Effect of V-9-M, a peptide fragment derived from procholecystokinin, on memory processes in the rat

It has been reported that a nonapeptide (Val-Pro-Val-Glu-Ala-Val-Asp-Pro-Met) called V-9-M is produced from procholecystokinin in the brain. Since this peptide is particularly abundant in the hippocampus, septum, and amygdala. V-9-M may be involved in memory processes. The present study was attempted to observe the effect of V-9-M on memory processes of rat performing a one-trial passive avoidance task and a platform jumping active avoidance task. The results indicate that injection of V-9-M into the lateral ventricle of the rat prevents experimental amnesia induced by electroconvulsive shock in passive avoidance testing, and that this effect is not significantly affected by cholecystokinin-8 antagonists. V-9-M also causes a long-lasting enhancement of memory in the active avoidance task. These results suggest that V-9-M may participate in the facilitation of memory.     

Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells in culture

The release of peptides immunoreactive for a synthetic peptide (V-9-M) contained in the amino-terminal of pro-CCK was examined. The potassium-evoked release of V-9-M immunoreactive peptides from rat cerebral cortical slices in vitro was calcium dependent. Cholecystokinin-secreting rat medullary thyroid carcinoma cells also secreted significant quantities of these peptides. Sephadex column chromatography of the release media from slices and cells showed two V-9-M immunoreactive peptides, one larger and one smaller than V-9-M itself. Previous behavioral studies have suggested that V-9-M has a distinct neuropharmacological profile. These results demonstrate that V-9-M-like peptides are released along with CCK-8 and are consistent with the hypothesis that V-9-M-like peptides may be neurotransmitters or neuromodulators or may be involved in the sorting or transport of CCK-8.     

The subcellular distribution of peptides immunoreactive for the amino-terminal of pro-cholecystokinin in rat brain

Antiserum 1942 raised against the synthetic peptide V-9-M is specific for the amino-terminus of pro-cholecystokinin (pro-CCK). It detects three major peptides in whole rat brain extracts with molecular weights of about 13 000 (peak 1), 8000 (peak 2) and 2700 (peak 3), of which the major one is peak 3. Rat brain was found to contain large quantities of these V-9-M-like peptides. Subcellular fractionation of whole rat brain was performed to determine what cellular component was enriched in these peptides. The molecular weight of the V-9-M-like and CCK-8-like peptides enriched in various subcellular fractions has been determined by Sephadex G-50 chromatography. Primary subcellular fractionation experiments indicated a significant enrichment of V-9-M-like peptides in the mitochondrial pellet (P2), a lesser amount in the microsomal pellet (P3), and a slight enrichment in the soluble fraction (S3). Further purification of the P2 fraction demonstrated an increase of V-9-M-like immunoreactivity in purified synaptosomes. With the exception of the enrichment in the soluble fraction, V-9-M-like peptides follow a similar distribution to that of CCK-8-like peptides. Sephadex chromatography of P2 and P3 fractions indicates that the major form of V-9-M present is the peak 3 (2700) form. This V-9-M-like peptide may represent an intermediate in the processing of CCK, and its presence in synaptosomes may indicate that the proteolytic cleavage of pro-CCK into CCK 58 and peak 3 takes place in synaptic vesicles.     

The effect of drugs which alter GABA-ergic function on cerebellar guanosine-3',5'-monophosphate content.

The actions of several classes of drugs, thought to be involved with gamma-amino-butyric acid (GABA) mechanisms, have been examined for effects on cerebellar cGMP content. Picrotoxin and TRH increased, while ethanol and diazepam decreased, cerebellar cGMP. Doses of gamma-hydroxybutyrate (GHB) and baclofen caused no significant effect at doses that caused behavioral changes. These cGMP actions were contrasted with those induced by the dopaminergic agents, apomorphine and haloperidol, which respectively, raised and lowered cerebellar cGMP. Apomorphine-induced increases in cGMP were blocked by haloperidol, but not by GHB or baclofen given eight min before sacrifice. However, baclofen given one hour before sacrifice caused effects similar to those of haloperidol. These results are discussed in terms of dopaminergic-GABAergic interactions.     

Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay)

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.     

