Phosphodiesterase 5 restricts NOS3/Soluble guanylate cyclase signaling to L-type Ca current in cardiac myocytes

Endothelial nitric oxide synthase (NOS3) regulates the functional response to β-adrenergic (β-AR) stimulation via modulation of the L-type Ca²⁺ current (I_Ca). However, the NOS3 signaling pathway modulating I_Ca is unknown. This study investigated the contribution of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5), a cGMP-specific PDE, in the NOS3-mediated regulation of I_Ca. Myocytes were isolated from NOS3 knockout (NOS3^−/−) and wildtype (WT) mice. We measured I_Ca (whole-cell voltage-clamp), and simultaneously measured Ca²⁺ transients (Fluo-4 AM) and cell shortening (edge detection). Zaprinast (selective inhibitor of PDE5), decreased β-AR stimulated (isoproterenol, ISO)-I_Ca, and Ca²⁺ transient and cell shortening amplitudes in WT myocytes. However, YC-1 (NO-independent activator of sGC) only reduced ISO-stimulated I_Ca, but not cardiac contraction. We further investigated the NOS3/sGC/PDE5 pathway in NOS3^−/− myocytes. PDE5 is mislocalized in these myocytes and we observed dissimilar effects of PDE5 inhibition and sGC activation compared to WT. That is, zaprinast had no effect on ISO-stimulated I_Ca, or Ca²⁺ transient and cell shortening amplitudes. Conversely, YC-1 significantly decreased both ISO-stimulated I_Ca, and cardiac contraction. Further confirming that PDE5 localizes NOS3/cGMP signaling to I_Ca; YC-1, in the presence of zaprinast, now significantly decreased ISO-stimulated Ca²⁺ transient and cell shortening amplitudes in WT myocytes. The effects of YC-1 on I_Ca and cardiac contraction were blocked by KT5823 (a selective inhibitor of the cGMP-dependent protein kinase, PKG). Our data suggests a novel physiological role for PDE5 in restricting the effects of NOS3/sGC/PKG signaling pathway to modulating β-AR stimulated I_Ca, while limiting effects on cardiac contraction.     

Hepatitis G virus/GBV-C in serum,peripheral blood mononuclear cells and bone marrow in patients with hematological malignancies

BackgroundHGV/GBV-C is highly prevalent in the general population but its significance remains unclear. It is known that HGV/GBV-C is not primary hepatotropic and its replication was reported in PBMC, bone marrow and other tissues. To investigate a possible role of HGV/GBV-C 115 consecutive patients with hematological malignancies were analyzed for virus RNA presence and quasispecies composition in three compartments: serum, PBMC and bone marrow.MethodsRT-PCR was used to amplify 5′UTR HGV/GBV-C in serum, PBMC and bone marrow. Viral sequences obtained from three compartments were subjected for comparative molecular analysis performed by single strand conformational polymorphism (SSCP) and pyrosequencing.ResultsHGV/GBV-C RNA was detected in 23 out of 115 (20.0%) patients, most often in bone marrow (18 patients), followed by PBMC (11 patients) and serum (10 patients). Differences in SSCP bands distribution corresponding to different viral variants and confirmed by direct sequencing were observed in three patients.ConclusionHGV/GBV-C infection is frequent in patients with hematological malignancies. Common detection of HGV/GBV-C in bone marrow supports the hypothesis that it is a major replication site of this virus.     

Compartments in the striatum of the cat observed by retrograde cell labeling

Summary The distribution of neurons giving rise to striatal efferent projections has been studied in the cat by the aid of Mesulam's histochemical methods following large injections of horseradish peroxidase into the pallidum and substantia nigra. The findings suggest that neurons of medium size form a major fraction of the efferent neurons of the striatum and that these neurons are grouped into geometrically complex, three-dimensional networks.     

Method for application of drugs into separated parts of the cerebroventricular system

A method is described, which allows the spread of substances after intracerebroventricular application to be restricted to various parts of the ventricular space in unanaesthetized, unrestrained rabbits. After intracisternal injection also the ventricular system can be excluded from the spread of substance.     

