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The time of stimulation of support receptors in locomotor training was analyzed individually for 13 cosmonauts for each day of a long-term space flight. The time of support receptors stimulation with an intensity comparable to that under 1g conditions was shorter compared to Earth conditions. Negative correlation was found between average daily time of support stimulation and the magnitude of changes in the post-flight electromyographic response of soleus muscle to walking. It is proposed to consider the time of support receptors stimulation, along with such parameters as the axial loading and the proportion of the manual mode of treadmill belt movement, as a significant indicator for a personalized approach to the prevention of hypogravitational disorders during long-term space flights.

Ae4 transporters are critical for Cl(−) uptake across the basolateral membrane of acinar cells in the submandibular gland (SMG). Although required for fluid secretion, little is known about the physiological regulation of Ae4. To investigate whether Ae4 is regulated by the cAMP-dependent signaling pathway, we measured Cl(−)/HCO(3)(−) exchanger activity in SMG acinar cells from Ae2(−/−) mice, which only express Ae4, and found that the Ae4-mediated activity was increased in response to β-adrenergic receptor stimulation. Moreover, pretreatment with H89, an inhibitor of the cAMP-activated kinase (PKA), prevented the stimulation of Ae4 exchangers. We then expressed Ae4 in CHO-K1 cells and found that the Ae4-mediated activity was increased when Ae4 is coexpressed with the catalytic subunit of PKA (PKAc), which is constitutively active. Ae4 sequence analysis showed two potential PKA phosphorylation serine residues located at the intracellular NH(2)-terminal domain according to a homology model of Ae4. NH(2)-terminal domain Ser residues were mutated to alanine (S173A and S273A, respectively), where the Cl(−)/HCO(3)(−) exchanger activity displayed by the mutant S173A was not activated by PKA. Conversely, S273A mutant kept the PKA dependency. Together, we conclude that Ae4 is stimulated by PKA in SMG acinar cells by a mechanism that probably depends on the phosphorylation of S173. NEW & NOTEWORTHY We found that Ae4 exchanger activity in secretory salivary gland acinar cells is increased upon β-adrenergic receptor stimulation. The activation of Ae4 was prevented by H89, a nonselective PKA inhibitor. Protein sequence analysis revealed two residues (S173 and S273) that are potential targets of cAMP-dependent protein kinase (PKA). Experiments in CHO-K1 cells expressing S173A and S273A mutants showed that S173A, but not S273A, is not activated by PKA.

International audience; Background and PurposeDopamine in the striatum plays a crucial role in reward processes and action selection. Dopamine signals are transduced by D1 and D2 dopamine receptors which trigger mirror effects through the cAMP/PKA signalling cascade in D1 and D2 medium-sized spiny neurones (MSNs). Phosphodiesterases (PDEs), which determine the profile of cAMP signals, are highly expressed in MSNs, but their respective roles in dopamine signal integration remain poorly understood.Experimental approachWe used genetically-encoded FRET biosensors to monitor at the single cell level the functional contribution of PDE2A, PDE4 and PDE10A in the changes of the cAMP/PKA response to transient and continuous dopamine in mouse striatal brain slices.Key ResultsWe found that PDE2A, PDE4 and PDE10A operate on the moderate to high cAMP levels elicited by D1 or A2A receptor stimulation. In contrast, only PDE10A is able to reduce cAMP down to baseline in both type of neurones, leading to the dephosphorylation of PKA substrates.Conclusion and ImplicationsIn both MSN types, PDE10A inhibition blunts the responsiveness to dopamine, whereas PDE2A or PDE4 inhibition reinforces dopamine action.

Cell-bound ligands are often viewed as moving passively in response to displacement of their cognate receptors. Verron et al provide an example of the distribution of ligands influencing the functional outcome of receptor stimulation.

The majority of colon adenomas harbor genetic mutations in the APC gene. APC mutation leads to changes in Wnt signalling and cell-cell adhesion: as a consequence, intestinal crypt budding increases and the excess crypts accumulate to form adenomas that progress to colon cancer. When cultured with Wnt, R-spondin, EGF, Noggin, myofibroblast conditioned medium and Matrigel, crypts from normal mouse colon mucosa form crypt-producing organoids and can be passaged continuously. Under the same culture and passage conditions, crypts isolated from colon adenomas derived from Apcmin/+ mice typically grow as spheroidal cysts and do not produce crypts. The adenoma organoid growth requires EGF, but not Wnt, R-spondin or Noggin. However, when mouse colon adenoma spheroids are grown for more than 10 days in the presence of EGF, crypt formation occurs. EGF, EREG, β-cellulin, Neuregulin-1 or AREG are sufficient for initiating crypt formation, however, neuregulin-1 is more potent than the other EGF-family members. EGFR and ErbB2 inhibitors both prevent crypt formation in adenoma cultures. Either EGFR:ErbB2 or ErbB3:ErbB2 signalling is sufficient to initiate adenoma crypt budding and elongation. ErbB2 inhibitors may provide a therapeutic avenue for controlling and ablating colon adenomas.

