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Behavioral consequences of intracerebral vasopressin and oxytocin: focus on learning and memory.

Since the pioneering work of David de Wied and his colleagues, the neuropeptides arginine vasopressin and oxytocin have been thought to play a pivotal role in behavioral regulation in general, and in learning and memory in particular. The present review focuses on the behavioral effects of intracerebral arginine vasopressin and oxytocin, with particular emphasis on the role of these neuropeptides as signals in interneuronal communication. We also discuss several methodological approaches that have been used to reveal the importance of these intracerebral neuropeptides as signals within signaling cascades. The literature suggests that arginine vasopressin improves, and oxytocin impairs, learning and memory. However, a critical analysis of the subject indicates the necessity for a revision of this generalized concept. We suggest that, depending on the behavioral test and the brain area under study, these endogenous neuropeptides are differentially involved in behavioral regulation; thus, generalizations derived from a single behavioral task should be avoided. In particular, recent studies on rodents indicate that socially relevant behaviors triggered by olfactory stimuli and paradigms in which the animals have to cope with an intense stressor (e.g., foot-shock motivated active or passive avoidance) are controlled by both arginine vasopressin and oxytocin released intracerebrally.

Animals↗

Delta-9-tetrahydrocannabinol treatment results in a suppression of interleukin-2-induced cellular activities in human and murine lymphocytes.

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been shown to suppress a variety of interleukin-2-(IL-2)-dependent cellular functions in both murine and human lymphocytes. These effects were examined in both human peripheral blood lymphocytes (hPBL) and the IL-2-dependent murine cytotoxic T-cell line CTLL-2. Interleukin-2-induced thymidine uptake and uridine uptake were suppressed in a dose related manner when cells were co-incubated for 48 h with 100 U rhIL-2/ml and 1-10 micrograms THC/ml. Interleukin-2-induced protein synthesis was also suppressed in a dose related manner over this THC concentration range, with the hPBL being more susceptible to the suppressive effect of THC than the CTLL-2 cells. Autoradiographic analysis of the synthesized proteins from hPBL cell lysates reveals a generalized suppression of all nascent proteins in THC-treated cultures. Human natural killer cell activity is only affected at the highest concentration tested (10 micrograms THC/ml) while lymphokine-(IL-2)-activated natural killer cell activity is affected throughout the range of 1-10 micrograms THC/ml. Together these results suggest that THC interferes with the IL-2:IL-2 receptor signaling cascade at one or possibly many points causing a decrease in IL-2-induced metabolic activity and cytolytic function.

Animals↗

NF-kappaB is activated in cholestasis and functions to reduce liver injury.

Selected bile acids activate a nuclear factor-kappa B (NF-kappaB)-dependent survival signaling cascade in cultured hepatocytes. These data suggest that in cholestasis where liver tissue bile acid concentrations are increased, NF-kappaB should be activated and inhibition of NF-kappaB should potentiate liver injury. Our aims were to test these two predictions. Cholestasis was obtained by common bile duct ligation in mice. NF-kappaB activation was demonstrated in nuclear extracts by the electrophoretic mobility gel shift assay from 3-day bile duct-ligated (BDL) mice but not in controls. Immunohistochemistry for NF-kappaB demonstrated nuclear localization in hepatocytes of BDL mice consistent with its activation in this liver cell type. Electrophoretic mobility gel shift assay and immunohistochemistry for NF-kappaB in BDL tumor necrosis factor-receptor 1 knockout mice demonstrated hepatocyte NF-kappaB activation, suggesting that tumor necrosis factor-alpha was not responsible for the activation of this transcription factor. Liver injury was assessed in BDL mice after administration of the adenovirus 5 inhibitor of kappa B superrepressor (Ad5IkappaBsr) to inhibit NF-kappaB. TUNEL-positive cells and serum alanine aminotransferase values were increased at least threefold in mice treated with the Ad5IkappaBsr versus the empty virus. Liver histology also demonstrated increased liver injury in the BDL mice treated with the Ad5IkappaBsr. In conclusion, NF-kappaB is activated in hepatocytes during obstructive cholestasis and functions to reduce liver injury.

Alanine Transaminase↗

Platelet function assays.