Molecular diversity, trafficking and subcellular localization of GABAB receptors

GABA_B receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, γ-aminobutyric acid (GABA). While native studies predicted pharmacologically distinct GABA_B receptor subtypes, molecular studies failed to identify the expected receptor varieties. Mouse genetic experiments therefore addressed whether the cloned receptors can account for the classical electrophysiological, biochemical and behavioral GABA_B responses or whether additional receptors exist. Among G-protein coupled receptors, GABA_B receptors are unique in that they require 2 distinct subunits for functioning. This atypical receptor structure triggered a large body of work that investigated the regulation of receptor assembly and trafficking. With the availability of molecular tools, substantial progress was also made in the analysis of the receptor protein distribution in neuronal compartments. Here, we review recent studies that shed light on the molecular diversity, the subcellular distribution and the cell surface dynamics of GABA_B receptors.     

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca²⁺ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca²⁺ transients, increased L-type Ca²⁺ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca²⁺ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.     

Diversity of HIV-1 RNA and DNA in breast milk from HIV-1-infected mothers

We compared human immunodeficiency virus type 1 (HIV-1) RNA and DNA populations in the different fractions of breast milk (lactoserum, lipid layer, cell pellet) and between right and left breasts in four HIV-1-infected mothers by analyzing the hypervariable env C2-V5 region. Phylogenetic analyses of the viral quasispecies revealed that RNA populations and DNA populations were clearly distinct and that viral RNA sequences were similar in lipid layer and lactoserum in the milk of 3 out of 4 mothers. Comparison of viral DNA between milk from right and left breast showed a differential distribution of variants in three mothers. In contrast, RNA variants detected from milk of the two breasts were mixed in 3 out of 4 mothers. This study suggests that each mammary gland is subjected to microenvironmental pressure that may differ from the contralateral breast.     

人三角肌亚部化,肌纤维型分布及面积的研究

根据人三角肌的肌纤维起止、排列和神经支配特征,将该肌分为前、中、后3个亚部。用新鲜男性尸体标本16侧三角肌,按上述3个亚部的浅、深区分别取材,作恒冷箱冰冻横向切片,肌球蛋白ATP酶染色,将肌纤维分为Ⅰ型和Ⅱ型。检测各区的肌纤维型构成比例,并用图像分析仪测量各区两型肌纤维的横切面积和直径。结果发现,肌中部的I型纤维比例明显高于前、后两部,各亚部深区的I型纤维比例均比浅区高,而左、右侧之间无差异。各亚部及浅、深区。Ⅰ、Ⅱ型肌纤维的直径都相似(57～6lμm),仅中部深区Ⅱ型纤维直径(52.9μm)较其他各区肌纤维明显细小;除中部深区外,右侧两型纤维的直径均比左侧稍大,但统计学分析左右侧差异不显著。作者认为,三角肌纤维型分布的肌内差异,与该肌功能的分化密切相关。中部深区的Ⅰ型纤维比例较高,Ⅱ型纤维直径较细,可能提示该区的主要功能是维持肩关节稳定。     

Increased interleukin 6 production by bronchoalveolar lavage cells in patients with active sarcoidosis

Alveolitis of sarcoidosis is characterized by activated alveolar macrophages (AMs) and T cells. The mediators interleukin-1 (IL-1) and interleukin 6 (IL-6) released by AMs represent essential factors for the progression of the T cells in the cell cycle. The role of IL-1 in pulmonary sarcoidosis has previously been studied; however, the relevance of other mediators (i.e. IL-6) has not yet been evaluated. We measured the spontaneous and lipopolysaccharide (LPS)-induced release of IL-6 and tumor necrosis factor a (TNF) by bronchoalveolar lavage cells (BAL) and peripheral blood mononuclear cells (PBMNC) in 6 control subjects (group A) and in 15 patients with sarcoidosis, 10 with active (group B), 5 with inactive disease (group C). IL-6 as well as TNF were spontaneously released by BAL cells of the active group in significantly greater amounts compared to both other groups; IL-6: A, 165.5 pg/ml/24 hr/10⁶ cells (range, 0–604), B, 946 (0–2467), C, 16.6 (0–83); TNF: A, 162 pg/ml/24 hr/10⁶ cells (0–523), B, 803 (100–17352), C, 100 (0–379). In all groups autologous PBMNC proved to be quiescent, releasing only baseline levels of the cytokines tested. After stimulation with LPS all these cells released great quantities of IL-6 and TNF. In active disease a positive correlation between IL-6 and TNF release was observed (r = 0.77, p < 0.02). The present study documents that in active sarcoidosis the spontaneous release of IL-6 by BAL cells parallels the spontaneous release of TNF. IL-6 is capable of initiating the proliferation and activation of T cells in the lung. Offprint requests to: J. Müller-Quernheim