NPY, ET-1, and Ang II nuclear receptors in human endocardial endothelial cells

Neuropeptide Y (NPY), endothelin-1 (ET-1), and angiotensin II (Ang II) are peptides that are known to play many important roles in cardiovascular homeostasis. The physiological actions of these peptides are thought to be primarily mediated by plasma membrane receptors that belong to the G-protein-coupled receptor superfamily. However, there is increasing evidence that suggests the existence of functional G-protein-coupled receptors at the level of the nucleus and that the nucleus could be a cell within a cell. Here, we review our work showing the presence in the nucleus of the NPY Y(1) receptor, the ET(A) and ET(B) receptors, as well as the AT(1) and AT(2) receptors and their respective ligands. This work was carried out in 20-week-old fetal human endocardial endothelial cells. Our results demonstrate that nuclear Y1, AT(1), and ET(A) receptors modulate nuclear calcium in these cells.     

Involvement of extracellular Ca2+ influx through voltage-independent Ca2+ channels in endothelin-1 function

This article reviews the types and roles of voltage-independent Ca(2+) channels involved in the endothelin-1 (ET-1)-induced functional responses such as vascular contraction, cell proliferation, and intracellular Ca(2+)-dependent signaling pathways and discusses the molecular mechanisms for the activation of voltage-independent Ca(2+) channels by ET-1. ET-1 activates some types of voltage-independent Ca(2+) channels, such as Ca(2+)-permeable nonselective cation channels (NSCCs) and store-operated Ca(2+) channels (SOCC). Extracellular Ca(2+) influx through these voltage-independent Ca(2+) channels plays essential roles in ET-1-induced vascular contraction, cell proliferation, activation of epidermal growth factor receptor tyrosine kinase, regulation of proline-rich tyrosine kinase, and release of arachidonic acid. The experiments using various constructs of endothelin receptors reveal the importance of G(q) and G(12) families in activation of these Ca(2+) channels by ET-1. These findings provide a potential therapeutic mechanism of a functional interrelationship between G(q)/G(12) proteins and voltage-independent Ca(2+) channels in the pathophysiology of ET-1, such as in chronic heart failure, hypertension, and cerebral vasospasm.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

TREK-1 K+ channels couple angiotensin II receptors to membrane depolarization and aldosterone secretion in bovine adrenal glomerulosa cells

Bovine adrenal glomerulosa (AZG) cells were shown to express bTREK-1 background K(+) channels that set the resting membrane potential and couple angiotensin II (ANG II) receptor activation to membrane depolarization and aldosterone secretion. Northern blot and in situ hybridization studies demonstrated that bTREK-1 mRNA is uniformly distributed in the bovine adrenal cortex, including zona fasciculata and zona glomerulosa, but is absent from the medulla. TASK-3 mRNA, which codes for the predominant background K(+) channel in rat AZG cells, is undetectable in the bovine adrenal cortex. In whole cell voltage clamp recordings, bovine AZG cells express a rapidly inactivating voltage-gated K(+) current and a noninactivating background K(+) current with properties that collectively identify it as bTREK-1. The outwardly rectifying K(+) current was activated by intracellular acidification, ATP, and superfusion of bTREK-1 openers, including arachidonic acid (AA) and cinnamyl 1-3,4-dihydroxy-alpha-cyanocinnamate (CDC). Bovine chromaffin cells did not express this current. In voltage and current clamp recordings, ANG II (10 nM) selectively inhibited the noninactivating K(+) current by 82.1 +/- 6.1% and depolarized AZG cells by 31.6 +/- 2.3 mV. CDC and AA overwhelmed ANG II-mediated inhibition of bTREK-1 and restored the resting membrane potential to its control value even in the continued presence of ANG II. Vasopressin (50 nM), which also physiologically stimulates aldosterone secretion, inhibited the background K(+) current by 73.8 +/- 9.4%. In contrast to its potent inhibition of bTREK-1, ANG II failed to alter the T-type Ca(2+) current measured over a wide range of test potentials by using pipette solutions of identical nucleotide and Ca(2+)-buffering compositions. ANG II also failed to alter the voltage dependence of T channel activation under these same conditions. Overall, these results identify bTREK-1 K(+) channels as a pivotal control point where ANG II receptor activation is transduced to depolarization-dependent Ca(2+) entry and aldosterone secretion.     

