Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Expression of type I and II receptors for transforming growth factor β in the adult rat testis

 After having established the specificity of the antibodies for the rat testis by western blot analysis, the potential target cells for transforming growth factors (TGFβs) were identified by immunohistochemical detection of both type I (TβRI) and type II (TβRII) transducing receptors for TGFβs in the adult rat testis in situ. Leydig cells showed a strong TβRII immunoreactivity whereas the TβRI staining was weak. Only TβRII was detectable in Sertoli cells. In germ cells, staining for TβRI was stronger than for TβRII and the expression of both receptors depended on the seminiferous cycle stage. TβRI first appeared in pachytene spermatocytes and was absent in elongated spermatids from stage XIV onwards. Labelling for TβRII was observed as early as the spermatogonia stage; it increased in pachytene spermatocytes at the onset of TβRI and disappeared in elongating spermatids from stage XI onwards. These results show that TGFβs can affect somatic cells functions and suggest that these factors are involved in the control of meiosis and early spermiogenesis, exerting a direct effect on germ cells. Accepted: 18 June 1998     

β-Amyloid enhances intracellular calcium rises mediated by repeated activation of intracellular calcium stores and nicotinic receptors in acutely dissociated rat basal forebrain neurons

β-Amyloid, a 39–43 amino acid peptide, may exert its biological effects via neuronal nicotinic acetylcholine receptors. Using the ratiometric dye, fura-2, we examined the effect of soluble β-amyloid_1–42 on the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in acutely dissociated rat basal forebrain neurons. Focal applications of nicotine (0.5–20 mM), evoked dose-dependent increases in intracellular [Ca²⁺]_i that were mediated by the entry of extracellular Ca²⁺ via nicotinic acetylcholine receptors, and the release of intracellular Ca²⁺ from stores. With repeated nicotine challenges, the nicotinic responses were potentiated by 98 ± 12% (P < 0.05) while β-amyloid_1–42 (100 nM) was present for ∼5 min. This potentiation became larger during the subsequent washout of β-amyloid_1–42, which was associated with a gradual rise in baseline [Ca²⁺]_i. Application of β-amyloid_1–42 by itself did not alter [Ca²⁺]_i, and β-amyloid_1–42 also had no significant effect on the response to repeated KCl challenges. Therefore, β-amyloid_1–42 caused neither gross disturbance of cellular Ca²⁺ homeostasis nor enhancement of voltage-gated Ca²⁺ channels. Interestingly, β-amyloid_1–42 transiently potentiated the response to repeated caffeine challenges, which was also associated with a transient rise in baseline [Ca²⁺]_i. β-amyloid_1–42 potentiation of nicotine-evoked rises in [Ca²⁺]_i was reversed by the SERCA pump inhibitor, thapsigargin, and the mitochondrial Na⁺/Ca²⁺ exchanger inhibitor, CGP-37157. These results suggest that the dysregulation of [Ca²⁺]_i by β-amyloid_1–42 during multiple challenges with nicotine or caffeine involved the sensitization or overfilling of intracellular stores that are maintained by SERCA pump and Ca²⁺ efflux from the mitochondria.     

Site-specific radical formation in DNA induced by Cu(II)-H₂O₂ oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)–H₂O₂ oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.     

The WD40 repeat protein,WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G_q-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed G_q regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gα_q to phospholipase C (PLC)-β and the thromboxane A₂ receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M₃ and bradykinin B₂ receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca²⁺]_i increases. Surprisingly, WDR36 also augmented M₃ receptor-induced [Ca²⁺]_i increases, which was due to increased Ca²⁺ mobilization while the Ca²⁺ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by G_q-coupled M₃, B₂ and H₁ receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B₂ receptors. It is concluded that WDR36 reduces signalling of G_q-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gα_q and PLCβ. Our results support a role of WDR36 in orchestration of G_q signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.     

Localization of α1-adrenoceptors in rat and human hearts by immunocytochemistry

The localization of the ai adrenoceptors (₁-AR) in the heart tissues from rat and human and in the cultured heart cells from neonatal rats was studied by indirect immunofluorescence and postembedding electronmicroscopical immuno-gold technique. With antipeptide antibodies directed against the second extracellular loop of the human ₁-AR (AS sequence 192–218), this receptor was found to be localized along the sarcolemma in both human and rat hearts. Similar localization sites were detected in cultivated rat neonatal cardiomyocytes. Beside the localization in cardiomyocytes, ₁-AR were identified in endothelial cells of capillaries and smooth muscle cells of coronary vessels, in neuronal endings, in mast cells of cultivated heart cells but not, or in less amount in fibroblasts. Interestingly, in the right atrium of rat heart the localization of ₁-AR was found to be near or on atrial natriuretic factor (ANF) granules, providing the basis for the -adrenergic influence on ANF release. The immunocytochemical studies further confirm and complete the findings known by using autoradiographic binding studies with specific ligands.     