Dopamine in the striatum plays a crucial role in reward processes and action selection. Dopamine signals are transduced by D1 and D2 dopamine receptors which trigger mirror effects through the cAMP/PKA signalling cascade in D1 and D2 medium-sized spiny neurones (MSNs). Phosphodiesterases (PDEs), which determine the profile of cAMP signals, are highly expressed in MSNs, but their respective roles in dopamine signal integration remain poorly understood. We used genetically-encoded FRET biosensors to monitor at the single cell level the functional contribution of PDE2A, PDE4 and PDE10A in the changes of the cAMP/PKA response to transient and continuous dopamine in mouse striatal brain slices. We found that PDE2A, PDE4 and PDE10A operate on the moderate to high cAMP levels elicited by D1 or A2A receptor stimulation. In contrast, only PDE10A is able to reduce cAMP down to baseline in both type of neurones, leading to the dephosphorylation of PKA substrates. PDE10A is therefore critically required for dopamine signal integration in both D1 and D2 MSNs.

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is one of the most important transcription factors involved in the regulation of inflammatory signaling pathways. Inappropriate activation of these pathways has been linked to autoimmunity and cancers. Emerging experimental evidences have been showing the existence of elaborate spatial organizations for various molecular components in the pathways. One example is the scaffold protein tumor necrosis factor receptor associated factor (TRAF). While most TRAF proteins form trimeric quaternary structure through their coiled-coil regions, the N-terminal region of some members in the family can further be dimerized. This dimerization of TRAF trimers can drive them into higher-order clusters as a response to receptor stimulation, which functions as a spatial platform to mediate the downstream poly-ubiquitination. However, the molecular mechanism underlying the TRAF protein clustering and its functional impacts are not well-understood. In this article, we developed a hybrid simulation method to tackle this problem. The assembly of TRAF-based signaling platform at the membrane-proximal region is modeled with spatial resolution, while the dynamics of downstream signaling network, including the negative feedbacks through various signaling inhibitors, is simulated as stochastic chemical reactions. These two algorithms are further synchronized under a multiscale simulation framework. Using this computational model, we illustrated that the formation of TRAF signaling platform can trigger an oscillatory NF-κB response. We further demonstrated that the temporal patterns of downstream signal oscillations are closely regulated by the spatial factors of TRAF clustering, such as the geometry and energy of dimerization between TRAF trimers. In general, our study sheds light on the basic mechanism of NF-κB signaling pathway and highlights the functional importance of spatial regulation within the pathway. The simulation framework also showcases its potential of application to other signaling pathways in cells.

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule- organizing centre (MTOC) and the polarization oftheMTOCtotheimmunesynapse(IS).Theseevents lead to exosome secretion at the IS. We describe herethat upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1βphosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1βphosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization;nonetheless, FMNL1βre-expres- sion restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδregulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1βregulation and controlling F-actin reorganiza- tion at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization., This work was supported by the Ministerio de Ciencia, Innovación y Universidades [SAF2016-77561-R]; Ministerio de Economía y Competitividad [SAF2016-77561-R].

The β-adrenergic receptor stimulation improves endurance in fast twitch muscles and these effects are sensitive to extracellular Ca2+ influx. Present study is aimed to determine the effects of adrenaline, with different concentrations of extracellular Ca2+ $$\left( {{\text{Ca}}_{\text{ECF}}^{ 2+ } } \right)$$ , on the contractility and endurance of slow twitch muscles during high frequency stimulations (HFS). Isolated soleus of rabbit was electrically stimulated (strength; 50 Hz, duration; 0.5 ms) in the presence (Test) of adrenaline (1 × 10−7 mM) or without adrenaline (CTL). Fatigue was induced with HFS (80 Hz) for the duration of 20 s. Contractions were recorded through isometric transducer connected with Powerlab. Kreb’s buffer was used with three compositions: standard with 2.5 mM Ca2+ (Ca-S), Ca2+ free buffer (Ca-F) and buffer with raised Ca2+ i.e., 10 mM (Ca-R). Muscles endurance was assessed by measuring the decline in tetanic tension in the terms of percentage (%Pmax) and rate of decline in tetanic tension (dP/dt). During 20 s, %Pmax showed reduction of only 10% in Ca-S. This decline was enhanced in Ca-F (50%) and reduced in Ca-R (6%). Effect of adrenaline was observed only in Ca-F where %Pmax was about 20% greater in Test than CTL. These effects were not observed in both Ca-S and Ca-R during 20 s. However, when duration of stimulation was increased to 120 or 150 s in Ca-S and Ca-R respectively, decline in %Pmax was less in Test as compared to CTL. Thus, $${\text{Ca}}_{\text{ECF}}^{ 2+ }$$ plays protective role against fatigue during continuous HFS in slow twitch muscles. In addition, adrenaline improves the muscles endurance during fatiguing contraction but these effects are not mediated through $${\text{Ca}}_{\text{ECF}}^{ 2+ }$$ influx.

