Role of cyclic nucleotides in the control of cytosolic Ca2+ levels in vascular endothelial cells.

1. Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca(2+) concentrations ([Ca(2+)](i)) to some extent and Ca2+ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca(2+)](i) is maintained by a well-balanced Ca(2+) flux across the endoplasmic reticulum and plasma membrane. 2. Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca(2+)](i) directly or indirectly (via the actions of protein kinase (PK) A or PKG-mediated phosphorylation) by regulating Ca(2+) mobilization and Ca(2+) influx. Fine-tuning of [Ca(2+)](i) is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca(2+). 3. Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases. 4. The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca(2+)](i) in endothelial cells; (iii) the impact of excessive [Ca(2+)](i) in endothelial cells; and (iv) the balanced control of [Ca(2+)](i) in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

Survival prediction in patients with chronic obstructive pulmonary disease following a pulmonary rehabilitation programme in Hong Kong.

BACKGROUND AND OBJECTIVE: Pulmonary rehabilitation programme (PRP) is an important component in the management of chronic obstructive pulmonary disease (COPD). However, to date so far there has been limited literature on the survival outcomes of patients with COPD after a PRP in Hong Kong. This study aimed to investigate the outcomes of a pulmonary rehabilitation programme on the survival rates of a retrospective cohort of patients with COPD. METHODS: This was a retrospective study that included subjects who participated in the PRP in a rehabilitation hospital from the year 2003 to 2015. A total of 431 patients with chronic obstructive pulmonary disease were identified from the electronic record system of the hospital. The dataset were split into two age groups for reporting and analysis using the mean age of 72 as the cut-off. Their median survival times were calculated using Kaplan-Meier analysis. Cox-proportional regression model was used to explore factors that predicted better survival. The most significant predictors were used as strata, and their respective effects on survival functions were analysed with Kaplan-Meier analysis again. RESULTS: The overall median survival of the cohort was 4.3 years. The median survival times of the younger patient group (aged <72) and the older patient group (aged ≥72) were 5.3 and 3.6 years, respectively. For the patients, aged <72 years old, Moser's Activities of Daily Living class and the pulmonary rehabilitation programme completion rate were the most significant survival predictors. For the patients aged ≥72 years old, Monitored Functional Task Evaluation score was the most significant survival predictor. CONCLUSION: Moser's Activities of Daily Living class ≥2 and non-completion of PRP for younger group, low exercise capacity with Monitored Functional Task Evaluation score <17 for older group were identified as significant predictors of poor survival. The findings of this study helped identifying those patients with COPD who have the needs to be more intensively treated and closely monitored.

Cyclic nucleotides and Ca2+ influx pathways in vascular endothelial cells.

Ca(2+) mobilizing agonists and hemodynamic shear stress both elicit a rise in endothelial cytosolic Ca(2+) [Ca(2+)](i), which then acts to stimulate nitric oxide synthase and phospholipase A(2), leading to the production and release of nitric oxide (NO) and other vascular substances such as prostacyclin and endothelium-derived hyperpolarizing factors (EDHF). In this article, regulatory mechanisms of agonist-induced and mechanosensitive Ca(2+) influx pathways in vascular endothelial cells will be discussed. Special emphasis will be placed on the regulation of agonist-induced Ca(2+) influx by protein kinase G (PKG). Flow-induced Ca(2+) influx in relation to vascular dilation and the vasodilator produced will also be discussed.

Rod-type cyclic nucleotide-gated cation channel is expressed in vascular endothelium and vascular smooth muscle cells.

OBJECTIVES: Ca(++)-permeable nonselective cation channels mediate the entry of extracellular Ca++ in vascular endothelium. They are also partly responsible for Ca++ entry in vascular smooth muscle cells (SMCs). The molecular identities of these channels have not been identified. The aim of this study is to examine whether rod-type nucleotide-gated nonselective cation (CNG1) channel, a channel which has been molecularly cloned, is related to the nonselective channels in vascular cells. METHODS: We used RT-PCR, molecular cloning, northern Blot and in situ hybridization to examine the expression of CNG1 mRNA in a variety of guinea pig and rat blood vessels with different diameters and in cultured vascular endothelial cells and vascular smooth muscle cells. RESULTS: We have cloned a 402-bp partial cDNA of CNG1 channel from guinea pig mesenteric arteries. RT-PCR and southern blot results indicate that the CNG1 mRNA is expressed in both cultured vascular endothelial and cultured vascular SMCs. Northern blot revealed the transcripts of approximately 3.2 kb, approximately 5.0 kb, and approximately 1.8 kb in cultured endothelial cells. In situ hybridization yielded strong labeling in endothelium layer of aorta, medium-sized mesenteric arteries, and small mesenteric arteries. CONCLUSION: Our findings suggest a potential role of CNG protein for Ca++ entry in vascular endothelium and vascular smooth muscles. The high expression of CNG1 mRNA in the endothelium of medium-sized arteries and small-sized arteries implicates a possible involvement of CNG1 protein in the regulation of blood supply to different regions and in the regulation of arterial blood pressure.