The B°AT1 amino acid transporter from rat kidney reconstituted in liposomes: kinetics and inactivation by methylmercury

The neutral amino acid transporter B°-like from rat kidney, previously reconstituted in liposomes, was identified as B°AT1 by a specific antibody. Collectrin was present in the brush-border extract but not in functionally active proteoliposomes, indicating that it was not required for the transport function. Neutral amino acids behaved as competitive inhibitors of the glutamine transport mediated by B°AT1 with half saturation constants ranging from 0.13 to 4.74mM. The intraliposomal half saturation constant for glutamine was 2.0mM. By a bisubstrate kinetic analysis of the glutamine-Na(+) cotransport, a random simultaneous mechanism was found. Methylmercury and HgCl(2) inhibited the transporter; the inhibition was reversed by dithioerythritol, Cys and, at a lower extent, N-acetylcysteine but not by S-carboxymethylcysteine. The IC(50) of the transporter for methylmercury and HgCl(2) was 1.88 and 1.75μM, respectively. The reagents behaved as non-competitive inhibitors toward both glutamine and Na(+) and no protection by glutamine or Na(+) was found for the two inhibitors.     

Ubiquitin-specific protease 2 regulates Ang Ⅱ–induced cardiac fibroblasts activation by up-regulating cyclin D1 and stabilizing β-catenin in vitro

Cardiac fibrosis, featuring abnormally elevated extracellular matrix accumulation, decreases tissue compliance, impairs cardiac function and accelerates heart failure. Mounting evidence suggests that the ubiquitin proteasome pathway is involved in cardiac fibrosis. In the present study, ubiquitin-specific protease 2 (USP2) was identified as a novel therapeutic target in cardiac fibrosis. Indeed, USP2 expression was increased in angiotensin II–induced primary cardiac fibroblasts (CFs) from neonatal rats. In addition, USP2 inhibition suppressed CFs proliferation, collagen synthesis and cell cycle progression. Furthermore, USP2 interacted with β-catenin, thereby regulating its deubiquitination and stabilization in CFs. To sum up, these findings revealed that USP2 has a therapeutic potential for the treatment of cardiac fibrosis.     

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at − 40 mV produced inward currents which displayed biphasic desensitization. The EC₅₀ of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC₅₀ increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

Postnatal expression of neurotransmitters, receptors, and cytochrome oxidase in the rat pre-B?tzinger complex.

The pre-B?tzinger complex (PBC) is postulated as the center of respiratory rhythmogenesis. Previously, we found a reduction or plateau of cytochrome oxidase (CO) activity in the PBC and other respiratory nuclei at postnatal days 3-4, despite a general increase of CO with age, suggesting a period of synaptic readjustment. The present study examined the expression of CO and a number of neurochemicals in the PBC at closer time intervals. At postnatal days 3-4 and, more prominently, at postnatal day 12, expression of CO, glutamate, and N-methyl-D-aspartate receptor subunit 1 was reduced, whereas expression of GABA, GABA(B) receptor, glycine receptor, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit 2 was increased. These findings are consistent with our hypothesis that decreased CO activity is associated with an increase in inhibitory drive (mediated by GABA and glycine, their receptors, and possibly blockage of Ca(2+) entry by glutamate receptor subunit 2) and a decrease in excitatory drive (mediated by glutamate and its receptors). Our findings point to two critical periods during postnatal development of the rat when their respiratory system may be more vulnerable to respiratory insults.     