Facial expressions are in reaction to basic tastes by the response to receptor stimulation. The objective of this study was to assess the autonomic nervous system responses to basic tastes in chocolates and to identify relationships between conscious and unconscious responses from participants. Panelists (n = 45) tasted five chocolates with either salt, citric acid, sugar, or monosodium glutamate, which generated four distinctive basic tastes plus bitter, using dark chocolate. An integrated camera system, coupled with the Bio-Sensory application, was used to capture infrared thermal images, videos, and sensory responses. Outputs were used to assess skin temperature (ST), facial expressions, and heart rate (HR) as physiological responses. Sensory responses and emotions elicited during the chocolate tasting were evaluated using the application. Results showed that the most liked was sweet chocolate (9.01), while the least liked was salty chocolate (3.61). There were significant differences for overall liking (p &lt, 0.05) but none for HR (p = 0.75) and ST (p = 0.27). Sweet chocolate was inversely associated with angry, and salty chocolate positively associated with sad. Positive emotion-terms were associated with sweet samples and liking in self-reported responses. Findings of this study may be used to assess novel tastes of chocolate in the industry based on conscious and emotional responses more objectively.

The majority of research has indicated a role for serotonin in inhibiting subsequent eating behavior. However, the effect of serotonergic activity on feeding is moderated by the specific serotonin receptor being stimulated, the location of receptor stimulation, the nutritional state of the animal, and other environmental factors. The current chapter discusses the effects of serotonergic activity on feeding under each of these moderating conditions as well as the clinical implications of these findings.

International audience; Quick responses to fast changes in the environment are crucial in animal behaviour and survival,for example to seize prey, escape predators, or negotiate obstacles. Here, we study the ‘simpleresponse time’ that is the time elapsed between receptor stimulation and motor activation astypically shown in escape responses, for mobile organisms of various taxa ranging from bacteria tolarge vertebrates. We show that 95% of these simple response times lie within one order ofmagnitude of the overall geometric mean of about 25 ms, which is similar to that of a well-studiedsensory time scale, the inverse of the critical flicker fusion frequency in vision, also lying withinclose bounds for all the organisms studied. We find that this time scale is a few times smaller thanthe minimum time to move by one body length, which is known to lie also within a relativelynarrow range for all moving organisms. The remarkably small 102-fold range of the simpleresponse time among so disparate life forms varying over 1020-fold in body mass suggests that it isdetermined by basic physicochemical constraints, independently on the structure and scale of theorganism. We thus propose first-principle estimates of the simple response and sensory time scalesin terms of physical constants and a few basic biological properties common to mobile organismsand constraining their responses.

Whether the treatment of Parkinson's disease has to be initiated with levodopa or a D2 agonist like pramipexole remains debatable. Levodopa is more potent against symptoms than D2 agonists, but D2 agonists are less prone to induce motor complications and may have neuroprotective effects. Although regulation of plastic changes in striatal circuits may be the key to their different therapeutic potential, the gene expression patterns induced by de novo treatments with levodopa or D2 agonists are currently unknown. By studying the whole striatal transcriptome in a rodent model of early stage Parkinson's disease, we have identified the gene expression patterns underlying therapeutically comparable chronic treatments with levodopa or pramipexole. Despite the overall relatively small size of mRNA expression changes at the level of individual transcripts, our data show a robust and complete segregation of the transcript expression patterns induced by both treatments. Moreover, transcripts related to oxidative metabolism and mitochondrial function were enriched in levodopa-treated compared to vehicle-treated and pramipexole-treated animals, whereas transcripts related to olfactory transduction pathways were enriched in both treatment groups compared to vehicle-treated animals. Thus, our data reveal the plasticity of genetic striatal networks possibly contributing to the therapeutic effects of the most common initial treatments for Parkinson's disease, suggesting a role for oxidative stress in the long term complications induced by levodopa and identifying previously overlooked signaling cascades as potentially new therapeutic targets. Fil: Taravini, Irene Rita Eloisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Larramendy, Celia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Gomez, Gimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Saborido, Mariano Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentina Fil: Spaans, Floor. University of Groningen; Países Bajos Fil: Fresno Rodríguez, Cristóbal. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: González, Germán A.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; Argentina Fil: Fernandez, Elmer Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Area de Cs. Agrarias, Ingeniería, Cs. Biológicas y de la Salud de la Universidad Catolica de Córdoba; Argentina Fil: Murer, Mario Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina Fil: Gershanik, Oscar Samuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; Argentina

The helicase DHX9 is required for production of the cytokine IL-6 in response to Toll-like receptor stimulation.

