Phenylephrine preconditioning involves modulation of cardiac sarcolemmal K(ATP) current by PKC delta, AMPK and p38 MAPK

Preconditioning of hearts with the α₁-adrenoceptor agonist phenylephrine decreases infarct size and increases the functional recovery of the heart following ischaemia-reperfusion. However, the cellular mechanisms responsible for this protection are not known. We investigated the role of protein kinase C ε and δ (PKCε and PKCδ), AMP-activated protein kinase (AMPK), p38 MAPK (p38) and sarcolemmal ATP-sensitive potassium (sarcK_ATP) channels in phenylephrine preconditioning using isolated rat ventricular myocytes. Preconditioning of ventricular myocytes with phenylephrine increased the recovery of contractile activity following metabolic inhibition and re-energisation from 30.1 ± 1.9% to 66.5 ± 5.2% (P < 0.01) and increased the peak sarcK_ATP current activated during metabolic inhibition from 32.1 ± 1.8 pA/pF to 46.0 ± 5.0 pA/pF (P < 0.05), which was required for protection. Phenylephrine preconditioning resulted in a sustained activation of PKCε and PKCδ, and transient activation of AMPK, which was dependent upon activation of PKCδ but not PKCε. P38 was also activated by phenylephrine preconditioning and this was blocked by inhibitors of PKCε, PKCδ or AMPK. Inhibition of PKCδ, AMPK or p38 was sufficient to prevent the increase in current, suggesting that these kinases are involved in modulation of sarcK_ATP channel current by phenylephrine preconditioning. However, whilst inhibition of AMPK and p38 prevented the protection from phenylephrine preconditioning, PKCδ inhibition paradoxically had no effect. The increase in sarcK_ATP current induced by phenylephrine preconditioning requires PKCδ, AMPK and p38 and may contribute to the observed improvement in contractile recovery.     

Rapid component I(Kr) of the guinea-pig cardiac delayed rectifier K(+) current is inhibited by beta(1)-adrenoreceptor activation, via cAMP/protein kinase A-dependent pathways

OBJECTIVE: The antiarrhythmic potential of betablockers contributes to their beneficial effects in the treatment of cardiac diseases, although the molecular basis of their class II antiarrhythmic action has not been clarified yet. METHODS: To investigate a putative functional link between beta-adrenoreceptors and the fast component of cardiac delayed rectifier K(+) channels (I(Kr)), whole-cell patch-clamp experiments were performed with isolated guinea-pig ventricular myocytes. Tail currents of I(Kr) were measured at -40 mV after short (200 ms) test pulses to +40 mV. RESULTS: After application of the unspecific beta-receptor agonist isoproterenol (10 microM) for 12 min, the I(Kr) tail current was decreased by 72%, with an IC(50) of 1.4 microM. The specific beta(1)-blocker CGP207120A (10 microM) significantly attenuated the isoproterenol effect (net 24% decrease). The specific beta(1)-agonist xamoterol (10 microM), could mimic the isoproterenol effect (58% decrease). Modulators of beta(2)- or beta(3)-adrenoreceptors were far less effective. When isoproterenol or xamoterol were combined with KT5720 (2.5 microM), a specific inhibitor of protein kinase A (PKA), their effects were drastically reduced, indicating that PKA presumably mediates the beta(1)-adrenergic inhibition of I(Kr). Tail current reductions by cAMP, forskolin, PKA catalytic subunit and a combination of PKA holoenzyme and cAMP support an involvement of PKA in the regulation of I(Kr). CONCLUSIONS: The functional link between I(Kr) and the beta(1)-adrenergic receptor involving PKA may play an important role in arrhythmogenesis and contribute to the antiarrhythmic action of clinically used beta(1)-blockers.     

Transcription factor 3',5'-cyclic adenosine 5'-monophosphate-responsive element-binding protein (CREB) is decreased during human adrenal cortex tumorigenesis and fetal development

