Stim, ORAI and TRPC channels in the control of calcium entry signals in smooth muscle

Ca2+ entry signals are crucial in the control of smooth muscle contraction. Smooth muscle cells are unusual in containing plasma membrane (PM) Ca2+ entry channels that respond to voltage changes, receptor activation and Ca2+ store depletion. Activation of these channel subtypes is highly coordinated. The TRPC6 channel, widely expressed in most smooth muscle cell types, is largely non-selective to cations and is activated by diacylglycerol arising from receptor-induced phosholipase C activation. Receptor activation results largely in Na+ ion movement through TRPC6 channels, depolarization and subsequent activation of voltage-dependent L-type Ca2+ channels. The TRPC6 channels also appear to be activated by mechanical stretch, resulting again in depolarization and L-type Ca2+ channel activation. Such a coupling may be crucial in mediating the myogenic tone response in vascular smooth muscle. The emptying of stores mediated by inositol 1,4,5-trisphosphate receptors triggers the endoplasmic reticulum (ER) Ca2+ sensing protein stromal-interacting molecule (STIM) 1 to translocate into defined ER-PM junctional areas in which coupling occurs to Orai proteins, which serve as highly Ca2+-selective low-conductance Ca2+ entry channels. These ER-PM junctional domains may serve as crucial sites of interaction and integration between the function of store-operated, receptor-operated and voltage-operated Ca2+ channels. The STIM, Orai and TRPC channels represent highly promising new pharmacological targets through which such control may be induced.     

New target molecules in the drug control of blood pressure and circulation

Ion channels play a pivotal role in blood pressure regulation. Amongst them, much attention has been directed to dihydropyridine (DHP)-sensitive (L-type) voltage-dependent Ca(2+) channels (VDCCs) and iberiotoxin-sensitive Ca(2+)-dependent K(+) channels which are distributed over the whole vascular tree and contribute to vascular tone regulation. Recent advances in vascular electrophysiology have, however, added novel and interesting molecules to this repertoire. In small mesenteric arterioles, the predominant VDCC phenotype is not L-type but DHP-insensitive, high voltage-activated VDCCs that exhibit unique properties distinguishable from those of hitherto-known VDCCs. Surprisingly, mibefradil, a well-known T-type selective blocker potently inhibits these channels, and the use of this blocker has indicated that Ca(2+) entry through these channels may be one of the important determinants of peripheral vascular tone. Another new candidate likely involved in blood pressure control is the mammalian homologue of Drosophila transient receptor potential (TRP) protein, including TRPC4 and TRPC6. Experiments in genetically engineered TRPC4-deficient mice have suggested that expression of TRPC4 is indispensable for agonist-induced Ca(2+) entry in endothelial cells and production of nitric oxide and vasorelaxation. TRPC6 is likely to contribute to sustained Ca(2+) entry into vascular smooth muscle cells activated by stimulation of sympathetic nerves and elevation of intravascular pressure. Antisense oligonucleotide experiments have suggested that this protein is an essential component of alpha1-adrenoceptor activated and mechanosensitive cation channels in some vascular tissues. This review overviews what is known about the role of ionic channels in blood pressure control with main focus on the above-mentioned new molecules as promising targets for drug discovery and development.     

Transient receptor potential channel activation and endothelium-dependent dilation in the systemic circulation

