD8-deficient mice and that stimulation of CD8+ T cells in vivo with TCR-specific Ag does not convert CD8+ T cells into DN Tregs. In addition, we found that DN T cells are present in the spleens and lymph nodes of thymectomized mice that are irradiated and reconstituted with T cell-depleted bone marrow cells. Interestingly, DN Tregs that develop in thymectomized mice can suppress syngeneic CD8+ T cells more effectively than those that develop in sham-thymectomized mice. Taken together, our data suggest that DN Tregs are not derived from CD8+ T cell precursors and that functional DN Tregs may preferentially develop outside of the thymus. These data suggest that DN Tregs may represent a developmentally and functionally unique cell population.     

Effect of highly purified thymus factor on the cellular and humoral indices of immunity in thymectomized mice]

Effect of homogeneous polypeptide thymus factor of mol weight about 5000 (thymarin-III) on cellular and humoral immune responses of thymectomized adult CBA mice was studied. Thymectomy proved to greatly decrease the number of T-cells in the spleen. Accordingly, the capability of these mice to produce both IgM and IgG antibody-forming cells in response to the thymus-dependent antigen (sheep red blood cells) was significantly depressed. Subcutaneous injections of thymarin-III (1 microgram per g of body weight) for 7 days completely restored the T-cell spleen population and normalized the animals' immune response.     

Ethanol sensitizes NF-kappaB activation in pancreatic acinar cells through effects on protein kinase C-epsilon

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-kappaB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-kappaB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-kappaB and PKC-delta, -epsilon, and -zeta activation, whereas 0.1 nM CCK-8 did not increase PKC-epsilon, PKC-zeta, or NF-kappaB activity. CCK-8 (0.1 nM) did activate PKC-delta. PKC-epsilon activator alone did not cause NF-kappaB activation; however, together with 0.1 nM CCK-8, it caused NF-kappaB activation. Ethanol activated PKC-epsilon without affecting other PKC isoforms or NF-kappaB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-delta, PKC-epsilon, and NF-kappaB. The NF-kappaB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-delta inhibitor and PKC-epsilon inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-kappaB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-epsilon and -delta is required for NF-kappaB activation. These results suggest that ethanol enhances the CCK-8-induced NF-kappaB activation at least in part through its effects on PKC-epsilon.     

Reversed behavioral effect of cholecystokinin after frontal decortication in rats

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

CCK-8 induces fever-like regulated hyperthermia and symptoms of sickness behavior in mice: A biotelemetric study

In earlier studies it has been found that rats respond to intracerebroventricular (i.c.v.) injection of cholecystokinin-octapeptide (CCK-8) with a febrile response characterized by rises of heat production and core temperature together with tail-skin vasoconstriction mediated by CCK2 receptors. Biotelemetric investigations of the same species have additionally shown that CCK-induced fever is accompanied by decreased locomotor activity. Similar data for mice have not been reported so far. In the present studies C57BL/6 mice were infused i.c.v. for 3 days with CCK-8 to see effects on body core temperature, locomotor activity, food intake and body weight. Biotelemetric monitoring disclosed a rise in daylight core temperature and a fall of night-time locomotor activity both lasting beyond the time of i.c.v. infusions. Food intake was suppressed only during infusion, while a significant decrease of body weight was sustained after the end of CCK-8 infusion. It is concluded that similar to rats mice also respond to i.c.v. infusion of CCK-8 with a fever-like (regulated) hyperthermia and some components of sickness behavior as measured by biotelemetry, and thus a CCK-mediated mechanism may contribute to fever genesis also in mice.     

Specificity of nucleus accumbens to activities related to cholecystokinins in rats

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