Small molecule of sphingosine as a rescue of dopaminergic cells: A cell therapy approach in neurodegenerative diseases therapeutics

Multiple sclerosis (MS) patients should take medication such as fingolimod (FTY-720) for a long time, hence pharmaceutical effects on other neural cells such as dopaminergic cells are important. Dopaminergic cell line, BE(2)-M17, was treated by FTY-720 and then cell viability and genes involve in neurosurvival were investigated. It was disclosed that FTY-720 significantly stimulates Bcl2 overexpression. Whereas, it decreased intracellular reactive oxygen species production and cell membrane damage of dopaminergic cells. The increase in Bcl2/Bax ratio increased the cell metabolic activity and decreased propidium iodide-positive cells. Besides, FTY-720 induced the overexpression of CACNA1C, nNOS gene, and nitric oxide production. However, FTY-720 induced GABARA1 overexpression and eventually it could overcame to the cytotoxic effect of intracellular calcium. This cascade led to tyrosine hydroxylase and BDNF genes overexpression whereas FTY-720 did not change GDNF concentration in BE(2)-M17 cells. Concluding, it might be said that taking FTY-720 in MS patients did not induce adverse effect on dopaminergic cells.     

Influence of D-1 receptor system on the D-2 receptor-mediated hypothermic response in mice

The hypothermia induced by apomorphine, a mixed dopamine (DA) agonist in male Swiss-Webster mice, was not blocked by the selective D-1 antagonist SCH 23390 but was completely blocked by the selective D-2 antagonists haloperidol, sulpiride and YM-09151-2. The selective D-1 agonist SKF 38393 did not elicit hypothermic response but the selective D-2 agonist quinpirole caused a marked lowering of rectal temperature. D-2 antagonists blocked this response to quinpirole. SCH 23390 enhanced and SKF 38393 attenuated the hypothermia induced by quinpirole. Ineffective doses of haloperidol and SKF 38393, when given together, completely blocked the effect of quinpirole. It was concluded that hypothermia is a D-2 receptor mediated response but modulated by the D-1 receptor system. In another series of experiments the influence of neuroleptics and antidepressants on the hypothermic effect of apomorphine and quinpirole was investigated. The hypothermic effect of a low dose (1 mg/kg) of apomorphine was blocked by the D-2 receptor antagonists, but not by classical antidepressants. However, the response to a high dose (10 mg/kg) of apomorphine was blocked by both classical antidepressants and D-2 antagonists (except haloperidol). These drugs did not show similar effect on quinpirole-induced hypothermia. It is clear that the hypothermic response, especially that of quinpirole, is not a suitable model for testing either neuroleptics or antidepressants.     

Role of serotonin2 receptors in regulating aggressive behavior

Quipazine and pirenperone , the drugs interacting with serotonin2 -receptors, more readily displaced 3H-spiroperidol from its binding sites in the frontal cortex than in the striatum. Pirenperone (0,07-0,3 mg/kg), antagonist of serotonin2 -receptors, selectively decreased the intensity of apomorphine aggressiveness. The antiaggressive action of haloperidol (0,01-0,2 mg/kg) was in correlation with its antistereotypic activity. Long-term administration of naloxone (0,5; 15,0 mg/kg), together with apomorphine (0,5 mg/kg) reduced the number of head-twitches caused by quipazine (2,5 mg/kg). The administration of quipazine 48 hours after the last injection of naloxone and apomorphine caused spontaneous aggressiveness that did not differ from apomorphine aggressiveness. Intracerebroventricular injection of cholecystokinin tetrapeptide (CCK-4) markedly enhanced the foot-shock aggression. The same dose of CCK-4 also decreased the intensity of quipazine (2,5 mg/kg) head-twitches. Compared to haloperidol, pirenperone was a more selective antagonist of CCK-4. After long-term apomorphine treatment (0,5 mg/kg during 10 days, twice daily), the effect of CCK-4 on aggressive behaviour was markedly enhanced. It is possible that two subtypes of serotonin2 -receptors exist in the brain and have opposite action on the aggressive behaviour. CCK-4 may play the role of an endogenous modulator of sensitivity of serotonin2 -receptors involved in the control of aggressiveness.     