The roles of platelets in hemostasis and arterial thrombosis involve their adherence to sites of vessel injury or ruptured atherosclerotic plaques, aggregation to form hemostatic plugs or thrombi, and acceleration of the coagulation cascade leading to the formation of thrombin. These roles of platelets are described in this review, hereditary platelet defects and other abnormalities associated with bleeding disorders are listed, and the various aggregating agents are discussed. A number of tests of platelet function are reviewed, including a description of their advantages and disadvantages: bleeding time determination; measurement of platelet aggregation in citrated platelet-rich plasma by recording changes in light transmission; measurement of platelet aggregation in citrated whole blood by impedance aggregometry; measurement of platelet-related hemostasis with the high shear Platelet Function Analyzer (PFA-100) system and the Ultegra Rapid Platelet Function Assay; use of the Cone and Plate(let) Analyzer to measure platelet adherence and aggregation under conditions of high shear; measurement of secretion of granule contents (ATP, 14C-serotonin, platelet factor 4, beta-thromboglobulin) and the formation of thromboxane B(2); and use of flow cytometry to assess the state of platelet activation (including conformational changes in membrane glycoproteins and surface expression of P-selectin and phosphatidylserine) ex vivo and in vitro following addition of agonists, and to measure levels of platelet membrane glycoproteins in the detection of inherited deficiencies.

Blood Platelet Disorders↗

Alteration of B-cell antigen receptor signaling by CD19 co-ligation. A study with bispecific antibodies.

The activation of B-cell antigen receptor-associated protein tyrosine kinases is an early and crucial event in B-cell signaling. Apart from the B-cell antigen receptor (BCR), the B-cell-specific transmembrane glycoprotein CD19 has also been shown to directly activate intracellular signaling cascades. In addition, because CD19 and the BCR are associated on the surface of activated B-cells, it has been proposed that close approximation between these two entities is crucial for optimal B-cell triggering. To test this hypothesis, bispecific antibodies were generated that bind membrane IgM and CD19 simultaneously. Although CD19 bispecific antibodies strongly induced tyrosine phosphorylation, they were, in contrast to muF(ab)2 fragments, unable to induce a proliferative response. Detailed analysis of the early signaling events showed that compared with muF(ab)2 fragments CD19 bispecific antibodies potently raised the intracellular [Ca2+], which was correlated with an efficient tyrosine phosphorylation of syk. Strikingly, the assembly of Grb2 complexes that may couple the BCR to p21(ras) was clearly altered by the CD19 bispecific antibody. In addition to the reported Shc and 145-kDa phosphoproteins, a prominent 90-95-kDa phosphoprotein resembling CD19 was detected in the Grb2 complexes. Thus, studies with CD19 bispecific antibodies show that CD19 co-ligation both quantitatively and qualitatively alters BCR signaling.

Adaptor Proteins, Signal Transducing↗

Caveolin scaffolding peptide-1 interferes with norepinephrine-induced PLC-beta activation in cultured rat vascular smooth muscle cells.

Caveolins are a family of integral membrane proteins implicated in various cell functions, including the organization and inactivation of signaling molecules of G protein-coupled receptors. We tested the ability of human caveolin scaffolding peptide-1 (CSP-1) to regulate norepinephrine- (NE) or histamine (HIS)-induced increases on intracellular calcium concentrations ([Ca(2+)]i). In cultured rat vascular smooth muscle cells (VSMC), CSP-1 inhibited in a concentration-dependent manner NE- and HIS-induced increases in [Ca(2+)]i. This effect can be explained by the fact that CSP-1 inhibited a common signaling pathway. We tested the ability of this peptide to decrease the activation of PLC-beta3 and MAPK. CSP-1 inhibited the expression of the activated form of both enzymes, suggesting a direct effect of the peptide on the signaling cascade. CSP-1 readily enters VSMC in culture, as observed when FITC-conjugated CPS-1 is added to cell culture media. Taken together, these data suggest that CSP-1 blocks the effects of NE and HIS on [Ca(2+)]i of VSMC by inhibiting the activation of PLC-beta3 and MAPK.

Animals↗

Complement activation by cold agglutinins.