Various cellular and molecular alterations of the cAMP pathway have been observed in adrenal Cushing syndrome. We recently reported the loss of cAMP-responsive element-binding protein (CREB) expression in the adrenocortical cancer cell line H295R. CREB is the major nuclear target of the cAMP pathway. This study therefore aimed to analyze the status of the CREB protein in various types of human adrenocortical tumors and normal fetal adrenal cortex. CREB protein status was studied by Western blotting in adrenocortical adenomas (AAs, n = 27) and adrenocortical carcinomas (ACs, n = 24). A decrease of CREB protein was noticed in the majority of the adrenocortical tumors. The dramatic decrease in CREB protein levels was more pronounced in ACs than in AAs. Levels of the phosphorylated form of CREB were also low in adrenocortical tumors, with a greater decrease in ACs than in AAs. EMSAs also showed decreases in the amounts of CREB- containing complexes in nuclear extracts from adrenocortical tumors. The secretory status of adenomas was strongly correlated with CREB levels, significantly lower in nonfunctioning AAs (n = 9) than in functioning AAs (n = 9). CREB levels, determined by Western blotting and immunohistochemistry, were very low in the fetal zone of human fetal adrenal cortex, whereas they were normal in the definitive zone. In tumors, adrenocortical cells in several zones were weakly immunohistochemically stained for CREB, whereas CREB was uniformly detected in nonendocrine cell nuclei (e.g. vascular cells, fibroblasts). These results suggest that the absence of CREB may be linked to the development of a highly aggressive tumor with a dedifferentiated benign (nonfunctioning AA) or malignant (AC) phenotype. These findings highlight the similarities between the normal human fetal adrenal gland and adrenal cancers previously observed in terms of parallelism in IGF-II production.     

Availability of mesalazine (5-aminosalicylic acid) from enemas and suppositories during steady-state conditions

The local and systemic bioavailability of a mesalazine enema (Pentasa, Ferring A/S, Denmark) and a mesalazine suppository (Pentasa, Ferring) was assessed during steady-state conditions. Eleven healthy subjects took 1 g of the enema or the suppository twice daily for 1 week, with a drug-free period of at least 1 week in between. At the end of each treatment period the urine and faeces were collected for 48 h, and the concentrations of mesalazine and the metabolite acetyl-mesalazine were measured. Plasma concentrations of drug and metabolite were measured hourly during a 12-h dose interval. The faecal water concentration of mesalazine was significantly higher after suppository treatment (55.7 mmol/l) compared with enema treatment (31.7 mmol/l) (p less than 0.01). The systemic absorption was low; 15% of daily mesalazine dose was recovered in urine after enema treatment and 10% after suppositores (p less than 0.01). Plasma concentrations were low, and no accumulation of either mesalazine or acetyl-mesalazine occurred. In conclusion, the enema and the suppository can be continuously administered as 1 g of mesalazine twice daily, respectively, giving high faecal water concentrations of mesalazine and a low systemic absorption.     

Resistance in vitro to low-dose aspirin is associated with platelet PlA1 (GP IIIa) polymorphism but not with C807T(GP Ia/IIa) and C-5T Kozak (GP Ibalpha) polymorphisms

OBJECTIVES: We investigated whether three platelet gene polymorphisms, Pl(A1/A2), C807T, and C-5T Kozak (encoding, respectively, for platelet membrane glycoproteins (GP) IIIa, GP Ia/IIa, GP Ibalpha), could contribute to the resistance to a low dose of aspirin (160 mg/day). BACKGROUND: Aspirin antiplatelet effect is not uniform in all patients, and the mechanism by which some patients are in vitro resistant to aspirin remains to be determined. However, it has been suggested that polymorphisms of platelet membrane glycoproteins might contribute to aspirin resistance. METHODS: Ninety-eight patients on aspirin (160 mg/day) for at least one month were enrolled. Aspirin resistance was measured by the platelet function analyzer (PFA)-100 analyzer; genotyping of the three polymorphisms was performed using a polymerase chain reaction-based restriction fragment-length polymorphism analysis. RESULTS: Using a collagen/epinephrine-coated cartridge on the PFA-100, the prevalence of aspirin resistance was 29.6% (n = 29). Aspirin-resistant patients were significantly more often Pl(A1/A1) (86.2%; n = 25) than sensitive patients (59.4%; n = 41; p = 0.01). Of the 29 patients, 25 were reevaluated after having taken 300 mg/day aspirin for at least one month. Only 11 patients still have nonprolonged collagen epinephrine closure time, and these were all Pl(A1/A1). No relation was found between resistance status and C-5T Kozak or C807T genotypes. CONCLUSIONS: Platelets homozygous for the Pl(A1) allele appear to be less sensitive to inhibitory action of low-dose aspirin. This differential sensitivity to aspirin may have potential clinical implications whereby specific antiplatelet therapy may be best tailored according to the patient's Pl(A) genotype.     