Ca2+ responses to thyrotropin-releasing hormone and angiotensin II: the role of plasma membrane integrity and effect of G11alpha protein overexpression on homologous and heterologous desensitization

The molecular mechanisms involved in GPCR-initiated signaling cascades where the two receptors share the same signaling cascade, such as thyrotropin-releasing hormone (TRH) and angiotensin II (ANG II), are still far from being understood. Here, we analyzed hormone-induced Ca(2+) responses and the process of desensitization in HEK-293 cells, which express endogenous ANG II receptors. These cells were transfected to express exogenously high levels of TRH receptors (clone E2) or both TRH receptors and G(11)alpha protein (clone E2M11). We observed that the characteristics of the Ca(2+) response, as well as the process of desensitization, were both strongly dependent on receptor number and G(11)alpha protein level. Whereas treatment of E2 cells with TRH or ANG II led to significant desensitization of the Ca(2+) response to subsequent addition of either hormone, the response was not desensitized in E2M11 cells expressing high levels of G(11)alpha. In addition, stimulation of both cell lines with THR elicited a clear heterologous desensitization to subsequent stimulation with ANG II. On the other hand, ANG II did not affect a subsequent response to TRH. ANG II-mediated signal transduction was strongly dependent on plasma membrane integrity modified by cholesterol depletion, but signaling through TRH receptors was altered only slightly under these conditions. It may be concluded that the level of expression of G-protein-coupled receptors and their cognate G-proteins strongly influences not only the magnitude of the Ca(2+) response but also the process of desensitization and resistance to subsequent hormone addition.     

Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis-dependent mechanisms

Bovine adrenocortical cells express bTREK-1 K+ channels that set the resting membrane potential (V(m)) and couple angiotensin II (AngII) and adrenocorticotropic hormone (ACTH) receptors to membrane depolarization and corticosteroid secretion. In this study, it was discovered that AngII inhibits bTREK-1 by separate Ca2+- and ATP hydrolysis-dependent signaling pathways. When whole cell patch clamp recordings were made with pipette solutions that support activation of both Ca2+- and ATP-dependent pathways, AngII was significantly more potent and effective at inhibiting bTREK-1 and depolarizing adrenal zona fasciculata cells, than when either pathway is activated separately. External ATP also inhibited bTREK-1 through these two pathways, but ACTH displayed no Ca2+-dependent inhibition. AngII-mediated inhibition of bTREK-1 through the novel Ca2+-dependent pathway was blocked by the AT1 receptor antagonist losartan, or by including guanosine-5'-O-(2-thiodiphosphate) in the pipette solution. The Ca2+-dependent inhibition of bTREK-1 by AngII was blunted in the absence of external Ca2+ or by including the phospholipase C antagonist U73122, the inositol 1,4,5-trisphosphate receptor antagonist 2-amino-ethoxydiphenyl borate, or a calmodulin inhibitory peptide in the pipette solution. The activity of unitary bTREK-1 channels in inside-out patches from adrenal zona fasciculata cells was inhibited by application of Ca2+ (5 or 10 microM) to the cytoplasmic membrane surface. The Ca2+ ionophore ionomycin also inhibited bTREK-1 currents through channels expressed in CHO-K1 cells. These results demonstrate that AngII and selected paracrine factors that act through phospholipase C inhibit bTREK-1 in adrenocortical cells through simultaneous activation of separate Ca2+- and ATP hydrolysis-dependent signaling pathways, providing for efficient membrane depolarization. The novel Ca2+-dependent pathway is distinctive in its lack of ATP dependence, and is clearly different from the calmodulin kinase-dependent mechanism by which AngII modulates T-type Ca2+ channels in these cells.     

