PDE1 or PDE5 inhibition augments NO-dependent hypoxic constriction of porcine coronary artery via elevating inosine 3',5'-cyclic monophosphate level.

Hypoxic coronary vasospasm may lead to myocardial ischaemia and cardiac dysfunction. Inosine 3',5'-cyclic monophosphate (cIMP) is a putative second messenger to mediate this pathological process. Nevertheless, it remains unclear as to whether levels of cIMP can be regulated in living tissue such as coronary artery and if so, what is the consequence of this regulation on hypoxia-induced vasoconstriction. In the present study, we found that cIMP was a key determinant of hypoxia-induced constriction but not that of the subsequent relaxation response in porcine coronary arteries. Subsequently, coronary arteries were treated with various phosphodiesterase (PDE) inhibitors to identify PDE types that are capable of regulating cIMP levels. We found that inhibition of PDE1 and PDE5 substantially elevated cIMP content in endothelium-denuded coronary artery supplemented with exogenous purified cIMP. However, cGMP levels were far lower than their levels in intact coronary arteries and lower than cIMP levels measured in endothelium-denuded coronary arteries supplemented with exogenous cIMP. The increased cIMP levels induced by PDE1 or PDE5 inhibition further led to augmented hypoxic constriction without apparently affecting the relaxation response. In intact coronary artery, PDE1 or PDE5 inhibition up-regulated cIMP levels under hypoxic condition. Concomitantly, cGMP level increased to a comparable level. Nevertheless, the hypoxia-mediated constriction was enhanced in this situation that was largely compromised by an even stronger inhibition of PDEs. Taken together, these data suggest that cIMP levels in coronary arteries are regulated by PDE1 and PDE5, whose inhibition at a certain level leads to increased cIMP content and enhanced hypoxic constriction.

Seihai-to (TJ-90)-Induced Activation of Airway Ciliary Beatings of Mice: Ca2+ Modulation of cAMP-Stimulated Ciliary Beatings via PDE1.

Sei-hai-to (TJ-90, Qing Fei Tang), a Chinese traditional medicine, increases ciliary beat frequency (CBF) and ciliary bend angle (CBA) mediated via cAMP (3',5'-cyclic adenosine monophosphate) accumulation modulated by Ca2+-activated phosphodiesterase 1 (PDE1A). A high concentration of TJ-90 (≥40 µg/mL) induced two types of CBF increases, a transient increase (an initial increase, followed by a decrease) and a sustained increase without any decline, while it only sustained the CBA increase. Upon inhibiting increases in intracellular Ca2+ concentration ([Ca2+]i) by 10 µM BAPTA-AM (Ca2+-chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) or Ca2+/calmodulin-dependent PDE1 by 8MmIBMX (a selective PDE1 inhibitor), TJ-90 (400 µg/mL) induced only the sustained CBF increase without any transient CBF increase. The two types of the CBF increase (the transient increase and the sustained increase) induced by TJ-90 (≥40 µg/mL) were mimicked by the stimulation with both procaterol (100 pM) and ionomycin (500 nM). Thus, TJ-90 stimulates small increases in the intracellular cAMP concentration ([cAMP]i) and [Ca2+]i in airway ciliary cells of mice. These small increases in [cAMP]i and [Ca2+]i cause inducing a transient CBF increase or a sustained CBF increase in an airway ciliary cells, depending on the dominant signal, Ca2+-signal, or cAMP-signal.

Genetic and Targeted eQTL Mapping Reveals Strong Candidate Genes Modulating the Stress Response During Chicken Domestication.