The inner ring (5-) monodeiodination of thyroxine (T4) in cerebral cortex during fetal, neonatal, and adult life

Inner ring (-5) monodeiodination of T4 was studied by incubating T4 (approximately 0.26 mumol/L) with rat cerebral cortical homogenate (approximately 4 mg protein) in the presence of dithiothreitol (up to 400 mmol/L) and quantifying the amount of the product, rT3, by a specific radioimmunoassay. The production of rT3 was dependent on duration of incubation (up to 2 hours), amount of tissue protein (up to 8 mg), temperature (optimal at 37 degrees C) and pH (optimal, 7.0) of the incubation mixture and the concentration of DTT (maximally stimulated at 400 mmol/L). The apparent Km and Vmax of the T4-inner ring monodeiodinating activity were 36 nmol/L and 1.75 pmol/mg protein/h, respectively. The activity was inhibited by T3 and 3,5-T2, but not by 3'5'-T2, PTU, methimazole, sodium salicylate, or 8-anilino-I-naphthalene sulfonic acid. Ipodate weakly inhibited T4-to-rT3 monodeiodination. Hyperthyroidism induced by T4 (100 micrograms/d IP X 3 days), T3 (80 micrograms/d IP X 3 days) or DIMIT (45 micrograms/d IP X 3 days) significantly stimulated T4-to-rT3 conversion; DIMIT was the most potent agent. Hypothyroidism inhibited T4-to-rT3 converting activity in cerebral cortex. Fasting for three days had no appreciable effect on T4-to-rT3 conversion in cerebral cortex. Cerebral cortical T4 5-deiodinase activity in the pregnant rat at term was about 50% of that in the adult nonpregnant rat, whereas that in the fetus was about three-fold higher than that in the nonpregnant adult.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) used in a naturalistic group setting is associated with unintended improvements in depression and anxiety

Background: A recent epidemiological study suggested that 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) used for spiritual and recreational reasons is associated with subjective improvement in depression and anxiety. Further exploration of the potential psychotherapeutic effects of 5-MeO-DMT could inform future clinical trials. Objectives: We examined self-reported improvement in depression and anxiety among people who use 5-MeO-DMT in a group setting with structured procedures guiding dose and administration of 5-MeO-DMT. Such procedures also include activities for the preparation of, and support during/following sessions, which are similar to procedures used in clinical trials of hallucinogen administration. Next, we examined whether depression or anxiety were improved following use, and whether the acute subjective effects (mystical/challenging) or beliefs about the 5-MeO-DMT experience were associated with improvements in these conditions. Methods: Respondents (n = 362; M_age = 47.7; Male = 55%; White/Caucasian = 84%) completed an anonymous web-based survey. Results: Of those reporting having been diagnosed with depression (41%) or anxiety (48%), most reported these conditions were improved (depression = 80%; anxiety = 79%) following 5-MeO-DMT use, and fewer reported they were unchanged (depression = 17%; anxiety = 19%) or worsened (depression = 3%; anxiety = 2%). Improvement in depression/anxiety conditions were associated with greater intensity of mystical experiences and higher ratings of the spiritual significance and personal meaning of the 5-MeO-DMT experience. There were no associations between depression or anxiety improvement and the intensity of acute challenging physical/psychological effects during the 5-MeO-DMT experience. Conclusions: Future prospective controlled clinical pharmacology studies should examine the safety and efficacy of 5-MeO-DMT administration for relieving depression and anxiety.     

Relation between aspirin dose, all-cause mortality, and bleeding in patients with recent cerebrovascular or coronary ischemic events (from the BRAVO Trial)

Despite aspirin's established role in the treatment of atherosclerotic vascular disease, considerable controversy exists regarding its most effective dosing strategy. In a retrospective observational study, we examined the relation between prescribed aspirin dose (<162 mg vs > or =162 mg/day aspirin) and clinical outcome in 4,589 placebo-treated patients enrolled in the Blockage of the Glycoprotein IIb/IIIa Receptor to Avoid Vascular Occlusion (BRAVO) trial over a median follow-up of 366 days. Standard Cox regression analysis was employed because propensity analysis was not feasible. Compared with lower aspirin doses, higher doses were associated with lower unadjusted all-cause mortality (2.9 vs 1.6%, respectively; log rank chi-square 8.6, p = 0.0034). Higher aspirin dose remained independently predictive of lower all-cause mortality in a multivariable Cox proportional hazards model (hazard ratio 0.64, 95% confidence interval 0.42 to 0.97, p = 0.037). However, there was no significant difference in the incidence of the composite endpoint death, nonfatal myocardial infarction, or nonfatal stroke (6.1% vs 6.2%, p = 0.74). Higher aspirin dose was a significant independent predictor of any (hazard ratio 1.32, 95% confidence interval 1.12 to 1.55, p = 0.001) but not serious bleeding. In conclusion, our findings suggest that aspirin doses of > or =162 mg/day may be more beneficial than those <162 mg/day at preventing death.     