The endothelium plays a crucial role in the regulation of vascular tone by releasing a number of vasodilator mediators, including nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor(s). The production of these mediators is typically initiated by an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in endothelial cells. An essential component of this Ca(2+) signal is the entry of Ca(2+) from the extracellular space through plasma membrane Ca(2+)-permeable channels. Although the molecular identification of the potential Ca(2+) entry channel(s) responsible for the release of endothelial relaxing factors is still evolving, accumulating evidence indicates that the transient receptor potential (TRP) channels, a superfamily of Ca(2+)-permeable cation channels, serve as an important mechanism of Ca(2+) entry in endothelial cells and other nonexcitable cells. The activation of these channels has been implicated in diverse endothelial functions ranging from control of vascular tone and regulation of vascular permeability to angiogenesis and vascular remodeling. This review summarizes recent evidence concerning TRP channels and endothelium-dependent dilation in several systemic vascular beds. In particular, we highlight the emerging roles of several TRP channels from the canonical and vanilloid subfamilies, including TRPV4, TRPC4, and TRPC6, in vasodilatory responses to shear stress and receptor agonists and discuss potential signaling mechanisms linking the TRP channel activation and the initiation of endothelium-derived hyperpolarizing factor-mediated responses in endothelial cells.     

Agonist-evoked calcium entry in vascular smooth muscle cells requires IP3 receptor-mediated activation of TRPC1

Transient receptor potential canonical (TRPC) proteins have been proposed to function as plasma membrane Ca2+ channels activated by store depletion and/or by receptor stimulation. However, their role in the increase in cytosolic Ca2+ activated by contractile agonists in vascular smooth muscle is not yet elucidated. The present study was designed to investigate the functional and molecular properties of the Ca2+ entry pathway activated by endothelin-1 in primary cultured aortic smooth muscle cells. Measurement of the Ca2+ signal in fura-2-loaded cells allowed to characterize endothelin-1-evoked Ca2+ entry, which was resistant to dihydropyridine, and was blocked by 2-aminoethoxydiphenylborate (2-APB) and micromolar concentration of Gd3+. It was not activated by store depletion, but was inhibited by the endothelin ETA receptor antagonist BQ-123, and by heparin. On the opposite, thapsigargin-induced store depletion activated a Ca2+ entry pathway that was not affected by 2-APB, BQ-123 or heparin, and was less sensitive to Gd3+ than was endothelin-1-evoked Ca2+ entry. Investigation of the gene expression of TRPC isoforms by real-time RT-PCR revealed that TRPC1 was the most abundant. In cells transfected with TRPC1 small interfering RNA sequence, TRPC1 mRNA and protein expression were decreased by 72+/-3% and 86+/-2%, respectively, while TRPC6 expression was unaffected. In TRPC1 knockdown cells, both endothelin-1-evoked Ca2+ entry and store-operated Ca2+ entry evoked by thapsigargin were blunted. These results indicate that in aortic smooth muscle cells, TRPC1 is not only involved in Ca2+ entry activated by store depletion but also in receptor-operated Ca2+ entry, which requires inositol (1,4,5) triphosphate receptor activation.     

TRP channels in lymphocytes

TRP proteins form ion channels that are activated following receptor stimulation. Several members of the TRP family are likely to be expressed in lymphocytes. However, in many studies, messenger RNA (mRNA) but not protein expression was analyzed and cell lines but not primary human or murine lymphocytes were used. Among the expressed TRP mRNAs are TRPC1, TRPC3, TRPM2, TRPM4, TRPM7, TRPV1, and TRPV2. Regulation of Ca2+ entry is a key process for lymphocyte activation, and TRP channels may both increase Ca2+ influx (such as TRPC3) or decrease Ca2+ influx through membrane depolarization (such as TRPM4). In the future, linking endogenous Ca2+/cation channels in lymphocytes with TRP proteins should lead to a better molecular understanding of lymphocyte activation.     