Objective: Accumulating evidences and previous our research suggest that angiotensin II type 2 (AT 2 ) receptor stimulation could contribute to protection against ischemic brain damage. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and reported that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on ischemic brain damage is still unclear. Therefore, we investigated the effects of the ATIP and compound 21 (C21), a selective non-peptidic AT2 receptor agonist, on focal cerebral ischemia. Method: Ten-week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to middle cerebral artery occlusion (MCAO) with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCAO. Twenty-four hours after MCAO, ischemic area was determined. Cerebral blood flow (CBF) before and after MCAO was measured by laser speckle flowmetry. Collateral circulation was evaluated by the perfusion of India ink. Expression of mRNA was determined by real-time RT-PCR. Results: There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size in both strains. Interestingly, this protective effect of C21 was more marked in ATIP-Tg compared with WT mice. In CBF of core region of ischemic area, there were no significant differences among all groups. However, the reduction of CBF in penumbra region just after MCA occlusion was attenuated in ATIP-Tg mice with C21 administration. Treatment with C21 tended to increase the cerebral collateral number before MCA occlusion in ATIP-Tg mice. Expression of vascular endothelial growth factor (VEGF) mRNA in the cortex before MCA occlusion did not differ among all groups. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the attenuation of CBF reduction and increase of MMS2 expression after ischemia.

Objective: We previously reported that the ischemic brain area was significantly larger in angiotensin II type 2 (AT 2 ) receptor-deficient mice after middle cerebral artery (MCA) occlusion compared to wild-type mice. We have cloned ATIP (AT 2 receptor interacting protein) as a protein interacting specifically with the C-terminal tail of the AT 2 receptor, and suggest that ATIP might play key roles in diverse mechanisms of AT 2 receptor signaling. However, the effect of ATIP on brain damage has not been clarified. We investigated the possibility that ATIP could enhance the protective effects of compound 21 (C21), a selective direct non-peptidic AT 2 receptor agonist, on focal cerebral ischemia. Method: Ten week-old male ATIP-transgenic (ATIP-Tg) and littermate (WT) mice were subjected to permanent MCA occlusion with silicon-coated micro-filament. C21 (10 μg/kg/day) was administered 2 weeks before MCA occlusion. Twenty-four hours after MCA occlusion, ischemic area and neurological deficit was assessed. Cerebral blood flow (CBF) was measured by laser speckle flowmetry. Expression of methyl methanesulfonate sensitive 2 (MMS2) as a neuroprotective factor were measured by real-time RT-PCR. Collateral circulation was evaluated by the perfusion of India ink. Results: Systolic blood pressure did not differ between ATIP-Tg and WT mice with or without C21. There was no significant difference in ischemic size without C21 treatment between two strains. Treatment with C21 decreased ischemic size and improved neurological deficit in both strains. These protective effects by C21 were more marked in ATIP-Tg mice compared with WT mice. Treatment of C21 did not affect CBF in the core region of ischemic area after MCA occlusion in both strains; however, the reduction of CBF in penumbra region was markedly attenuated in ATIP-Tg mice treated with C21. MMS2 expression increased in ipsilateral hemisphere of ATIP-Tg mice compared with contralateral hemisphere. C21 treatment tended to increase collateral number in ATIP-Tg mice. Conclusions: These results suggested that ATIP could enhance the cerebral protective effects of AT 2 receptor stimulation at least in part due to the improvement of CBF and increase of MMS2 expression.

Timing performance maintained under the free-operant psychophysical procedure (FOPP) is sensitive to 5-hydroxytryptamine (5-HT)1A and 5-HT2A receptor stimulation. Agonists of these receptors displace the psychometric function towards shorter durations, reducing , the index of central tendency of timing. Here we report the effects of Ro-600175, a selective 5-HT2C receptor agonist, and mCPP, a 5-HT receptor agonist with high affinity for 5-HT2C receptors and lower affinity for 5-HT1A, 5-HT1B and 5-HT2A receptors, on timing behaviour. Rats were trained under the FOPP to press two levers (A and B) in 50-s trials in which reinforcers were provided intermittently for responding on A in the first half, and B in the second half of the trial. Percent responding on B (%B) was recorded in successive 5-s epochs of the trials; logistic psychometric curves were fitted to the data from each rat under each treatment condition for derivation of timing indices [ (time corresponding to %B = 50%), Weber fraction]. The rats received systemic treatment with Ro-600175, mCPP, and mCPP in combination with antagonists of 5-HT1A (WAY-100635), 5-HT1B (isamoltane), 5-HT2A (MDL-100907) or 5-HT2C (SB-242084) receptors. mCPP (2.5 mg kg−1 i.p.), but not Ro-600175 (1, 2, 4 mg kg−1 i.p.), reduced . SB-242084 (0.6 mg kg−1 i.p.) potentiated mCPP’s effect on . mCPP’s effect on was not altered by isamoltane (8.0 mg kg−1 i.p.), but was attenuated by MDL-100907 (1.0 mg kg−1 i.p.) and WAY-100635 (0.1 mg kg−1 s.c.). The results suggest that mCPP’s effect on timing is mediated by an agonistic action at 5-HT1A and 5-HT2A, but not 5-HT1B, receptors. The role of 5-HT2C receptors is unclear, in view of SB-242084’s ability to potentiate the effect of mCPP, while Ro-600175 had no effect on . The possibility is considered that 5-HT2C receptors may counteract 5-HT1A and/or 5-HT2A receptor-mediated effects on timing performance.