Activity of voltage-gated K+ channels is associated with cell proliferation and Ca2+ influx in carcinoma cells of colon cancer.

Cell proliferation of carcinoma cells DLD-1 derived from colon cancer as measured by [3H] thymidine incorporation was drastically reduced in the presence of 4-aminopyridine, an inhibitors of voltage-gated K channel. A number of nonspecific K+ channel inhibitors including TPeA, TEA, verapamil and diltiazem also inhibited [3H] incorporation at the concentration reported to inhibit voltage-gated K+ channels. The presence of voltage-gated K+ channels was confirmed by reverse transcription-PCR and cDNA sequencing. Charybdotoxin and iberiotoxin, inhibitors for Ca2+-sensitive K+ channel, and glibenclamide, a specific inhibitor for ATP-sensitive K+ channel, did not have effect on cell proliferation. These experiments suggested a critical role of voltage-gated K+ channels in proliferation of colon cancer cells. Mechanism of action of K+ channel activity in cell proliferation was explored by studying the relationship between the K+ channel activity and Ca2+ entry. The results from experiments indicated that K+ channel inhibitors blocked [Ca2+]i influx. Therefore, it is likely that K+ channel activity may modulate Ca2+ influx into colon cancer cells, and subsequently modulate the proliferation of these cells.

A mechanosensitive cation channel in endothelial cells and its role in vasoregulation.

Ca2+ is an important intracellular second messenger in signal transduction of endothelial cells. It has long been recognized that a mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel may increase intracellular Ca2+ level in endothelial cells. A recent finding is that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells. Another important finding is that the opening of this mechanosensitive channel by KT5823 leads to endothelium-dependent vascular dilation. Therefore, it appears that this channel may play a crucial role in the regulation of vascular tone.

The first transcriptome and genetic linkage map for Asian arowana.

Asian arowana or dragonfish (Scleropages formosus) is an important fish species due to its unusual breeding biology and high economic value in the ornamental fish markets. In the present study, we aimed to (i) create the first transcriptome by Roche 454 pyrosequencing of Asian arowana brain and gonad samples; (ii) identify differentially expressed genes between the two sexes and develop microsatellite (SSR) markers; and (iii) construct a first-generation SSR-based genetic linkage map. A total of over 1.3 million reads were obtained from the brain and gonad of adult Asian arowana individuals through pyrosequencing. These reads were assembled into 16,242 contigs that were further grouped into 13,639 isogroups. BLASTX annotation identified a total of 8316 unique proteins from this data set. Many genes with sexually dimorphic expression levels and some putatively involved in sex development were identified. A total of 316 EST-SSRs and 356 new genomic-SSRs were developed by screening through the current transcriptome data set and SSR-enriched genomic libraries. The first genetic linkage map of the species was constructed based on these markers. Linkage analysis allowed for mapping of 308 markers to 28 linkage groups (LGs), ranging in size from 14.9 to 160.6 cM. The potentially sex-associated gene sox9 was mapped to LG4 on the consensus linkage map. Pairwise putative conserved syntenies between the Asian arowana, zebrafish, and three-spined stickleback were also established. These resources will help the conservation of the species through better understanding of its phylogenetics, genomics and biology, and comparative genome analysis within the Osteoglossidae family.

A mechanosensitive cation channel in endothelial cells.

A mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel increases in response to hemodynamic blood flow. Recently, it has been found that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells.

Store-operated calcium entry in vascular endothelial cells is inhibited by cGMP via a protein kinase G-dependent mechanism.