Lack of antiemetic effects of delta9-tetrahydrocannabinol in apomorphine-induced emesis in the dog

Clinical studies have suggested that Δ⁹-tetrahydrocannabinol (THC) may be a clinically useful antiemetic. However, the ability of THC to decrease experimentally induced emesis in animals has not been extensively studied. The present study compares the antiemetic effects of THC with chlorpromazine on apomorphine-induced emesis in the dog. THC, chlorpromazine, THC vehicle, or saline was administered i.v. 30 min prior to an i.v. infusion of apomorphine; apomorphine was infused until emesis occurred. THC had no effect on the dose of apomorphine required to produce emesis, whereas chlorpromazine increased this dose approximately 75%. Moreover, THC nearly doubled the time from the first to the last occurrence of emesis relative to control values, while chlorpromazine greatly reduced this value. In addition, THC had no effect on the stimulation of pulse rate produced by apomorphine; chlorpromazine potentiated this effect, probably through indirect mechanisms. These findings demonstrate that THC is not an antagonist of the emetic agent apomorphine in the dog.     

Prolonged modulation of FGF-2 expression in astrocytic cultures induced by O,O'-diacetyl-apomorphine

Apomorphine (APO) is an anti-parkinsonian drug currently in use, which provides relief of Parkinson’s symptoms. However, the utility of APO is greatly hampered by its poor bioavailability and rapid metabolism. In the present study, O,O′-diacetyl-apomorphine, a prodrug of apomorphine, was synthesized and its biological activity was examined. The prodrug induced fibroblast growth factor-2 production in astrocytic cultures similarly to apomorphine. However, its duration of action was significantly prolonged, and its resistance to oxidation was markedly enhanced compared to APO. O,O′-Diacetyl-apomorphine also induced MEK/MAPK signaling. These results suggest that O,O′-diacetyl-apomorphine can efficiently counteract oxidation and thereby enhance FGF-2 production in astrocytes.     

Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway

Flavokawain B (FKB) possesses strong anti-neoplastic activity against many cancer cells. Here we assessed its antitumor activity and molecular mechanisms in lung cancer H460 cells in vitro. FKB significantly inhibited cell proliferation and caused arrest of the cell cycle G2-M of H460 cells in a dose-dependent manner. FKB also inducted apoptosis, which was associated with cytochrome c release, caspase-7 and caspase-9 activation and Bcl-xL/Bax dys-regulation. FKB significantly down-regulated survivin and XIAP, and the inhibitory effect induced by FKB was greatly attenuated by through over-expression of survivin or Bax(-/-) MEFs. Furthermore, FKB activated the mitogen-activated protein kinases and the JNK inhibitor SP600125 significantly decreased the growth-inhibitory and apoptotic effects of FKB. Together, these results suggest the anti-lung cancer potential of flavokawain B for the prevention and treatment of lung cancer.     

Serotonergic modulation of footshock induced aggression in paired rats.

Footshock induced aggression (FIA) was induced in paired rats and three paradigms of aggressive behaviour were recorded, namely, latency to fight (LF), total period of physical contact (TPP) and cumulative aggression scores (CAS). The effects of increasing or decreasing central serotonergic activity, by using a number of pharmacological agents with well defined effects on rat brain serotonin, were investigated on FIA and on FIA augmented by apomorphine, a dopamine receptor agonist. The results show that centrally administered serotonin, the serotonin precursor, 5-hydroxytryptophan administered with clorgyline, a selective MAO A inhibitor, quipazine, a serotonin receptor agonist, and fluoxetine, a selective inhibitor of neuronal re-uptake of serotonin, attenuated all paradigms of FIA and apomorphine induced potentiation of FIA. On the contrary, the other re-uptake inhibitor used, citalopram, appeared to have a dual effect and decreased LF and CAS, while increasing TPP. The serotonin synthesis inhibitor, p-chlorophenylalanine and the selective serotonin receptor (5-HT2) antagonist, ketanserin, augmented all paradigms of FIA per se and apomorphine induced augmentation of FIA. However, the other serotonin receptor antagonist used, metergoline, which blocks both 5-HT1 and 5-HT2 receptor subtypes, attenuated FIA per se but decreased only CAS in apomorphine induced increase in FIA. The data confirm the inhibitory effect of the central serotonergic system on aggressive behaviour and the inverse relationship existing between it and the central dopaminergic system in the modulation of FIA, as has also been confirmed in earlier biochemical investigations from this laboratory. The data has been discussed in the light of existing knowledge on serotonin receptor subtypes and the presence of modulatory serotonergic heteroreceptors on central dopaminergic neurones.     