This presentation will cover the methods used to investigate neuronal circuitries between peripheral receptors and skeletal muscles in human subjects. There are a number of problems regarding reflex studies using experimental animals. There are also problems in the recording and analysis aspects of these experiments. To overcome these problems we have utilized precisely-controlled mechanical or electrical stimuli to activate receptors and single motor units from human muscles. We also used classical and novel methods to analyze the results to indicate neuronal networks.

Pera T, Atmaj C, van der Vegt M, Halayko AJ, Zaagsma J, Meurs H. Role for TAK1 in cigarette smoke-induced proinflammatory signaling and IL-8 release by human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 303: L272-L278, 2012. First published April 20, 2012; doi:10.1152/ajplung.00291.2011.-Chronic obstructive pulmonary disease (COPD) is an inflammatory disease, characterized by a progressive decline in lung function. Airway smooth muscle (ASM) mass may be increased in COPD, contributing to airflow limitation and proinflammatory cytokine production. Cigarette smoke (CS), the major risk factor of COPD, causes ASM cell proliferation, as well as interleukin-8 (IL-8)-induced neutrophilia. In various cell types, transforming growth factor-beta-activated kinase 1 (TAK1) plays a crucial role in MAP kinase and NF-kappa B activation, as well as IL-8 release induced by IL-1 beta, TNF-alpha, and lipopolysaccharide. The role of TAK1 in CS-induced IL-8 release is not known. The aim of this study was to investigate the role of TAK1 in CS-induced NF-kappa B and MAP kinase signaling and IL-8 release by human ASM cells. Stimulation of these cells with CS extract (CSE) increased IL-8 release and ERK-1/2 phosphorylation, as well as I kappa-B alpha degradation and p65 NF-kappa B subunit phosphorylation. CSE-induced ERK-1/2 phosphorylation and I kappa-B alpha degradation were both inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). Similarly, expression of dominant-negative TAK1 inhibited CSE-induced ERK-1/2 phosphorylation. In addition, inhibitors of TAK1 and the NF-kappa B (SC-514; 50 mu M) and ERK-1/2 (U-0126; 3 mu M) signaling inhibited the CSE-induced IL-8 release by ASM cells. These data indicate that TAK1 plays a major role in CSE-induced ERK-1/2 and NF-kappa B signaling and in IL-8 release by human ASM cells. Furthermore, they identify TAK1 as a novel target for the inhibition of CS-induced inflammatory responses involved in the development and progression of COPD.

We investigated whether the PTPN22 C1858T polymorphism is associated with the autoimmune conditions present in the family of a child affected by type 1 diabetes (T1D) carrying the TT genotype (index patient) and the potential immunological effect of the variant. We found that nine family members carried the CT genotype and five suffered from autoimmunity. Interestingly, anti-ZnT8 antibodies were detected in T1D patients and in three healthy relatives. In the TT patient, we showed diminished T-cell proliferation and reduced interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production. A marked reduction of IL-2 was also observed for all CT relatives with autoimmunity and a lack of IFN-γ production was observed for the younger brother of the index patient, heterozygous for the polymorphism. In this family, the C1858T variant might confer a high risk of autoimmunity. Moreover, our data confirm that impaired IL-2 production upon T-cell receptor stimulation is associated with autoimmunity in the carriers of the polymorphism. This study might prompt to extend the panel of risk markers in relatives of subjects affected by T1D.

Baarsma HA, Meurs H, Halayko AJ, Menzen MH, Schmidt M, Kerstjens HA, Gosens R. Glycogen synthase kinase-3 regulates cigarette smoke extract- and IL-1 beta-induced cytokine secretion by airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 300: L910-L919, 2011. First published March 18, 2011; doi:10.1152/ajplung.00232.2010.-Glycogen synthase kinase-3 (GSK-3) is a constitutively active kinase that regulates multiple signaling proteins and transcription factors involved in inflammation. Its role in inflammatory lung diseases, including chronic obstructive pulmonary disease (COPD), is largely unknown. We investigated the role of GSK-3 in the secretion of chemokines and growth factors by human airway smooth muscle cells after exposure to cigarette smoke extract (CSE) or interleukin-1 beta (IL-1 beta), important factors involved in the development of COPD. Cultured human airway smooth muscle cells were exposed to increasing concentrations of CSE (1-15%) and IL-1 beta (0.01-1.0 ng/ml), which induced the secretion of VEGF-A and IL-8, whereas eotaxin secretion was induced by IL-1 beta only. Inhibition of GSK-3 by the selective inhibitor SB216763 or CHIR/CT99021 attenuated the cytokine and growth factor release induced by CSE and/or IL-1 beta, without affecting the basal release. Secretion of the cytokines by airway smooth muscle partially depends on NF-kappa B signaling, and GSK-3 has been implicated in regulating multiple steps in activating the NF-kappa B signaling pathway. IL-1 beta treatment induced degradation of the NF-kappa B inhibitory protein I kappa-B alpha followed by nuclear translocation and DNA binding of p65 NF-kappa B, which were unaffected by inhibition of GSK-3. However, induction of NF-kappa B-dependent transcriptional activity by IL-1 beta and CSE was largely reduced upon GSK-3 inhibition by SB216763. Collectively, we demonstrate that CSE and IL-1 beta activate airway smooth muscle cells to secrete the proinflammatory cytokines IL-8, eotaxin, and VEGF-A. Furthermore, we show that GSK-3 regulates the release of these cytokines induced by CSE and IL-1 beta by promoting NF-kappa B-dependent gene transcription.