Reversed Na+/Ca2+ exchange contributes to Ca2+ influx and respiratory burst in microglia

Phagocytosis and the ensuing NADPH-mediated respiratory burst are important aspects of microglial activation that require calcium ion (Ca(2+)) influx. However, the specific Ca(2+) entry pathway(s) that regulates this mechanism remains unclear, with the best candidates being surface membrane Ca(2+)-permeable ion channels or Na(+)/Ca(2+) exchangers. In order to address this issue, we used quantitative real-time RT-PCR to assess mRNA expression of the Na(+)/Ca(2+) exchangers, Slc8a1-3/NCX1-3, before and after phagocytosis by rat microglia. All three Na(+)/Ca(2+) exchangers were expressed, with mRNA levels of NCX1 > NCX3 > NCX2, and were unaltered during the one hour phagocytosis period. We then carried out a biophysical characterization of Na(+)/Ca(2+) exchanger activity in these cells. To investigate conditions under which Na(+)/Ca(2+) exchange was functional, we used a combination of perforated patch-clamp analysis, fluorescence imaging of a Ca(2+) indicator (Fura-2) and a Na(+) indicator (SBFI), and manipulations of membrane potential and intracellular and extracellular ions. Then, we used a pharmacological toolbox to compare the contribution of Na(+)/Ca(2+) exchange with candidate Ca(2+)-permeable channels, to the NADPH-mediated respiratory burst that was triggered by phagocytosis. We find that inhibiting the reversed mode of the Na(+)/Ca(2+) exchanger with KB-R7943, dose dependently reduced the phagocytosis-stimulated respiratory burst; whereas, blockers of store-operated Ca(2+) channels or L-type voltage-gated Ca(2+) channels had no effect. These results provide evidence that Na(+)/Ca(2+) exchangers are potential therapeutic targets for reducing the bystander damage that often results from microglia activation in the damaged CNS.     

Nuclear membrane receptors for ET-1 in cardiovascular function

Plasma membrane endothelin type A (ET(A)) receptors are internalized and recycled to the plasma membrane, whereas endothelin type B (ET(B)) receptors undergo degradation and subsequent nuclear translocation. Recent studies show that G protein-coupled receptors (GPCRs) and ion transporters are also present and functional at the nuclear membranes of many cell types. Similarly to other GPCRs, ET(A) and ET(B) are present at both the plasma and nuclear membranes of several cardiovascular cell types, including human cardiac, vascular smooth muscle, endocardial endothelial, and vascular endothelial cells. The distribution and density of ET(A)Rs in the cytosol (including the cell membrane) and the nucleus (including the nuclear membranes) differ between these cell types. However, the localization and density of ET-1 and ET(B) receptors are similar in these cell types. The extracellular ET-1-induced increase in cytosolic ([Ca](c)) and nuclear ([Ca](n)) free Ca(2+) is associated with an increase of cytosolic and nuclear reactive oxygen species. The extracellular ET-1-induced increase of [Ca](c) and [Ca](n) as well as intracellular ET-1-induced increase of [Ca](n) are cell-type dependent. The type of ET-1 receptor mediating the extracellular ET-1-induced increase of [Ca](c) and [Ca](n) depends on the cell type. However, the cytosolic ET-1-induced increase of [Ca](n) does not depend on cell type. In conclusion, nuclear membranes' ET-1 receptors may play an important role in overall ET-1 action. These nuclear membrane ET-1 receptors could be targets for a new generation of antagonists.     

Influence of membrane ion channel in pituitary somatotrophs by hypothalamic regulators

The secretion of growth hormone (GH) from somatotrophs located within the anterior pituitary gland is stimulated by endogenous hypothalamic growth hormone-releasing hormone (GHRH) and the GH secretagogue (GHS) ghrelin, and inhibited by somatotropin-releasing inhibitory factor (SRIF, also known as somatostatin). These factors bind to specific G-protein-coupled receptors on the cell membrane, and directly or indirectly modify the properties of ion channels and second messenger systems. Ultimately this results in a change in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and the secretion of GH. Somatotrophs possess a variety of ion channels on their membranes, and modification of these ion channels, especially Ca(2+), K(+), and Na(+) channels, is tightly linked to intracellular Ca(2+) levels and therefore hormone secretion. Various issues regarding receptor distribution, role of ion channels, alteration of membrane potential, and involvement of intracellular signaling system in the control of GH secretion are discussed in this review. In particular, this work will focus on ion channels and [Ca(2+)](i) in somatotrophs.     