The hypolipidemic peroxisome-proliferating drug, bis(carboxymethylthio)-1.10 decane, a dicarboxylic metabolite of tiadenol, is activated to an acylcoenzyme A thioester

Bis(carboxymethylthio)-1.10 decane (BCMTD), a thiodicarboxylic acid, was shown to be a hypolipidemic peroxisome-proliferating drug as it: (a) decreased the total serum triacylglycerols and cholesterol; (b) induced hepatomegaly; (c) increased the peroxisomal beta-oxidation and catalase activity and the activities of the multiorganelle localized enzymes: palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, glycerophosphate acyltransferase; (d) decreased the carnitine palmitoyltransferase and urate oxidase activities; and (e) induced the bifunctional eonyl-CoA hydratase in peroxisomes. The present study has confirmed the effect of tiadenol administration on the activities of key enzymes involved in hepatic fatty acid metabolism in male rats. However, the hepatic pleiotropic response was more marked with the dicarboxylic acid than with its alcohol. In a separate dose-response study BCMTD was found to be a more potent inducer of peroxisomal beta-oxidation compared to tiadenol. BCMTD can be activated in vitro to its coenzyme A thioester by a dicarboxyl-CoA synthetase. In control and BCMTD-treated animals, the synthetase activity was found in all cellular fractions except the cytosolic. Whether the acyl-CoA thioesters of peroxisome-proliferating drugs may be mediators of peroxisomal proliferation should be considered.     

The effect of spermine on the disaccharidase activities in suckling rats of different age.

The influence of the age of the rat on the maturation of disaccharidase activities induced by spermine was studied. Three-day- and 9-day-old rats were used in the experiment. Spermine was administered orally or directly into the stomach using thin tubing daily for 3 days, and disaccharidase activities in the jejunum were measured. While spermine caused maturation of not only lactase activity but also maltase and sucrase activities in 9-day-old rats, it only caused maturation of lactase activity in 3-day-old rats. Histological studies showed no significant changes in the jejunum of 3-day-old rats treated with spermine.     

Virion envelope content, infectivity, and neutralization sensitivity of simian immunodeficiency virus

A truncating E767stop mutation was introduced into the envelope glycoprotein of simian immunodeficiency virus (SIV) strain SIV239-M5 (moderately sensitive to antibody-mediated neutralization and lacking five sites for N-linked carbohydrate attachment) and strain SIV316 (very sensitive to neutralization, with eight amino acid changes from the neutralization-resistant parental molecular clone, SIV239). The truncating mutation increased Env content in virions, increased infectivity, and decreased sensitivity to antibody-mediated neutralization in both strains. However, the magnitude of the effect on infectivity and neutralization sensitivity differed considerably between the two strains. In the context of strain SIV239-M5, truncation increased Env content in virions approximately 10-fold and infectivity in a reporter cell assay 24-fold. The truncated SIV239-M5 was only slightly more resistant to neutralization by polyclonal monkey sera and by monoclonal antibodies than SIV239-M5 with a full-length envelope glycoprotein. In the context of strain SIV316, truncation increased infectivity a dramatic 480-fold, while envelope content in virions was increased only about 14-fold. This dramatic increase in infectivity cannot be simply explained by the increase in envelope content and is likely due to an increase in inherent infectivity, i.e., infectivity per spike, that results from truncation. The truncated SIV316 was extremely resistant to antibody-mediated neutralization. In fact, it was not neutralized by any of the antibodies tested. When increasing amounts of SIV316 envelope glycoprotein (full length) were provided in trans to SIV316, infectivity was increased and sensitivity to neutralization was decreased, but to nowhere near the degree that was obtained when truncated SIV316 envelope glycoprotein was used. Truncated forms of SIV239 and SIV239-M5 required higher levels of soluble CD4 for inhibition of infection than their nontruncated forms; truncated SIV316 did not. Our results suggest that envelope content in SIV virions, infectivity, and resistance to antibody-mediated neutralization can be increased not only by truncation of the cytoplasmic domain but also by provision of excess envelope in trans. The striking increase in infectivity that results from truncation in the context of SIV316 appears to be due principally to an increase in inherent infectivity per spike.     