The stress response has been largely modified in all domesticated animals, offering a strong tool for genetic mapping. In chickens, ancestral Red Junglefowl react stronger both in terms of physiology and behavior to a brief restraint stress than domesticated White Leghorn, demonstrating modified functions of the hypothalamic-pituitary-adrenal (HPA) axis. We mapped quantitative trait loci (QTL) underlying variations in stress-induced hormone levels using 232 birds from the 12th generation of an advanced intercross between White Leghorn and Red Junglefowl, genotyped for 739 genetic markers. Plasma levels of corticosterone, dehydroepiandrosterone (DHEA), and pregnenolone (PREG) were measured using LC-MS/MS in all genotyped birds. Transcription levels of the candidate genes were measured in the adrenal glands or hypothalamus of 88 out of the 232 birds used for hormone assessment. Genes were targeted for expression analysis when they were located in a hormone QTL region and were differentially expressed in the pure breed birds. One genome-wide significant QTL on chromosome 5 and two suggestive QTL together explained 20% of the variance in corticosterone response. Two significant QTL for aldosterone on chromosome 2 and 5 (explaining 19% of the variance), and one QTL for DHEA on chromosome 4 (explaining 5% of the variance), were detected. Orthologous DNA regions to the significant corticosterone QTL have been previously associated with the physiological stress response in other species but, to our knowledge, the underlying gene(s) have not been identified. SERPINA10 had an expression QTL (eQTL) colocalized with the corticosterone QTL on chromosome 5 and PDE1C had an eQTL colocalized with the aldosterone QTL on chromosome 2. Furthermore, in both cases, the expression levels of the genes were correlated with the plasma levels of the hormones. Hence, both these genes are strong putative candidates for the domestication-induced modifications of the stress response in chickens. Improved understanding of the genes associated with HPA-axis reactivity can provide insights into the pathways and mechanisms causing stress-related pathologies.

Schisandrin enhances dendrite outgrowth and synaptogenesis in primary cultured hippocampal neurons.

BACKGROUND: Schisandra chinensis, commonly used in Asia for tea material and traditional Chinese medicine, is presumed to enhance mental and intellectual functions. In this study, the effects and signalling mechanisms of a purified compound schisandrin, one of the lignan of Schisandra chinensis, on primary cultured hippocampal neurons were investigated. RESULTS: Schisandrin treatment enhanced total dendritic length and branching complexity, both of which were significantly suppressed in the presence of specific blockers for calmodulin-dependent kinase II (CaMKII), protein kinase C epsilon (PKCε), and mitogen activated protein kinase kinase (MEK). Moreover, schisandrin induced calcium influx, and phosphorylation of CaMKII, PKCε, and MEK. Inhibition of CAMKII and PKCε attenuated the schisandrin-induced phosphorylation of PKCε and MEK, and the phosphorylation of MEK, respectively. Moreover, schisandrin also stimulated the phosphorylation of cyclic AMP responsive-element binding protein (CREB) at Ser-133, an effect that was blocked by KN93. In addition to its neuritogenic effects, schisandrin increased the numbers of postsynaptic density-95-positive and FM1-43-positive puncta in dendrites and synaptic boutons, respectively. CONCLUSION: In hippocampal neurons, schisandrin exhibits neurotrophic properties that are mediated by the CaMKII-PKCε-MEK pathway.

Levels of phosphodiesterase 4A and 4B are altered by chronic treatment with psychotropic medications in rat frontal cortex.

Our laboratory has recently demonstrated altered expression of phosphodiesterase (PDE) 4A and 4B in subjects with autism, bipolar disorder, and schizophrenia, suggesting disrupted cAMP signaling in these diagnostic groups. In the current study, we measured expression of PDEs in rat frontal cortex (FC) following chronic treatment with clozapine, fluoxetine, haloperidol, lithium, olanzapine, valproic acid (VPA), or sterile saline for 21 days. Western blotting experiments showed decreased expression of PDE4A subtypes in FC following treatment with clozapine, haloperidol, lithium, and VPA. PDE4B subtypes were similarly reduced in FC following treatment with clozapine, fluoxetine, and lithium. We also measured levels of nine PDE subtypes via qRT-PCR in FC and found significant upregulation of PDE1A and PDE8B following treatment with olanzapine, while treatment with lithium reduced expression of mRNA for PDE8B. Our results demonstrate altered expression of PDE4A and PDE4B in response to a variety of psychotropic medications suggesting potentially new therapeutic avenues for treatment of neuropsychiatric diseases.

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: an in vitro and in vivo analysis.

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.