Alterations in the anterior hypothalamic dopamine system in aggressive adolescent AAS-treated hamsters

Anabolic androgenic steroid (AAS) treatment throughout adolescence facilitates offensive aggression in male Syrian hamsters (Mesocricetus auratus). The present study was conducted to investigate the role of the dopaminergic system in the modulation of AAS-induced aggressive behavior. Hamsters were administered AAS during adolescence, scored for offensive aggression using the resident-intruder paradigm, and then examined for alterations in DA immunoreactivity in brain regions implicated in the aggressive phenotype, including the anterior hypothalamus (AH), the bed nucleus of the stria terminalis (BNST), the medial and central amygdala (MeA and CeA), the lateral septum (LS) and the ventrolateral hypothalamus (VLH). When compared with non-aggressive sesame-oil-treated controls, aggressive AAS-treated animals showed increased tyrosine hydroxylase immunoreactivity in anterior hypothalamic subnuclei, namely the nucleus circularis (NC) and medial supraoptic nucleus (mSON). In addition, AAS-treated animals showed altered D₂ receptor expression in the AH and the VLH, as measured by D₂-immunoreactivity. Together these results suggest that alterations in DA synthesis and function together with modifications in D₂ receptor expression in the AH may underlie neuroplastic events which facilitate AAS-induced aggression.     

Oxytocin hyperpolarizes cultured duodenum myenteric intrinsic primary afferent neurons by opening BK(Ca) channels through IP₃ pathway

Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca2? concentration ([Ca2?](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10?? to 10?? M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10?? M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca2?](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10?? M) or CdCl?. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP? receptor antagonist 2-APB (10?? M) or the PLC inhibitor U73122 (10?? M). OT (10?? M) also increased the IP? concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP?-Ca2? signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner.     

Over-expression of PKGIα inhibits hypoxia-induced proliferation, Akt activation, and phenotype modulation of human PASMCs: the role of phenotype modulation of PASMCs in pulmonary vascular remodeling

The proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a role in pulmonary vascular remodeling (PVR). Recently, it was shown that vascular smooth muscular cell phenotype modulation is important for their proliferation in other diseases. However, little is known about the role of human PASMC phenotype modulation in the proliferation induced by hypoxia and its molecular mechanism during PVR. In this study, we found using primary cultured human PASMCs that hypoxia suppressed the expression of endogenous PKGIα, which was reversed by transfection with a recombinant adenovirus containing the full-length cDNA of PKGIα (Ad-PKGIα). Ad-PKGIα transfection significantly attenuated the hypoxia-induced downregulation of the expression of smooth muscle α-actin (SM-α-actin), myosin heavy chain (MHC) and calponin in PASMCs, indicating that hypoxia-induced phenotype modulation was blocked. Furthermore, flow cytometry and ³H-TdR incorporation demonstrated that hypoxia-induced PASMC proliferation was suppressed by upregulation of PKGIα. These results suggest that enhanced PKGIα expression inhibited hypoxia-induced PASMC phenotype modulation and that it could reverse the proliferation of PASMCs significantly. Moreover, our previous work has demonstrated that Akt protein is activated in the process of hypoxia-induced proliferation of human PASMCs. Interestingly, we found that Akt was not activated by hypoxia when PASMC phenotype modulation was blocked by Ad-PKGIα. This result suggests that blocking phenotype modulation might be a key up-stream regulatory target.     

Identification of novel splice variants of ARNT and ARNT2 in the rat

Most of the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the bHLH/PAS protein AH receptor (AHR). For regulation of gene activities, AHR dimerizes with another member of the bHLH/PAS protein family, AHR nuclear translocator (ARNT). A substrain of Wistar rats, Han/Wistar (Kuopio) (H/W), is about 1000-fold more resistant to the acute lethality of TCDD than other strains, exemplified by Long-Evans (Turku/AB) (L-E); the LD50 values for these two strains are >9600 and 10-20 microg/kg, respectively. Previous studies have demonstrated that the major reason for the exceptional TCDD resistance of H/W rats lies in their AHR, which is remodeled at its C-terminal transactivation domain, but there appears to be another contributing gene product. The present study set out to compare the primary structure of ARNT and the closely related ARNT2 proteins in H/W and L-E rats by cDNA cloning. To our surprise, we found several isoforms of these proteins only one of which has previously been reported in rats. All of the isoforms appeared to arise from alternative splicing. For ARNT, isoforms with deletions at exon 5, 3(') end of exon 6 or 5(') end of exon 11, or with an insertion at 5(') end of exon 20 were discovered. There was also interindividual variation in the number of glutamine-encoding codons at 5(') end of exon 16. The most exciting new variant was revealed for ARNT2, because the insertion found at 5(') end of exon 19 disrupts the functionally critical transactivation domain in the protein, implying a dominant negative role for this isoform. The relative expression levels of the variants did not differ in the two rat strains, nor did TCDD modify the ratios, suggesting that the variants do not contribute to TCDD resistance. However, the regulation of ARNT and ARNT2 activities may be more intricate than previously assumed.     