Characterization of gp120 and its single-chain derivatives, gp120-CD4D12 and gp120-M9: implications for targeting the CD4i epitope in human immunodeficiency virus vaccine design

Single-chain derivatives of JRFL gp120 linked to the first two domains of human CD4 (gp120-CD4D12) or to the CD4 miniprotein analog CD4M9 (gp120-M9), have been constructed. Biacore studies revealed that gp120-CD4D12 and gp120-M9 bound to antibody 17b with dissociation constants of 0.8 and 25 nM, respectively, at pH 7.0, while gp120 alone did not bind. The binding of gp120-CD4D12 to 17b is not affected by the addition of excess soluble CD4D12, while the binding of gp120-M9 is enhanced. This finding indicates that the M9 component of the single chain interacts relatively weakly with gp120 and can be displaced by soluble CD4D12. Immunogenicity studies of gp120, gp120-CD4D12, and gp120-M9 were carried out with guinea pigs. All three molecules were highly immunogenic. The resulting antisera were examined for neutralizing activities against various human immunodeficiency virus type 1 isolates. Broadly neutralizing activity was observed only with sera generated against gp120-CD4D12. These antisera were depleted of anti-CD4D12 antibodies by being passed over a column containing immobilized CD4D12. The depleted sera showed a loss of broadly neutralizing activity. Sera that were affinity purified over a column containing immobilized gp120-M9 also lacked such neutralizing activity. This finding suggests that the broadly neutralizing response observed is exclusively due to anti-CD4 antibodies. Competition experiments showed that only antisera generated against gp120-CD4D12 competed with the CD4i antibody 17b and that this activity was not affected by depletion of anti-CD4 antibodies. The data indicate that although antibodies targeting the CD4i epitope were generated by the gp120-CD4D12 immunogen, these antibodies were nonneutralizing.     

The potentiating effect of naloxone upon apomorphine-induced hyperthermia

Intravenous or intracerebroventricular pretreatment with the narcotic antagonist naloxone in rabbits significantly enhanced the magnitude of the hyperthermic response to the dopaminergic agonist apomorphine. Naloxone did not potentiate the hyperthermic action of either amphetamine or lysergic acid diethylamide. Apomorphine-in induced hyperthermia was sensitive to antagonism by haloperidol, cyproheptadine and p-chlorophenylalanine. However in rabbits pretreated with any of the above antagonists, administration of naloxone five minutes prior to apomorphine challenge restored the hyperthermic effect of apomorphine. Increasing the dose of the apomorphine challenge likewise surmounted the antagonism. It was concluded from these data that naloxone exerts a potentiating influence upon apomorphine drug effect in naive rabbits as well as rabbits pretreated with antagonists of apomorphine-induced hyperthermia.     

α-Mangostin promotes apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes by reactive oxygen species-dependent activation of ERK1/2 mitogen-activated protein kinase

α-Mangostin (α-M) is a commonly used traditional medicine with various biological and pharmacological activities. Our study aimed to explore the effects and mechanism of α-M in regulating apoptosis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). α-M of 10 to 100 μM was used to treat RA-FLS for 24 hours, followed by measuring cell viability and apoptosis. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinases was detected. Treatment of α-M promoted apoptosis and reduced viability of RA-FLS in a dose-dependent manner. The mitochondrial membrane potential in RA-FLS was remarkably reduced by α-M treatment, accompanied by the cytochrome c accumulation in the cytosol and increased activities of caspase-3 and caspase-9. Moreover, we found that α-M treatment promoted ROS production and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. The proapoptotic activity of α-M in RA-FLS was markedly reversed by the co-induction with the ERK1/2 inhibitor LY3214996 or ROS scavenger N-acetyl-l -cysteine. In conclusion, our studies found that α-M had remarkable proapoptotic activities in RA-FLS, which is regulated by the induction of ROS accumulation and ERK1/2 phosphorylation. α-M may thus have potential therapeutic effects for rheumatoid arthritis.     

Naloxone potentiation of apomorphine-induced stereotypic climbing in mice and interaction with mu-, sigma- and kappa-opiate drugs

Pretreatment with the narcotic antagonist naloxone produced a dose-related potentiation of mouse stereotypic climbing behavior induced by the dopaminergic agonist apomorphine. In further experiments, mice were also pretreated with various drugs specific for mu-opiate receptors (morphine), sigma-opiate receptors (N-allylnormetazocine) and kappa-opiate receptors (ketocyclazocine). Doses of morphine that alone did not affect apomorphine-induced climbing antagonized naloxone potentiation of apomorphine. Doses of N-allylnormetazocine that did not influence apomorphine stereotypy also reversed naloxone potentiation of apomorphine. On the other hand, ketocyclazocine alone exerted a behavioral suppressant effect upon apomorphine- induced stereotypic climbing, however, these same doses failed to prevent naloxone potentiation of apomorphine.