Valsartan, independently of AT1 receptor or PPARγ, suppresses LPS-induced macrophage activation and improves insulin resistance in cocultured adipocytes

Macrophages are integrated into adipose tissues and interact with adipocytes in obese subjects, thereby exacerbating adipose insulin resistance. This study aimed to elucidate the molecular mechanism underlying the insulin-sensitizing effect of the angiotensin II receptor blocker (ARB) valsartan, as demonstrated in clinical studies. Insulin signaling, i.e., insulin receptor substrate-1 and Akt phosphorylations, in 3T3-L1 adipocytes was impaired markedly by treatment with tumor necrosis factor-α (TNFα) or in the culture medium of lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages, and valsartan had no effects on these impairments. However, in contrast, when cocultured with RAW 264.7 cells using a transwell system, the LPS-induced insulin signaling impairment in 3T3-L1 adipocytes showed almost complete normalization with coaddition of valsartan. Furthermore, valsartan strongly suppressed LPS-induced productions of cytokines such as interleukin (IL)-1β, IL-6, and TNFα with nuclear factor-κB activation and c-Jun NH(2)-terminal kinase phosphorylation in RAW 264.7 and primary murine macrophages. Very interestingly, this effect of valsartan was also observed in THP-1 cells treated with angiotensin II type 1 (AT1) siRNA or a peroxisome proliferator-activated receptor-γ (PPARγ) antagonist as well as macrophages from AT1a receptor-knockout mice. We conclude that valsartan suppresses the inflammatory response of macrophages, albeit not via PPARγ or the AT1a receptor. This suppression appears to secondarily improve adipose insulin resistance.     

Crosstalk between adenosine A1 and β1-adrenergic receptors regulates translocation of PKCε in isolated rat cardiomyocytes

Adenosine A(1) receptor (A(1)R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β(1)-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β(1)-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A(1)R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β(1)-adrenergic stimulation on PKCε translocation elicited by A(1)R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A(1)R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β(1)-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A(1)R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A(1)R. This increase was attenuated by the A(1)R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A(1)R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A(1)R, it is apparent that the interactive effects of adenosine and β(1)-adrenergic agonists on function are complex in the heart.     

Activation of rat brain adenylate cyclase by proteases: involvement of distinct protein components in the activation by α-chymotrypsin and trypsin

Adenylate cyclase in synaptic plasma membranes from rat brain is activated by α-chymotrypsin or trypsin. These proteases also activate adenylate cyclase reconstituted from the catalytic subunit of adenylate cyclase and the partially purified fraction of the GTP-binding proteins containing both the stimulatory and inhibitory GTP-binding proteins. Properties of the activation of reconstituted adenylate cyclase by the proteases are as follows. (1) The proteases do not directly activate the catalytic subunit. However, the pre-treatment of the partially purified GTP-binding proteins with α-chymotrypsin (100 μg/ml) increases the subsequently reconstituted cyclase activity at least 3-fold. Trypsin (10–30 μg/ml) much more weakly enhances the cyclase activity. (2) α-Chymotrypsin and trypsin synergistically activate the cyclase. (3) Trypsin but not α-chymotrypsin no longer activates the cyclase when the purified stimulatory GTP-binding protein (Gs) replaces the partially purified GTP-binding proteins. (4) The stimulatory effects of α-chymotrypsin and trypsin on the cyclase activity are little or slight unless 5′-guanylylimidodiphosphate (Gpp(NH)p) is present in the reconstitution. (5) The purified βγ-subunits of the GTP-binding proteins markedly inhibit adenylate cyclase. This inhibition is nearly completely attenuated by treating the βα-subunits with α-chymotrypsin (> 10 μg/ml). (6) Trypsin (1–10 μg/ml) inactivates the GTPase of the α-subunit of the inhibitory GTP-binding protein (Gi). This inactivation of the GTPase seems to correlate with the activation of the reconstituted adenylate cyclase by trypsin.We conclude that two distinct protein components are involved in the activation of adenylate cyclase by α-chymotrypsin and trypsin. One component sensitive to α-chymotrypsin is probably the βγ-subunits of the GTP-binding proteins. The other component sensitive to trypsin may be the α-subunit of Gi.     

Bone defect repair in rat tibia by TGF-β1 and IGF-1 released from hydrogel scaffold

Bone repair is one of the major challenges facing reconstructive surgery. Bone regeneration is needed for the repair of large defects and fractures. The ability of TGF-β1 and IGF-1 incorporated into hydrogel scaffold to induce bone regeneration was evaluated in a rat tibia segmental defect model. External fixation was performed prior to the induction of the segmental bone defect in order to stabilize the defect site. Hydrogel scaffold containing either TGF-β, IGF-1, TGF-β + IGF-1, hydrogel containing saline or saline, were inserted in the defect. Calcified material was observed in the defects treated with TGF-β 2 weeks following the start of treatment. Bone defects treated with TGF-β, IGF-1 or TGF-β + IGF-1 revealed significant bone formation after 4 and 6 weeks when compared to the control specimens. X-ray images showed that solid bone was present at the defect site after 6 weeks of treatment with TGF-β or TGF-β + IGF-1. A less pronounced bone induction was observed in the control specimens and bones treated with IGF-1. Percent closure ratio of bone defects after 6 weeks were 40, 80, 89, and 97% for saline, hydrogel, IGF-1, TGF-β and IGF-1 + TGF-β groups, respectively. It is concluded that hydrogel scaffold can serve as a good osteoconductive matrix for growth factors, and that it provides a site for bone regeneration and enhances bone defect healing and could be used as alternative graft material. This revised version was published online in July 2006 with corrections to the Cover Date.     