Pitavastatin increases ABCA1-mediated lipid efflux from Fu5AH rat hepatoma cells

ATP binding cassette A1 (ABCA1) is responsible in vivo for the formation of HDL by promoting the lipidation of apoprotein A-I (apoA-I) via cholesterol and phospholipid efflux from the liver. Treatment of patients with statins produces an increase in HDL plasma level, but the underlying mechanism is not completely understood. In this work we investigated the ability of pitavastatin to modulate ABCA1-mediated efflux from Fu5AH rat hepatoma cells, that here we demonstrate to express functional ABCA1 upon treatment with 22OH/cRA. In both basal and ABCA1 expressing cells pitavastatin 0.1-50microM induced a dose-dependent increase in cholesterol efflux to apoA-I; this effect was reversed by mevalonate or geranyl geraniol. A stimulatory effect was also observed on phospholipid efflux. Similar results were obtained with compactin, suggesting a class-related effect of statins. These results indicate a potential mechanism for the improvement in HDL plasma profile observed in patients treated with statins.     

Acetylcholine Mobilization in a Sympathetic Ganglion in the Presence and Absence of 2-(4-Phenylpiperidino)Cyclohexanol (AH5183)

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.     

Mobilization of a Vesamicol-Insensitive Pool of Acetylcholine from a Sympathetic Ganglion by Ouabain

Abstract: These experiments investigate the release of transmitter from the perfused superior cervical ganglia of cats induced by ouabain in the absence or presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a blocker of acetylcholine (ACh) uptake. Ouabain, perfused through the ganglia, released ACh in a Ca²⁺-dependent way. Vesamicol caused some inhibition of the release of ACh by ouabain; however, under this condition, the Na⁺, K⁺-ATPase inhibitor released five times more transmitter than did preganglionic stimulation at 5 Hz. Also, when ganglia exposed to vesamicol were depleted of the impulse-releasable pool of ACh, subsequent perfusion with ouabain released ACh, and this included ACh newly synthesized in the presence of vesamicol; this phenomenon could be inhibited by the lack of Ca²⁺ and presence of EGTA, and was completely abolished by perfusion with a medium containing 18 mM Mg²⁺. To test whether the release of this vesamicol-insensitive Ca²⁺-dependent pool by ouabain is associated with a decrease in the number of synaptic vesicles, ganglia treated with the ATPase inhibitor after the depletion of the impulse-releasable pool of ACh were fixed for electron microscopy. In the presence of Ca²⁺, coincident with the release of the vesamicol-insensitive pool of ACh, nerve terminals were almost depleted of synaptic vesicles; ganglia treated similarly, but with medium containing 18 mM Mg²⁺ instead of Ca²⁺, were not depleted of synaptic vesicles. These results suggest that ouabain releases a vesamicol-insensitive pool of ACh from the sympathetic ganglion and also support the notion that this compartment is vesicular and its exocytosis depends on extracellular Ca²⁺. It is suggested that empty-vesicle recycling in the presence of vesamicol restricts mobilization of full vesicles to release sites.     

Purification and Characterization of a Nonvesicular Vesamicol-Binding Protein from Electric Organ and Demonstration of a Related Protein in Mammalian Brain

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.     

AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

嗜水产气单胞菌诱导的草鱼免疫球蛋白分析

取草鱼经嗜水产气单胞菌疫苗免疫,获免疫血清分两组,一组样品经饮和硫酸铵分级沉淀,秀析和电泳分析,另一组经Sephadex G-200柱层析分离及电泳分析,各分离样品经特异性集活性测定,发现沉淀分离组样品中抗体凝集活性最强的主要为35％饱和硫酸铵沉淀的组分,而柱层析分离组样品的凝集抗体主要集中在第一个洗脱蜂里。在电泳图谱中,各免疫组均比对照组增加了一条大分子B带,分子量为309kD左右,后者经变性电泳分离表现为两条区带,相对分子量分别为52kD和26KD。因而推测它们是单体IgG解离的重链和经链,而聚集在B带的是IgG的聚合形式IgM。