Allelic loss on chromosome 4 (Lyr2/TLSR5) is associated with myeloid, B-lympho-myeloid, and lymphoid (B and T) mouse radiation-induced leukemias

The CBA/H mouse model of radiation-induced acute myeloid leukemia (AML) was re-examined using molecular approaches. In addition to the typical promyelocytic AMLs, 34% were reclassified as early pre-B lympho-myeloid leukemias (L-ML) based on leukemic blood cell morphology, immunoglobulin heavy-chain gene re-arrangements (IgH(R)), or expression of both lymphoid (Vpre-B1 and Rag1) and myeloid (myeloperoxidase and lysozyme M) genes. Allelic loss on chromosome 4 was frequently detected in AMLs (53%) and L-MLs (more than 95%), and the preferential loss of the maternally transmitted allele suggests the locus may be imprinted. A minimally deleted region (MDR) maps to a 3.4-cM interval, which is frequently deleted in radiation-induced thymic lymphomas (TLSR5) and contains a recessive, maternally transmitted genetic locus (Lyr2) that confers resistance to spontaneous and radiation-induced pre-B and T cell lymphomas, suggesting they are one and the same. Thus, the Lyr2/TLSR5 locus is frequently implicated in myeloid, lymphoid (B and T), and mixed-lineage mouse leukemias and lymphomas. Epigenetic inactivation of one Lyr2/TLSR5 allele during normal mouse development suggests that only a single hit is required for its inactivation during leukemogenesis, and this may be a significant contributing factor to the efficiency of the leukemogenic process in the mouse.     

Urinary adenosine 3',5'-monophosphate (cAMP) response to antidiuretic hormone in diabetes insipidus (DI): comparison between congenital nephrogenic DI type 1 and 2, and vasopressin sensitive DI

Urinary adenosine 3',5'-monophosphate (cAMP) excretion before and after administration of aqueous vasopressin (pitressin) and 1-deamino-8-D-arginine vasopressin (DDAVP) was measured in congenital nephrogenic and in vasopressin sensitive diabetes insipidus (VS-DI). Excretion of cAMP into the urine increased markedly in response to pitressin (676%) and to DDAVP (252%) in VS-DI. Nephrogenic diabetes insipidus (N-DI) could be divided into two categories (type 1 and type 2) in respect to urinary cAMP responsiveness. In type 1, cAMP excretion showed no definite change after stimulation with pitressin (102%) or DDAVP (127%). On the other hand, urinary excretion of cAMP was significantly elevated in response to DDAVP in familial cases of N-DI type 2 (1269%) without producing any concentrating effect on the urine. Two different defects are considered to be involved in the pathogenesis of N-DI.     

Differential regulation of interleukin (IL)-4, IL-5, and IL-10 during measles in Zambian children

To determine the effect of measles virus infection on cytokine production in children from sub-Saharan Africa, temporal changes in cytokine production in vivo were analyzed and the T cell sources of type 1 and type 2 cytokines were identified in Zambian children with measles. The immune response during measles involved early type 1 responses, with production of interferon-gamma by CD8(+) T cells and of interleukin (IL)-2 by CD4(+) T cells. Subsequently, more-prolonged increases were observed in the type 2 cytokines IL-4 and IL-13, both produced by CD4(+) T cells. IL-5 was regulated differently from IL-4 and IL-13: levels were low compared with levels in control children and were reflected in lower eosinophil counts during measles. Immunoglobulin E was lower in children with measles, despite high levels of IL-4 and IL-13. Plasma levels of IL-10 were elevated for weeks, potentially contributing to impaired cellular immunity and depressed hypersensitivity responses following measles.     

Changing concentrations of CO, CH(4), C(5)H(8), CH(3)Br, CH(3)I, and dimethyl sulfide during the Southern Ocean Iron Enrichment Experiments

Oceanic iron (Fe) fertilization experiments have advanced the understanding of how Fe regulates biological productivity and air-sea carbon dioxide (CO(2)) exchange. However, little is known about the production and consumption of halocarbons and other gases as a result of Fe addition. Besides metabolizing inorganic carbon, marine microorganisms produce and consume many other trace gases. Several of these gases, which individually impact global climate, stratospheric ozone concentration, or local photochemistry, have not been previously quantified during an Fe-enrichment experiment. We describe results for selected dissolved trace gases including methane (CH(4)), isoprene (C(5)H(8)), methyl bromide (CH(3)Br), dimethyl sulfide, and oxygen (O(2)), which increased subsequent to Fe fertilization, and the associated decreases in concentrations of carbon monoxide (CO), methyl iodide (CH(3)I), and CO(2) observed during the Southern Ocean Iron Enrichment Experiments.     