Store-operated Ca(2+) entry in vascular endothelial cells not only serves to refill the intracellular Ca(2+) stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca(2+) entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca(2+)-ATPase, were used to induce store-operated Ca(2+) entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca(2+) entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 microM) or H-8 (10 microM), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca(2+) entry. Addition of S-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca(2+) influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca(2+)-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca(2+) entry in vascular endothelial cells.

cGMP abolishes agonist-induced [Ca(2+)](i) oscillations in human bladder epithelial cells.

Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular Ca(2+) concentration ([Ca(2+)](i) ) while avoiding prolonged exposure to constantly elevated [Ca(2+)](i). In this study, we demonstrated that agonists could induce Ca(2+) oscillations in human bladder epithelial cells. Application of 10 microM acetylcholine or 200 nM bradykinin triggered an initial Ca(2+) transient that was followed by periodic [Ca(2+)](i) oscillations. The oscillations did not depend on extracellular Ca(2+). 8-Bromoguanosine 3',5'-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca(2+)](i) oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (IP(3)) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to IP(3) receptor-mediated Ca(2+) release.

Characterization of a regulatory region in the N-terminus of rabbit kv1.3.

The N-terminus of rabbit Kv1.3 contains a signal sequence which regulates expression of Kv1.3 proteins in the plasma membrane. Removal of an N-terminal region (aa3-39) produced an increase in expressed K+ current. Progressive deletion at the N-terminus demonstrated that the shortest deletion required for the elevation of K current is D6-34. Since the functional signal sequence must include both ends of the peptide segment aa6-34 where charged residues are densely distributed, it is conceivable that this N-terminal signal sequence is related to charge or its associated hydrophilicity. Removal of two charged residues (31R, 33E) through amino acid substitution which converts deletion construct D3-27 to D3-27* effectively raises the amplitude of expressed current, further indicating the importance of charged residues. With the use of a mutated dynamin and a soluble N-terminal peptide, we also revealed that the N-terminal signal sequence may not act through the endocytotic pathway.

A protein kinase G-sensitive channel mediates flow-induced Ca(2+) entry into vascular endothelial cells.

The hemodynamic force generated by blood flow is considered to be the physiologically most important stimulus for the release of nitric oxide (NO) and prostacyclin (PGI(2)) from vascular endothelial cells (1). NO and PGI(2) then act on the underlying smooth muscle cells, causing vasodilation and thus lowering blood pressure (2, 3). One critical early event occurring in this flow-induced regulation of vascular tone is that blood flow induces Ca(2+) entry into vascular endothelial cells, which in turn leads to the formation of NO (4, 5). Here we report a mechanosensitive Ca(2+)-permeable channel in vascular endothelial cells. The activity of the channel was inhibited by 8-Br-cGMP, a membrane-permeant activator of protein kinase G (PKG), in cell-attached membrane patches. The inhibition could be reversed by PKG inhibitor KT5823 or H-8. A direct application of active PKG in inside-out patches blocked the channel activity. Gd(3+), Ni(2+), or SK&F-96365 also inhibited the channel activity. A study of fluorescent Ca(2+) entry revealed a striking pharmacological similarity between the Ca(2+) entry elicited by flow and the mechanosensitive Ca(2+)-permeable channel we identified, suggesting that this channel is the primary pathway mediating flow-induced Ca(2+) entry into vascular endothelial cells.

Endothelial cell protein kinase G inhibits release of EDHF through a PKG-sensitive cation channel.

The release of dilator agents from vascular endothelial cells is modulated by changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). In this study, we demonstrate the presence of a Ca(2+)-permeable cation channel in inside-out membrane patches of endothelial cells isolated from small mesenteric arteries. The activity of the channel is increased by KT-5823, a highly selective inhibitor of protein kinase G (PKG), while it is decreased by direct application of active PKG. Application of KT-5823 induces Ca(2+) influx in the endothelial cells isolated from small mesenteric arteries, and it also causes endothelium-dependent relaxations in isolated small mesenteric arteries. KT-5823-induced relaxations in small mesenteric arteries are greatly reduced by 35 mM K(+) or 50 nM charybdotoxin + 50 nM apamin, suggesting that endothelium-derived hyperpolarizing factor (EDHF) is the participating dilator. The involvement of EDHF is further supported by experiments in which the relaxations of small mesenteric arteries are shown to be accompanied by membrane repolarization. These data strongly argue for a major role of a PKG-sensitive cation channel in modulating the release of EDHF from endothelial cells in rat small mesenteric arteries.