Emerging functions of 10 types of TRP cationic channel in vascular smooth muscle

1. The influx of Ca2+, Mg2+ and Na+ and the efflux of K+ have central importance for the function and survival of vascular smooth muscle cells, but progress in understanding the influx/efflux pathways has been restricted by a lack of identification of the genes underlying many of the non-voltage-gated cationic channels. 2. The present review highlights evidence suggesting the genes are mammalian homologues of the Transient Receptor Potential (TRP) gene of the fruit-fly Drosophila. The weight of evidence supports roles for TRPC1, TRPP2/1 and TRPC6, but recent studies point also to TRPC3, TRPC4/5, TRPV2, TRPM4 and TRPM7. 3. Activity of these TRP channels is suggested to modulate contraction and sense changes in intracellular Ca2+ storage, G-protein-coupled receptor activation and osmotic stress. Roles in relation to myogenic tone, actions of vasoconstrictors substances, Mg2+ homeostasis and the vascular injury response are suggested. 4. Knowledge that TRP channels are relevant to vascular smooth muscle cells in both their contractile and proliferative phenotypes should pave the way for a better understanding of vascular biology and provide the basis for the discovery of a new set of therapeutic agents targeted to vascular disease.     

TRPC6

TRPC6 is a Ca(2+)-permeable non-selective cation channel expressed in brain, smooth muscle containing tissues and kidney, as well as in immune and blood cells. Channel homomers heterologously expressed have a characteristic doubly rectifying current-voltage relationship and are six times more permeable for Ca2+ than for Na+. In smooth muscle tissues, however, Na+ influx and activation of voltage-gated calcium channels by membrane depolarization rather than Ca2+ elevation by TRPC6 channels is the driving force for contraction. TRPC6 channels are directly activated by the second messenger diacylglycerol (DAG) and regulated by specific tyrosine or serine phosphorylation. Extracellular Ca2+ has inhibitory effects, while Ca2+/calmodulin acting from the intracellular side has potentiator effects on channel activity. Given its specific expression, TRPC6 is likely to play a number of physiological roles. Studies with TRPC6(-/-) mice suggest a role for the channel in the regulation of vascular and pulmonary smooth muscle contraction. TRPC6 was identified as an essential component of the slit diaphragm architecture of kidney podocytes. Other functions in immune and blood cells, as well as in brain and in smooth muscle-containing tissues such as stomach, colon and myometrium, remain elusive.     

Activation of the melastatin-related cation channel TRPM3 by D-erythro-sphingosine [corrected

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-sphingosine, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of sphingosine. Sphingosine-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of sphingosine to sphingosine-1-phosphate. Although sphingosine-1-phosphate and ceramides had no effect, two structural analogs of sphingosine, dihydro-D-erythro-sphingosine and N,N-dimethyl-D-erythro-sphingosine, also activated TRPM3. Sphingolipids, including sphingosine, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.     

Newly emerging Ca2+ entry channel molecules that regulate the vascular tone

Local blood flow is critically determined by the arterial tone in which sustained Ca(2+) influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca(2+) entry channel molecules involved in this Ca(2+) influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).     

Role of Ca2+ signaling in the regulation of endothelial permeability

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.     

Canonical transient receptor potential channels in disease: targets for novel drug therapy?

The canonical transient receptor potential (TRPC) channels constitute one of the three major families within the large transient receptor potential (TRP) superfamily. TRPC channels are the closest mammalian homologues of Drosophila TRP, the light-activated channel in Drosophila photoreceptor cells. All TRPC channels (TRPC1-7) are activated via phospholipase-C-coupled receptors and were, therefore, proposed to encode elusive native receptor-activated cation channels in many cell types. A physiological role has been established for all of the known TRPC channels, including the control of vascular tone (TRPC1, TRPC4 and TRPC6) or lymphocyte activation, which is essential for immune competence (TRPC1 and TRPC3). The emergence of TRPC channels in controlling a variety of biological functions offers new and promising targets for drug development.     