The SGK1 inhibitor EMD638683, prevents Angiotensin II–induced cardiac inflammation and fibrosis by blocking NLRP3 inflammasome activation

Inflammation has emerged as a critical biological process contributing to hypertensive cardiac remodeling. Effective pharmacological treatments targeting the cardiac inflammatory response, however, are still lacking. Prior studies suggested that the serum- and glucocorticoid-inducible kinase (SGK1) plays a key role in inflammation and cardiac remodeling. Recently, a highly selective SGK1 inhibitor, EMD638683, was developed, though whether EMD638683 can prevent hypertension-induced cardiac fibrosis and the mechanisms by which this inhibitor may alter the disease process remain unknown. Using a murine Angiotension II (Ang II) infusion-induced hypertension model we found that EMD638683 treatment inhibited cardiac fibrosis and remodeling, with significant abatement of cardiac inflammation. EMD638683 was shown to suppress Ang II infusion-induced interleukin (IL)-1β release, and substantially reduce nucleotide-binding oligomerization domain-like receptor with pyrin domain 3 (NLRP3) expression and caspase-1 activation in cardiac tissues. In vitro experiments revealed that EMD638683 ameliorated Ang II-stimulated IL-1β secretion in macrophages by blocking NLRP3 inflammasome activation. By reducing IL-1β production in macrophages, the transformation of fibroblasts to myofibroblasts was inhibited. The effects of EMD638683 on cardiac fibrosis were abolished by supplementation with exogenous IL-1β. Administration of the NLRP3 inflammasome inhibitor MCC950 indicated that EMD638683 attenuated Ang II-induced cardiac inflammation and fibrosis by inhibiting the NLRP3 inflammasome/IL-1β secretion axis. These findings indicate that the SGK1 inhibitor EMD638683 can negatively regulate NLRP3 inflammasome activation, and may represent a promising approach to the treatment of hypertensive cardiac damage.     

Roxithromycin treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of Caveolin-1 in rat airway smooth muscle cells

Background

Roxithromycin (RXM) has been widely used in asthma treatment; however, the mechanism has not been fully understood. The aim of our study was to investigate the underlying mechanism of RXM treatment in mediating the effect of transforming growth factor (TGF)-β1 on airway smooth muscle cells (ASMCs) proliferation and caveolinn-1 expression.

Methods

Firstly, the rat ovalbumin (OVA) model was built according to the previous papers. Rat ASMCs were prepared and cultured, and then TGF-β1 production in ASMCs was measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the proliferation of ASMCs was determined using cell counting kit (CCK-8) assay. Additionally, the expressions of caveolin-1, phosphorylated-ERK1/2 (p-ERK1/2) and phosphorylated–AKT (p-AKT) in ASMCs treated with or without PD98059 (an ERK1/2 inhibitor), wortannin (a PI3K inhibitor), β-cyclodextrin (β-CD) and RXM were measured by Western blot. Finally, data were evaluated using t–test or one-way ANOVA, and then a P value < 0.05 was set as a threshold.

Results

Compared with normal control, TGF-β1 secretion was significantly increased in asthmatic ASMCs; meanwhile, TGF-β1 promoted ASMCs proliferation (P < 0.05). However, ASMCs proliferation was remarkably inhibited by RXM, β-CD, PD98059 and wortmannin (P < 0.05). Moreover, the expressions of p-ERK1/2 and p-AKT were increased and peaked at 20 min after TGF-β1 stimulation, and then suppressed by RXM. Further, caveolin-1 level was down-regulated by TGF-β1 and up-regulated by inhibitors and RXM.

Conclusion

Our findings demonstrate that RXM treatment inhibits TGF-β1-induced activation of ERK and AKT and down-regulation of caveolin-1, which may be the potential mechanism of RXM protection from chronic inflammatory diseases, including bronchial asthma.