Purified monoclonal human IgM cold agglutinins (CA) of different specificities (anti-I, anti-i, anti-Pr) were investigated for their complement-activating capacity in a homologous system. Incubation of human RBC with excess of IgM CA in the cold, and subsequently with human serum at 37 degrees C, resulted in striking differences in hemolysis. Hemolysis did not correlate to the amount of antibodies bound to RBC at 4 or 20 degrees C. Despite the hemolytic inefficiency of anti-i and anti-Pr CA tested, C1 fixation and subsequent activation of the classical pathway of complement could be assessed in all cases. Absolute numbers of C3 molecules bound to RBC, exceeding the critical level to initiate the terminal sequence of the complement cascade, could not fully explain the differences in the hemolytic activity of the CA. Since C8 binding protein (C8bp) carries I determinants it is hypothesized that anti-I-induced complement-mediated hemolysis might also be favored by the binding of the autoantibody to and probably steric hindrance of this major regulatory protein of the terminal complement sequence. The prominent role of homologous restriction of complement-mediated lysis as a protective mechanism can also be deduced from the fact that rabbit as well as rat serum as a source of heterologous complement lysed cold agglutinin-sensitized red blood cells more efficiently than human serum.

Agglutinins↗

Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.

Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.

Alkaline Phosphatase↗

[Depression and Alzheimer's disease].

Despite numerous studies the relationship between depression and Alzheimer's disease has not yet been clarified. The high prevalence of depression in Alzheimer's disease has been confirmed but the data on its incidence vary. Generally, depressed mood is the most prevalent symptom in 0-86% of dementia syndrome, minor depression, dysthymia is considered to be present in 20-30% of patients and major depression is least frequent. It seems confirmed that depression may be considered to be a risk factor for dementia, but the coincidence of these two diseases remains still unknown. Since the symptoms of depression and dementia are very similar, the clinical picture brings other controversies. Loss of energy, speech paucity, poor attention and concentration, diminished interest and psychomotor slowness cannot differentiate dementia from depression, the disability level seems to be the only differentiating factor. Depression may be suspected in case of changes in functional level, complaints about pain and diurnal variation of symptoms. From the practical point of view the type of contact and the willingness of perform tests are among the crucial symptoms. Sometimes, it is difficult to separate apathy and pathological crying from depression. The pathomechanism of depression in dementia is not known. The role of serotoninergic and cholinergic transmission changes, alterations of glucocorticoid cascade and presence of apoE are considered but without evident results.

Aged↗

Anti-inflammatory and antithrombotic effects of statins in the management of coronary artery disease.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins), a potent class of cholesterol-lowering drugs, exert a number of pleiotropic effects, including anti-inflammatory and antithrombotic properties. Evidence is now accumulating that these effects are not related to the reduction in lipid levels. In vitro studies, supported recently by in vivo data, indicate that treatment with statins results in a significant decrease in the levels of inflammation markers, such as C-reactive protein, interleukin 6, and tumor necrosis factor alpha, which appear to be predictors of acute coronary events and help stratify cardiovascular risk. Up to now, only high-sensitive C-reactive protein testing has the potential to become an adjunctive method to assess the risk of coronary events in low- and high-risk individuals. Statins can also inhibit tissue factor expression, leading to impaired activation of the blood coagulation cascade, as evidenced by a decrease in thrombin generation in vivo. Interrelated inhibition of inflammation and thrombosis induced by statins is believed to largely contribute to clinical benefits from statin therapy, regardless of cholesterol levels. Further studies will answer the question whether markers of inflammation, other than C-reactive protein and possibly indices of thrombin formation, might improve cardiovascular risk stratification.

Anti-Inflammatory Agents, Non-Steroidal↗

cAMP cascade leads to Ras activation in cortical neurons.

Monoaminergic G protein-coupled receptors (GPCRs) are highly expressed in the CNS at the cerebrocortical level, where they support a variety of behavioural responses. To elucidate possible intracellular signalling pathways coupled to these receptors, we have studied their ability to activate extracellular signal-regulated kinases (ERKs) in cultured cortical neurons. An increase in ERK activity was observed after stimulation of neurons with dopamine or serotonin, and with agonists selective for various GPCRs. In addition, ERK activation was also observed following treatment with phorbol dibutyrate (PdBu) and forskolin, activators of protein kinase C (PKC) and protein kinase A (PKA), respectively. Concomitant with ERK activation, all the monoaminergic agonists tested also increased the level of active Ras (Ras-GTP). Surprisingly, Ras activation was also observed after activation of cAMP pathway, and this effect was at least in part mediated by PKA. Ras activation by cAMP was unique for neurons, since in PC12 cells forskolin caused activation of ERK but did not increase Ras-GTP level. These results highlight the relevance of Ras as a target for multiple signalling cascades leading to activation of the ERK pathway in neurons.