DL-Metatyrosine (200 mg/kg intraperitoneally) administration after peripheral DOPA decarboxylase inhibition caused a temporary reversal of reserpine-induced (5 mg/kg intraperitoneally) suppression of a conditioned avoidance response in the rat, both with and without pretreatment with a dopamine-β-hydroxylase inhibitor, bis-(4-methyl-1-homopiperazinylthiocarbonyl)-disulfide, (FLA-63, 10 mg/kg intraperitoneally); but the duration of the reversal was shorter after FLA-63. After the injection of DL-metatyrosine there was a rapid and marked accumulation of metatyramine and a smaller accumulation of the β-hydroxylated metabolite, metaoctopamine. FLA-63 pretreatment prevented the metaoctopamine accumulation. Some of the behavioural effects observed after DL-metatyrosine may be due to displacement of endogenous noradrenaline not depleted by reserpine. The increase in locomotor activity after DL-metatyrosine was not affected by the dopamine-β-hydroxylase inhibitor. The results provide further support for the hypothesis that dopamine is important for elementary motor functions (e.g. locomotor activity) whilst additional noradrenaline receptor stimulation is essential for more complex and integrated hebaviour (e.g. a conditioned avoidance response).

The physical mechanisms responsible for cupulolithiasis and canalolithiasis have been investigated by two groups of experiments in isolated posterior semicircular canal (SCC) of frog (Rana esculenta L.). First, clouds of 10-30 isolated otoconia were let to fall (snowfall of otoconia) either through the ampulla onto the cupula, or inside the long arm of the canal, opposite to the cupula. Second, microspheres ranging 30 to 350 μm in diameter were gently moved to and fro inside the long arm of the canal by a micromanipulator. The effects were evaluated by recording the firing rate (Nfr) of the ampullary nerve. Snowfall of otoconia produced detectable changes of Nfr only when otoconia got in contact with the cupula, but not when falling through the endolymph. Movement of the microspheres in the canal long arm induced Nfr changes only if the microsphere diameter exceeded about 50 μm. Although the exact microsphere size needed for receptor stimulation may depend on the experimental conditions, these results strongly suggest that debris moving inside a SCC (canalolithiasis) can produce transcupular pressures able to stimulate ampullar receptors only if they have suitable size, whereas isolated otoconia cannot, except when lying on the cupula (cupulolithiasis).

Temporal differentiation of operant behaviour is sensitive to dopaminergic manipulations. Previous studies using the fixed-interval peak procedure implicated D(2)-like dopamine receptors in these effects. However, recent findings suggest that d-amphetamine alters timing performance on the free-operant psychophysical procedure via D(1)-like receptors. It is not known whether this effect of d-amphetamine is mimicked by direct D(1)-like receptor stimulation.The effects of a D(1)-like receptor agonist 6-chloro-2,3,4,5-tetrahydro-1-phenyl-1H-3-benzazepine (SKF-81297) on performance on the free-operant psychophysical procedure and the interaction between SKF-81297 and a D(1)-like receptor antagonist 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol (SKF-83566) and a D(2)-like receptor antagonist haloperidol, were examined.Rats were trained to respond on two levers (A and B) under a free-operant psychophysical schedule, in which sucrose reinforcement was provided intermittently for responding on A during the first half and on B during the second half of 50-s trials. Logistic psychometric functions were fitted to the relative response rate data (percent responding on B [%B] vs time from trial onset [t]) under each treatment condition, and quantitative indices of timing (T(50) [value of t corresponding to %B = 50] and the Weber fraction [(T(75)-T(25))/2T(50); T(25) and T(75) are values of t corresponding to %B = 25 and %B = 75] were compared among treatments.SKF-81297 (0.8 mg kg(-1)) reduced T(50); this effect was antagonized by SKF-83566 (0.03 mg kg(-1)) but not by haloperidol (0.05, 0.1 mg kg(-1)).Stimulation of D(1)-like dopamine receptors affects performance in the free-operant psychophysical procedure.