Newly emerging Ca2+ entry channel molecules that regulate the vascular tone

Local blood flow is critically determined by the arterial tone in which sustained Ca²⁺ influx, activated by a variety of mechanisms, plays a central regulatory role. Recent progress in molecular biological research has disclosed unexpectedly diverse and complex facets of Ca²⁺ entry channel molecules involved in this Ca²⁺ influx. Candidates include several transient receptor potential (TRP) superfamily members such as TRPC1, TRPC4, TRPC6, TRPV2, TRPV4 and TRPM4, none of which exhibit simple properties attributable to a single particular role. Rather, they appear to be multimodally activated or modulated by receptor stimulation, temperature, mechanical stress or lipid second messengers generated from various sources, and may be involved in both acute vasomotor control and long-term vascular remodelling. This paper provides an overview of existing knowledge of TRP proteins, and their possible relationships with principal factors regulating the arterial tone (i.e., autonomic nerves, various autocrine and paracrine factors, and intravascular pressure).     

血管平滑肌钾通道及其调节因素

血管张力是决定血管阻力和血流量的重要因素 ,而改变钾通道活性能直接影响血管张力。钾通道开放引起钾外流 ,细胞膜超极化 ,关闭电压依赖性钙通道 ,钙内流减少 ,血管舒张 ;当钾通道受抑制时 ,可使细胞膜去极化 ,从而使电压依赖的钙通道开放 ,细胞外钙内流 ,钙离子使肌球蛋白轻链磷酸化 ,粗细肌丝发生相对运动 ,血管收缩。本文介绍血管平滑肌上 4种钾通道的基因结构、电生理学与药理学特性     

Role of Ca2+ mobilization in muscarinic receptor-mediated membrane depolarization in guinea pig ileal smooth muscle cells

In single smooth muscle cells dispersed from guinea pig ileum, the muscarinic agonist carbachol (CCh) at 2 microM produced an oscillatory or sustained type of depolarization and at 100 microM, the latter type depolarization. Depletion of internal Ca2+ stores blocked the oscillatory response, but not the sustained responses to 2 microM and 100 microM CCh, although their decay after reaching the peak became faster. Blocking voltage-dependent Ca2+ channels (VDCCs) blocked both types of response to 2 microM CCh, but only slowed the initial rising phase of 100 microM CCh responses. Combination of Ca2+ store depletion and VDCC blockade abolished the responses to 2 microM CCh again and decreased those to 100 microM CCh in peak amplitude and persistency. Combination of Ca2+ store depletion with removal of extracellular Ca2+ markedly reduced or abolished the 100 microM CCh responses. The results suggest that muscarinic depolarization of the ileal cells requires Ca2+ mobilization for its generation and persistence; at weak muscarinic stimulation, both Ca2+ entry via VDCCs and Ca2+ release from internal stores may contribute to the Ca2+ mobilization; and under strong muscarinic stimulation, Ca2+ entry pathways resistant to VDCC blockers may also contribute to it.     

TRPC channels: interacting proteins

TRP channels, in particular the TRPC and TRPV subfamilies, have emerged as important constituents of the receptor-activated Ca2+ influx mechanism triggered by hormones, growth factors, and neurotransmitters through activation ofphospholipase C (PLC). Several TRPC channels are also activated by passive depletion of endoplasmic reticulum (ER) Ca2+. Although in several studies the native TRP channels faithfully reproduce the respective recombinant channels, more often the properties of Ca2+ entry and/or the store-operated current are strikingly different from that of the TRP channels expressed in the same cells. The present review aims to discuss this disparity in the context of interaction of TRPC channels with auxiliary proteins that may alter the permeation and regulation of TRPC channels.     

血管平滑肌和内皮细胞Ca2+内流机制及其与Cl-通道的关系

血管平滑肌和内皮细胞的Ｃａ２＋内流机制不同,前者是兴奋性细胞,Ｃａ２＋内流通过电压依赖性（ＶＤＣ）和非电压依赖性Ｃａ２＋通道;后者是非兴奋性细胞,Ｃａ２＋内流主要通过非ＶＤＣ途径。Ｃｌ－通道参与了这两种细胞的Ｃａ２＋调控,平滑肌细胞Ｃｌ－通道开放导致细胞膜去极化,促进ＶＤＣ开放,Ｃａ２＋内流增加;而内皮细胞Ｃｌ－通道开放导致细胞膜超极化,使Ｃａ２＋进入细胞内的电化学趋势增加,胞外Ｃａ２＋经非ＶＤＣ途径内流增加。目前对血管平滑肌和内皮细胞Ｃｌ－通道的分型、特性和功能还不清楚。     