Animals↗

Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1.

Participation of cytoskeletal elements in regulation of hormonal response and responsiveness has been suggested by several laboratories. Addition of dimeric tubulin to rat cerebral cortex synaptic membranes causes stable inhibition of adenylyl cyclase, and the molecular basis for this effect appears to require a direct interaction between tubulin and G proteins. To test whether such tubulin-G protein interaction occurred, several purified G proteins were bound to nitrocellulose, and 125I-tubulin overlay studies were performed. 125I-Tubulin bound to the alpha subunits of Gs and Gil with high specificity and an apparent Kd of approximately 130 nM. Other G protein alpha subunits (alpha i2, alpha i3, alpha 0, and transducin) displayed a much lower affinity for tubulin, despite the much closer relationship of those proteins to alpha il than to alpha s. Association of beta gamma subunits with alpha il or alpha s did not alter the binding of tubulin to these G protein heterotrimers, and the binding of a hydrolysis-resistant GTP analog to the alpha subunits was similarly without effect. These results suggest that tubulin forms complexes with specific G proteins and these complexes might provide a locus for the interaction of cytoskeletal components and signal transduction cascades. These results also provide evidence of a functional distinction among the closely related alpha i subtypes.

Animals↗

Synergistic antiproliferative and apoptotic effects induced by epidermal growth factor receptor and protein kinase a inhibitors in human prostatic cancer cell lines.

Our results revealed that the blockade of epidermal growth factor receptor (EGFR) tyrosine kinase and protein kinase A (PKA) signaling pathways by specific inhibitors (PD153035 and Rp-cAMPs) leads to a synergistic inhibition of EGF- and serum-stimulated growth of human prostatic cancer cells (LNCaP, DU145 and PC3) concomitant with an arrest in the G1 phase of cellular cycle. Of particular interest, the combination of PD153035 and Rp-cAMPs also caused a more substantial apoptotic/necrotic death of these prostatic cancer cells as compared to drugs alone. Moreover, we observed that the inhibition of acidic sphingomyelinase and caspase cascades results in a marked reduction of DNA fragmentation and apoptotic death induced by PD153035, alone or in combination with Rp-cAMPs, in EGF stimulated PC3 cells. This suggests that these agents might mediate their cytotoxic effects at least in part via the ceramide generation and activation of caspase signaling pathways. N-oleoylethanolamine (OE), an inhibitor of acidic ceramidase, consistently potentiated the apoptotic effects of PD153035 in all the prostatic cancer cell lines tested. Additionally, the cellular ceramide content estimated for PC3 cells was increased after treatment with PD153035, alone or in combination, at a lower dose with OE and Rp-cAMPs. The synergistic apoptotic effect of PD153035 plus Rp-cAMPs induced in PC3 was also accompanied by a significant rate of mitochondrial membrane depolarization and release of cytochrome c into cytosol as compared to drugs alone. Combined, the results indicated that the simultaneous inhibition of EGFR and PKA signaling cascades might lead to a more massive apoptotic death of metastatic prostatic cancer cells by increasing ceramide accumulation and activating of caspase cascade of a mitochondrial dependent manner.

Amino Acid Chloromethyl Ketones↗

The PIVKA II test: the first reliable coagulation test for autopsy investigations.

To date there is no routinely used reliable diagnostic test that can be performed in the post-mortem period to investigate whether a deceased had a coagulation disorder. This paper describes a series of experiments to assess the use of an antigen-based method to investigate the vitamin K-dependent factor II function in the deceased. It illustrates that by using this approach the functional status of factor II can be investigated in the post-mortem period. The abnormal proteins that are investigated by this method appear to remain stable for at least 72 h and potentially up to at least 7 days. The method that is illustrated could thus be reliably used in the post-mortem period to identify a natural or drug-induced factor II abnormality. The potential for other protein components of the coagulation cascade to be investigated by similar antigen-based methodology is suggested.

Aged↗

Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation.

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer's disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer's disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer's disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid beta-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.