The specificity of cellular responses to receptor stimulation is encoded by the spatial and temporal dynamics of downstream signalling networks. Computational models provide insights into the intricate relationships between stimuli and responses and reveal mechanisms that enable networks to amplify signals, reduce noise and generate discontinuous bistable dynamics or oscillations. These temporal dynamics are coupled to precipitous spatial gradients of signalling activities, which guide pivotal intracellular processes, but also necessitate mechanisms to facilitate signal propagation across a cell.

We used various orthostatic stimulus combinations to better understand the physiology and countermeasure potential of repeated change of body position in humans. The purpose of the investigations reported was threefold: To investigate cardiovascular and hormonal effects of repeated transition between partially antiorthostatic (-30 degrees HDT) and partially head-up passive body tilt (+30 degrees HUT). Protocol Y denotes the repeated transition between these two body positions; To apply, in the same test persons, repeated transition between supine and passive upright (Protocol X), and to compare the effect of the two protocols; To find out which stimulus pattern provides the largest physiological effects and, hence, presumably the largest countermeasure potential. We chose our tilt protocol according to tilt angle sine ranges: The sine difference is 1.0 both in Protocol X (sine=0 vs. sine=1.0) and Y (sine= -0.5 vs. sine= +0.5) since this difference, and not the angle change per se, determines hydrostatic effect intensities. Due to longer-lasting neurohormonal effects elicited by tilting procedures, they all should be a useful countermeasure against post-immobilization orthostatic instability, a conjecture not yet been tested in this specific form. Therefore, one of the questions asked in this study were if movement between the two defined body positions produces similar changes when employing Protocol X vs. Protocol Y.

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca 2 + overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after /3-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat (V O 2 ) intercept, which is composed of V O 2 for the total Ca 2 + handling in excitation-contraction coupling and basal metabolism, of the V O 2 and PVA linear relation was also significantly (P < 0.05) decreased (n = 8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, α-fodrin was identified (n = 7 of the 8 hearts with the decreased V O 2 intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-/3-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca 2 + handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, α-fodrin.

The sensitivity of the Cl−/HCO3−exchanger to dopamine D1- and D2-like receptor stimulation in immortalized renal proximal tubular epithelial cells from the spontaneous hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) was examined. The activity of the Cl−/HCO3−exchanger (in pH U/s) in SHR cells (0.00191) was greater than in WKY cells (0.00126). The activity of Cl−/HCO3−exchanger was exclusively observed at the apical cell side and probably occurs through the SLC26A6 anion transporter that is expressed in both WKY and SHR cells. Stimulation of D1-like receptors with SKF-38393 markedly attenuated the HCO3−-dependent intracellular pH recovery in WKY cells but not in SHR cells. Stimulation of D2-like receptors with quinerolane did not alter Cl−/HCO3−exchanger activity in both WKY and SHR cells. The selective D1-like receptor antagonist SKF-83566 prevented the effect of SKF-38393. Both WKY and SHR cells responded to dibutyryl-cAMP (DBcAMP) with inhibition of the Cl−/HCO3−exchanger, and downregulation of PKA (overnight exposure to DBcAMP) abolished the inhibitory effect of both DBcAMP and SKF-38393 in WKY cells. Both SHR and WKY cells responded to forskolin with increases in the formation of cAMP. However, only WKY responded to SKF-38393 with increases in the formation of cAMP that was prevented by SKF-83566. It is concluded that WKY cells respond to D1-like dopamine receptor stimulation with inhibition of the apical Cl−/HCO3−(SLC26A6) exchanger and SHR cells have a defective D1-like dopamine response.

The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5′-triphosphate, on Cl– transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 µM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl–, the Isc induced by 10 µM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na+, the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y2 antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y1 antagonist pyridoxal-phosphate-6-azophenyl-2′4′-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl– secretion from the serosal to the mucosal side via stimulation of P2Y2 receptors in the rabbit conjunctiva.

