Increased store-operated Ca²⁺ entry mediated by GNB5 and STIM1

Recent human genetic studies have shown that Gβ5 is related to various clinical symptoms, such as sinus bradycardia, cognitive disability, and attention deficit hyperactivity disorder. Although the calcium signaling cascade is closely associated with a heterotrimeric G-protein, the function of Gβ5 in calcium signaling and its relevance to clinical symptoms remain unknown. In this study, we investigated the in vitro changes of store-operated calcium entry (SOCE) with exogenous expression of Gβ5. The cells expressing Gβ5 had enhanced SOCE after depletion of calcium ion inside the endoplasmic reticulum. Gβ5 also augmented Stim1- and Orai1-dependent SOCE. An ORAI1 loss-of-function mutant did not show inhibition of Gβ5-induced SOCE, and a STIM1-ERM truncation mutant showed no enhancement of SOCE. These results suggested a novel role of GNB5 and Stim1, and provided insight into the regulatory mechanism of SOCE.

Sildenafil decreases rat tracheal hyperresponsiveness to carbachol and changes canonical transient receptor potential gene expression after antigen challenge

Inhibition of type-5 phosphodiesterase by sildenafil decreases capacitative Ca2+ entry mediated by transient receptor potential proteins (TRPs) in the pulmonary artery. These families of channels, especially the canonical TRP (TRPC) subfamily, may be involved in the development of bronchial hyperresponsiveness, a hallmark of asthma. In the present study, we evaluated i) the effects of sildenafil on tracheal rings of rats subjected to antigen challenge, ii) whether the extent of TRPC gene expression may be modified by antigen challenge, and iii) whether inhibition of type-5 phosphodiesterase (PDE5) may alter TRPC gene expression after antigen challenge. Sildenafil (0.1 µM to 0.6 mM) fully relaxed carbachol-induced contractions in isolated tracheal rings prepared from naive male Wistar rats (250-300 g) by activating the NO-cGMP-K+ channel pathway. Rats sensitized to antigen by intraperitoneal injections of ovalbumin were subjected to antigen challenge by ovalbumin inhalation, and their tracheal rings were used to study the effects of sildenafil, which more effectively inhibited contractions induced by either carbachol (10 µM) or extracellular Ca2+ restoration after thapsigargin (1 µM) treatment. Antigen challenge increased the expression of the TRPC1 and TRPC4 genes but not the expression of the TRPC5 and TRPC6 genes. Applied before the antigen challenge, sildenafil increased the gene expression, which was evaluated by RT-PCR, of TRPC1 and TRPC6, decreased TRPC5 expression, and was inert against TRPC4. Thus, we conclude that PDE5 inhibition is involved in the development of an airway hyperresponsive phenotype in rats after antigen challenge by altering TRPC gene expression.

Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca2+ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca2+ concentration ([Ca2+]i), and the increase of [Ca2+]i by OxLDL or by LPC was inhibited by La3+ or heparin. LPC failed to increase [Ca2+]i in the presence of an antioxidant tempol. In addition, store-operated Ca2+ entry (SOC), which was evoked by intracellular Ca2+ store depletion in Ca2+-free solution using the sarcoplasmic reticulum Ca2+ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca2+]i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La3+ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca2+ to 1 micrometer activated large-conductance Ca2+-activated K+ (BKCa) current spontaneously, and this activated BKCa current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca2+-permeable Ca2+-activated NSC current and BKCa current simultaneously, thereby increasing SOC.

Ca2+ signalling in endothelial cells: Role of ion channels

Ca2+-signals in endothelial cells are determined by release from intracellular stores and entry through the plasma membrane. In this review, the nature of Ca2+ entry and mechanisms of its control are reviewed. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identified as Kir2.1, a big-conductance, Ca2+-activated K+ channel (hslo) and at least two Cl- channels (a volume regulated Cl- channel, VRAC, and a Ca2+ activated Cl- channel, CaCC). At least two different types of Ca2+-entry channels exist: 1. A typical CRAC-like, highly selective Ca2+ channel is described. Current density for this Ca2+ entry is approximately 0.1 pA/pF at 0 mV and thus 10 times smaller than in Jurkat or mast cells. 2. Another entry pathway for Ca2+ entry is a more non-selective channel, which might be regulated by intracellular Ca2+. Although detected in endothelial cells, the functional role of trp1,3,4 as possible channel proteins is unclear. Expression of trp3 in macrovascular endothelial cells from bovine pulmonary artery induced non-selective cation channels which are probably not store operated or failed to induce any current. Several features as well as a characterisation of Ca2+ -oscillations in endothelial cells is also presented.

Capacitative Ca~(2+) entry mediate contraction in rat distal colon smooth muscle / 中国药理学通报

Aim To investigate whether capacitative Ca~(2+) entry involved in excitation-contraction coupling in rat distal colon smooth muscle.Methods Changes of isolated organ's tension were monitored with force-displacement transducer and Powerlab 4/25T recording system.Results Thapsigargin(10 nmol?L~(-1)～1 ?mol?L~(-1))produced slowly developing sustained contractions in isolated distal colon smooth muscle strips of rats.The timed contractile responses to thapsigargin(10 n mol?L~(1)-1 ?mol?L~(-1)) were significantly different.The contractile response to Ca~(2+) reintroduction following incubation of strips in a Ca~(2+)-free Krebs in the presense of thapsigargin was significantly higher than in its absence(99%?28% vs 70%?8%).Contractile responses to Ca~(2+)reintroduction following depletion of sarcoplasmic reticulum Ca~(2+) stores with thapsigargin were attenuated by La~(3+),while unaffected by verapamil.Conclusion Contractile responses to Ca~(2+)reintroduction following depletion of sarcoplasmic reticulum Ca~(2+)stores with thapsigargin,are mediated by capacitative Ca~(2+)entry.The results suggested that CCE provided activator Ca~(2+)for the contraction and participated in excitation-contraction process in rat distal colon smooth muscle.