Alzheimer Disease↗

Linking development with generation of novelty in mammalian teeth.

The evolution of mammalian teeth is characterized by the frequent and convergent evolution of new cusps. The evolution of new cusps can be linked to tooth development via population-level variation. This allows testing whether development increases the capacity to evolve, or evolvability, by facilitating and even directing morphological change. In a population sample of living seals, variation in cusp number of individual teeth is from three to five cusps, the variably present cusps being the shortest ones that also develop last. By factoring in recent evidence on development, I show that the variation in cusp number can be explained by a patterning cascade mode of cusp development that cumulatively increases and directs height variation in short cusps. The biased variation in seal tooth cusps supports the recognition of teeth as highly evolvable because only small developmental changes are needed to produce large changes in size and number of small cusps. This evolvability of tooth cusps may have facilitated the fast and independent acquisition of new cusps in mammalian evolution. In phylogenetic studies, small cusps may be unreliable as phylogenetic signals. Population level variation can be a powerful tool in testing and generating hypotheses in developmental evolution studies.

Animals↗

Comparison of chest pain, electrocardiographic changes and thallium-201 scintigraphy during varying exercise intensities in men with stable angina pectoris.

This study was performed to evaluate the presence of angina pectoris, electrocardiographic changes and reversible thallium-201 defects resulting from 2 different levels of exercise in 19 patients with known coronary artery disease and evidence of exercise-induced ischemia. The exercise protocols consisted of a symptom-limited incremental exercise test (Bruce protocol) followed within 3 to 14 days by a submaximal, steady-state exercise test performed at 70% of the maximal heart rate achieved during the Bruce protocol. The presence and time of onset of angina and electrocardiographic changes (greater than or equal to 0.1 mV ST-segment depression) as well as oxygen uptake, exercise duration and pressure-rate product were recorded. Thallium-201 (2.5 to 3.0 mCi) was injected during the last minute of exercise during both protocols, and the images were analyzed using both computer-assisted quantitation and visual interpretations. Incremental exercise resulted in anginal symptoms in 84% of patients, and electrocardiographic changes and reversible thallium-201 defects in all patients. In contrast, submaximal exercise produced anginal symptoms in only 26% (p less than 0.01) and electrocardiographic changes in only 47% (p less than 0.05), but resulted in thallium-201 defects in 89% of patients (p = not significant). The locations of the thallium-201 defects, when present, were not different between the 2 exercise protocols. These findings confirm the sequence of the ischemic cascade using 2 levels of exercise and demonstrate that the cascade theory is applicable during varying ischemic intensities in the same patient.

Angina Pectoris↗

An in-vitro technique for assessment of thrombogenicity in mechanical prosthetic cardiac valves: evaluation with a range of valve types.

BACKGROUND AND AIM OF THE STUDY: An in-vitro technique has been developed to assess the flow-induced thrombosis of artificial heart valves, using renneted milk as a blood analog. Previous studies have demonstrated similarities between the clotting of blood and milk on both microscopic and macroscopic scales. The study aim was to further validate the milk test by comparing the locations of milk clot to those of thrombus formation on a wide selection of mechanical heart valves. METHODS: Nine different valves were tested in the aortic position of a model heart chamber in the Edinburgh milk rig. These included caged-ball valves (Starr-Edwards silastic ball with bare struts and metal ball with cloth-covered struts), tilting-disc valves (Björk-Shiley Standard, Björk-Shiley Monostrut, Medtronic-Hall, and Ultracor), and bileaflet valves (St. Jude Medical, Edwards-Duromedics, and CarboMedics). Renneted milk was pumped through the valves for 30 min at 2 l/min, 70 bpm pulsatile flow. After each run, valves were photographed for comparison with documented sites of thrombosis. RESULTS: All valves developed milk clot in specific, reproducible locations when run in the aortic position. Milk clot was found on the struts of caged-ball valves and tilting-disc valves, and around the hinge mechanism of the bileaflet valves. This compared favorably to documented cases of thrombosis in vivo. CONCLUSION: Renneted milk may be used to model flow-induced thrombus formation and to predict the thrombogenic sites of mechanical heart valves. Whilst it is not suggested that milk mimics the entire blood coagulation cascade, these results indicate that such behavior may not be necessary for predicting fluid mechanically induced clotting.

Animals↗