Objectives: Stimulating central neurokinin type-3 (NK-3) receptors decreases ethanol intake in rats. Although paraventricular nucleus of the hypothalamus (PVN) has a high density of NK-3 receptors, their influence on ethanol reinforcement has not been examined. This study's purpose was to assess the effects of intra-PVN infusion of senktide, a NK-3 receptor agonist, on ethanol self-administration. In a follow-up study, senktide's effects on ethanol self-administration after intracerebroventricular (ICV) infusion were examined. Methods: Male Wistar rats were trained to self-administer 10% ethanol (10E) in the “Sipper Tube” model described by Samson and colleagues, Guide cannula were then aimed bilaterally at the PVN or unilaterally at the lateral cerebral ventricle. Intra-PVN (5–100 ng/side) or ICV (30–500 ng/rat) effects of senktide on 10E self-administration were also examined as a preliminary test of senktide's selectivity. Results: Intra-PVN and ICV infusion of senktide reduced the average number of consecutive lever presses and increased the time taken to complete the lever press requirement when 10E served as the reinforcer. Increased duration of the lever-pressing component was observed when senktide was administered prior to 2S self-administration sessions. Neither PVN nor ICV senktide administration significantly altered 10E or 2S consumption. Conclusions: These data suggest that stimulation of central neurokinin typ-3 receptors in the Wistar rat reduces appetitive behavior while having little or no impact on consummatory behavior. Ethanol “seeking” appeared more sensitive to disruption by senktide than sucrose “seeking.” However, further studies assessing the senktide's effects on sucrose-maintained behavior are needed to verify this hypothesis. Lastly, it is hypothesized that lack of effect of senktide on intake is in part related to the use of outbred Wistar rats in these studies instead of selectively bred rats.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a cancer therapeutic target. However, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL-targeted therapies. We show here that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through two signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 (DR5) and receptor-interacting protein 1 (RIP1), forms a plasma membrane bound preligand assembly complex (PLAC) under physiologic conditions. TRAIL treatment leads to the recruitment of caspase-8 to the PLAC for the assembly of a death-inducing signaling complex (DISC). In the DISC, the C-terminal Zinc finger (Znf) domain of A20 ubiquitin ligase mediates RIP1 ubiquitination through lysine (K)-63-linked polyubiquitin chains that bind the protease domain of caspase-8 and inhibits its dimerization, cleavage and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells.

Recently attempts have been made to analyse blood pressure (BP) and renal sympathetic nerve activity (RSNA) to determine how patterns contained within them might reflect control by the autonomic nervous system. To date, these studies have primarily used coherence analysis of BP and RSNA in the frequency domain. However, this analysis is unable to assess the non-linear properties of the underlying cardiovascular control system. In this study we employed not only coherence analysis but also cross-entropy analysis using balloon inflation ('balloon-on') to assess the influence of right atrial receptors on the relationship between BP and RSNA under two conditions in anaesthetised Wistar rats. Balloon-on stimulation alone inhibited RSNA by 28 +/- 4% in eight rats without changing BP. This effect on integrated nerve activity was not present when atrial stimulation was applied during stimulation of a site in the paraventricular nucleus (PVN) of the hypothalamus which increased RSNA by 158.7 +/- 58% and increased BP by 17.1 +/- 2.3 mmHg. However, the cross-entropy measurement was significantly decreased (P0.05) during balloon-on stimulation in both the conditions revealing that there is greater synchrony between the oscillating signals contained within the BP and RSNA time series. Thus during the enhanced RSNA elicited by stimulation of the PVN, the inhibitory influence of atrial receptors, although apparently blocked, was still effective in that it resulted in the energy of the RSNA spectrum becoming more evenly distributed over a range of frequencies. The data show that cross-entropy calculations are able to characterize the non-linearities of underlying cardiovascular control.

The normal heart beat intervals are neither strictly stationary nor completely random, and continuously shift from one period to another. Decoding the ECG identifies this “hidden” information that imparts inherent complexity to the heart-beating interval time series. Loss of this complexity in cardiovascular disease is manifested as a reduction in Heart Rate Variability (HRV) and this reduction correlates with an increase in both morbidity and mortality. Because HRV measurements are noninvasive and easy to perform, they have emerged as an important tool in cardiology. However, the identities of specific mechanisms that underline the changes in HRV that occur in cardiovascular diseases remain largely unknown. Changes in HRV have mainly been interpreted on a neural basis, i.e. due to changes in autonomic impulses to the heart: sympathetic activity decreases both the average heart beat interval and HRV, and parasympathetic activity increases both. It has now become clear, however, that the heart rate and HRV are also determined by intrinsic properties of the pacemaker cells that comprise the sinoatrial node, and the responses of these properties to autonomic receptor stimulation. Here we review how changes in the properties of coupled-clock mechanisms intrinsic to pacemaker cells that comprise the sinoatrial node and their impaired response to autonomic receptor stimulation are implicated in the changes of HRV observed in heart diseases.

The adapter protein RIP plays a crucial role in NF-kappaB activation by TNF. Here we show that triggering of the p55 TNF receptor induces binding of RIP to NEMO (IKKgamma), a component of the I-kappa-B-kinase (IKK) "signalosome" complex, as well as recruitment of RIP to the receptor together with the three major signalosome components, NEMO, IKK1 and IKK2, and some kind of covalent modification of the recruited RIP molecules. It also induces binding of NEMO to the signaling inhibitor A20, and recruitment of A20 to the receptor. Enforced expression of NEMO in cells revealed that NEMO can both promote and block NF-kappaB activation and dramatically augments the phosphorylation of c-Jun. The findings suggest that the signaling activities of the IKK signalosome are regulated through binding of NEMO to RIP and A20 within the p55 TNF receptor complex.