Nomenclature updates resulting from the evolution of avian influenza A(H5) virus clades 2.1.3.2a, 2.2.1, and 2.3.4 during 2013–2014

Aim

The A/goose/Guangdong/1/96-like hemagglutinin (HA) genes of highly pathogenic avian influenza (HPAI) A(H5) viruses have continued to rapidly evolve since the most recent update to the H5 clade nomenclature by the WHO/OIE/FAO H5N1 Evolution Working Group. New clades diverging beyond established boundaries need to be identified and designated accordingly.

Method

Hemagglutinin sequences deposited in publicly accessible databases up to December 31, 2014, were analyzed by phylogenetic and average pairwise distance methods to identify new clades that merit nomenclature changes.

Results

Three new clade designations were recommended based on division of clade 2·1·3·2a (Indonesia), 2·2·1 (Egypt), and 2·3·4 (widespread detection in Asia, Europe, and North America) that includes newly emergent HPAI virus subtypes H5N2, H5N3, H5N5, H5N6, and H5N8.

Conclusion

Continued global surveillance for HPAI A(H5) viruses in all host species and timely reporting of sequence data will be critical to quickly identify new clades and assess their potential impact on human and animal health.     

Decreased hepatic expression of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein in aging rats is associated with delayed clearance of chylomicrons from the circulation

Aging in both humans and rats is associated with the development of insulin resistance and the ensuing alterations in the plasma lipoprotein profile. In this study, young (2 months) and old (15 months) Sprague-Dawley (SD) rats were used to investigate age-related alterations in the chylomicron clearance pathway. Clearance from the blood of an intravenously injected bolus of 14C-labeled cholesterol ester (CE) and 3H-labeled triacylglycerol (TAG) lymph chylomicrons was markedly delayed in the old rats (P < .05). Hepatic expression of the two principal receptors of chylomicron remnant removal, the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP), was determined by ligand blotting and immunoblotting. The old rats expressed 43%+/-7% of the level of LDL receptor in the young animals (P < .05) and 45%+/-16% of the corresponding level of LRP (P < .05). The results suggest that the delayed clearance of chylomicron remnants in this animal model of aging and insulin resistance is due, at least in part, to a decrease in the hepatic expression of LDL receptor and LRP.     

Localization of K(+) channels in the tubules of cardiomyocytes as suggested by the parallel decay of membrane capacitance, IK(1) and IK(ATP) during culture and by delayed IK(1) response to barium

Adult ventricular myocytes lose T-tubules over few days in culture, which causes the loss of about 60% of the cell membrane capacitance (Cm) (Mitcheson et al., 1996). In this study, we have measured, in whole-cell voltage-clamped rabbit right ventricular myocytes at 0, 1, 2 and 3-5 days of culture (nine to 20 myocytes at each age) in a defined Dulbecco's modified Eagle's medium, the value of Cm and the magnitudes of the background inward rectifier current (IK(1)) and of the 2,4-dinitrophenol-induced ATP-sensitive potassium current (IK(ATP)). Cm, IK(1) and IK(ATP) all had decreased significantly by 51, 83 and 88%, respectively after 4 days of culture. Analysis using a single exponential decay function of time gave time constants of, 2.6+/-0.2, 2.2+/-0.5 and 2.4+/-0.4 days, respectively. Linear regressions of IK(1) and IK(ATP) versus Cm had regression coefficients of 0.93 and 0. 98, respectively. These observations are consistent with a strong link of the decay of IK(1) and IK(ATP) currents to that of Cm. Furthermore, the time course of changes in IK(1) when an external blocker (100 microm BaCl(2)) was applied and washed by local perfusion (95% change in 50 ms) agrees with a model including a diffusion time constant of 300 ms. This value is consistent with the known kinetics of diffusion of divalent cations in the T-tubules. Taken together, these results could be explained by the localization of a major part of the IK(1) and IK(ATP) currents of ventricular cardiomyocytes in the T-tubules. As a consequence, transient accumulation of K(+) ions in cardiac T-tubules may take place and modulate excitation-contraction coupling.