POTASSIUM CHANNELS IN VASCULAR SMOOTH MUSCLE

1. Regulation of smooth muscle membrane potential through changes in K⁺ channel activity and subsequent alterations in the activity of voltage-dependent calcium channels is a major mechanism of vasodilation and vasoconstriction, both in normal and pathophysiological conditions. The contribution of a given K⁺ channel type to this mechanism of vascular regulation depends on the vascular bed and species examined. 2. Multiple K⁺ channels are present in most vascular smooth muscle cells and these different K⁺ channels play unique roles in regulating vascular tone. Voltage-dependent K⁺ (Kv) channels are activated by depolarization, may contribute to steady state resting membrane potential and are inhibited by certain vasoconstrictors. Calcium-activated K⁺ (K_Ca) channels oppose the depolarization associated with intrinsic vascular tone and are activated by some endogenous vasodilators. Small-conductance, apamin-sensitive K_Ca channels may be activated by endothelium-derived hyperpolarizing factor. ATP-sensitive K⁺ (K_ATP) channels are activated by pharmacological and endogenous vasodilators. Inward rectifier K⁺ (K_ir) channels are activated by slight changes in extracellular K⁺ and may contribute to resting membrane potential. 3. Membrane potential and diameter are determined, in part, by the integrated activity of several K⁺ channels, which are regulated by multiple dilator and constrictor signals in vascular smooth muscle.     

Ca2+ signaling microdomains:platforms for the assembly and regulation of TRPC channels

The transient receptor potential canonical family (TRPC1-TRPC7) of ion channel proteins, which are activated in response to agonist-stimulated phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] hydrolysis, are proposed components of the elusive store-operated Ca(2+) (SOC) channel. TRPC channels display distinct properties and interact to form homomeric or heteromeric channels that differ in their function and regulation. Although the exact function of TRPC channels and how they are regulated has not been established, increasing data suggest that they are localized and regulated within Ca(2+) signaling microdomains. TRPC channels contribute to store-operated and store-independent Ca(2+) entry mechanisms, both of which are activated by agonist-stimulated PtdIns(4,5)P(2) hydrolysis. Elucidation of how cells achieve specificity and precise temporal and spatial coordination of channel activation is crucial for understanding the molecular basis of agonist-mediated stimulation of Ca(2+) entry and identifying downstream physiological functions. This review will address the assembly and localization of TRPC channels and how these processes impact their function.     

Potentiation of stretch-induced tone in the rabbit facial vein by an isoquinoline derivative, LOE 908

The rabbit facial vein exhibits extracellular Ca2+- and temperature-dependent spontaneous myogenic tone in response to stretch. The present study aimed to elucidate pharmacological characteristics of Ca2+ entry mechanisms responsible for the stretch-induced tension development of the rabbit facial vein. Ca2+- and temperature-sensitive vascular tone in response to stretch was refractory to L-type Ca2+ channel blockers such as nifedipine and diltiazem but was abolished by papaverine or SK&F 96365 which blocks both receptor- and store-operated Ca2+ channels. Interestingly, LOE 908, another type of voltage-independent Ca2+-permeable channel blocker, showed augmentation of the stretch-induced vascular tone instead of inhibition. Potentiation by LOE 908 of stretch-induced vascular tone was also extracellular Ca2+-dependent and counteracted by SK&F 96365. Membrane stretch-activated Ca2+ channels in the rabbit facial vein smooth muscle cells may have a unique characteristic that their opening is stimulated by LOE 908 and thus is distinguishable from other voltage-independent Ca2+-permeable channels.