Calcium channels, transporters and exchangers in placenta: a review

Calcium (Ca2+) entry in cells is crucial for development and physiology of virtually all cell types. It acts as an intracellular (second) messenger to regulate a diverse array of cellular functions, from cell division and differentiation to cell death. Among candidates for Ca2+ entry in cells are-voltage-dependant Ca2+ channels (VDCCs), transient receptor potential (TRP)-related Ca2+ channels and store-operated Ca2+ (SOC) channels. Plasma membrane Ca2+-ATPases (PMCA) and Na+/Ca2+ exchanger (NCX) are mainly responsible for Ca2+ extrusion. These different Ca2+channels/transporters and exchangers exhibit specific distribution and physiological properties. During pregnancy, the syncytiotrophoblast layer of the human placenta transfers as much as 30 g of Ca2+ from the mother to the fetus, especially in late gestation where Ca2+ transport through different channels must increase in response to the demands of accelerating bone mineralization of the fetus. The identification and characterization of the different Ca2+ channels/transporters and exchangers on the brush-border membrane (BBM) facing the maternal circulation, and the basal plasma membrane (BPM) facing the fetal circulation; placental membrane of the syncytiotrophoblasts have been the focus of numerous studies. This review discusses current views in this field regarding localization and functions during transcellular Ca2+ entry and extrusion from cells particularly in the placenta.     

Atorvastatin inhibits angiotensin II-induced T-type Ca2+ channel expression in endothelial cells

Ca2+ channels are involved in the regulation of vascular functions. Angiotensin II is implicated in the development of atherosclerosis and vascular remodeling. In this study, we demonstrated that angiotensin II preferentially increased the expression of alpha1G, a T-type Ca2+ channel subunit, via AT1 receptors in endothelial cells. Angiotensin II-induced expression of alpha1G was inhibited by pretreatment with atorvastatin and the MEK1/2 inhibitor, PD98059. The effect of atorvastatin was reversed by mevalonate and farnesyl pyrophosphate which implicates the activation of the small GTP-binding protein, Ras. Our data indicate that angiotensin II induces alpha1G expression in endothelial cells via AT1 receptors, Ras and MEK. Angiotensin II-induced migration of endothelial cells in a wound healing model was inhibited by incubation with mibefradil, a T-type Ca2+ channel blocker. Our data indicate that angiotensin II induces T-type Ca2+ channels in endothelial cells, which may play a role in the development of vascular disorders.     

Plasma membrane Ca2+ pumps and Na+/Ca2+ exchangers

Membrane-intrinsic transport systems play an essential role in intracellular Ca2+ homeostasis. ATP-driven Ca2+ pumps and carrier-mediated Na+/Ca2+ exchangers are the two specific Ca2+ transporting systems mainly responsible for Ca2+ extrusion across the plasma membrane. Ca2+ pumps operate in all eukaryotic cell types and are characterized by their high Ca2+ affinity and their specific regulation by direct interaction with Ca2+/calmodulin. Na+/Ca2+ exchangers are particularly abundant in excitable tissues and are responsible for the bulk Ca2+ efflux in these tissues. Recent success in the molecular characterization of the pumps has led to the determination of complete amino acid sequences for several isoforms and has allowed the identification and topological assignment of important functional and regulatory domains. Genetic evidence indicates that mammalian Ca2+ pump diversity is generated from a multigene family and via alternative RNA splicing. Different isoforms may vary in their regulatory properties, presumably reflecting different physiological requirements of the tissues of their expression. Although the molecular characterization of Na+/Ca2+ exchangers is not as far advanced as that of the pumps, recent studies have established detailed kinetic, stoichiometric and regulatory properties of these systems. Together with advances in expression cloning methods these studies promise to result in a rapid improvement of our knowledge of the functional properties of these ion transporters on a molecular level.     

Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes

We have examined the distribution of ryanodine receptors, L-type Ca(2+) channels, calsequestrin, Na(+)/Ca(2+) exchangers, and voltage-gated Na(+) channels in adult rat ventricular myocytes. Enzymatically dissociated cells were fixed and dual-labeled with specific antibodies using standard immunocytochemistry protocols. Images were deconvolved to reverse the optical distortion produced by wide-field microscopes equipped with high numerical aperture objectives. Every image showed a well-ordered array of fluorescent spots, indicating that all of the proteins examined were distributed in discrete clusters throughout the cell. Mathematical analysis of the images revealed that dyads contained only ryanodine receptors, L-type Ca(2+) channels, and calsequestrin, and excluded Na(+)/Ca(2+) exchangers and voltage-gated Na(+) channels. The Na(+)/Ca(2+) exchanger and voltage-gated Na(+) channels were distributed largely within the t-tubules, on both transverse and axial elements, but were not co-localized. The t-tubule can therefore be subdivided into at least three structural domains; one of coupling (dyads), one containing the Na(+)/Ca(2+) exchanger, and one containing voltage-gated Na(+) channels. We conclude that if either the slip mode conductance of the Na(+) channel or the reverse mode of the Na(+)/Ca(2+) exchanger are to contribute to the contractile force, the fuzzy space must extend outside of the dyad.     

Assessment of frequency-dependent alterations in the level of extracellular Ca2+ in the synaptic cleft.

The synaptic cleft may be represented as a very thin disk of extracellular fluid. It is possible that at high stimulation frequencies the interval between pulses would be insufficient for diffusion of Ca2+ from the periphery of the cleft to replace extracellular Ca2+ depleted at the center of the cleft as a result of activation of postsynaptic, Ca2(+)-permeable channels. Computer modeling was employed to assess the impact of activation of glutamate receptor channels (GRCs) in the postsynaptic membrane on the level of extracellular Ca2+ within the synaptic cleft. The model includes calcium influx from the synaptic cleft into the postsynaptic compartment through GRC and calcium efflux through calcium pumps and Na/Ca exchangers. Concentrations of extracellular Ca2+ inside the cleft are estimated by using a compartmental model incorporating flux across the postsynaptic membrane and radial diffusion from the edges of the cleft. The simulations suggest that substantial extracellular Ca2+ depletion can occur in the clefts during activation of GRCs, particularly at high stimulation frequencies used to induce long-term potentiation (LTP). Only minimal transitory changes in extracellular Ca2+ are observed at low frequencies. These frequency-dependent alterations in extracellular Ca2+ dynamics are a direct reflection of the activity of GRCs and could be involved in the modulation of presynaptic function via a retrograde messenger mechanism, if there are extracellular Ca2+ sensors on the presynaptic membranes. The recently cloned extracellular Ca2(+)-sensing receptors that are known to be present in nerve terminals in hippocampus and other areas of the brain could potentially play such a role.     

Physiological properties and functions of Ca(2+) sparks in rat intrapulmonary arterial smooth muscle cells

Ca(+) spark has been implicated as a pivotal feedback mechanism for regulating membrane potential and vasomotor tone in systemic arterial smooth muscle cells (SASMCs), but little is known about its properties in pulmonary arterial smooth muscle cells (PASMCs). Using confocal microscopy, we identified spontaneous Ca(2+) sparks in rat intralobar PASMCs and characterized their spatiotemporal properties and physiological functions. Ca(2+) sparks of PASMCs had a lower frequency and smaller amplitude than cardiac sparks. They were abolished by inhibition of ryanodine receptors but not by inhibition of inositol trisphosphate receptors and L-type Ca(2+) channels. Enhanced Ca(2+) influx by BAY K8644, K(+), or high Ca(2+) caused a significant increase in spark frequency. Functionally, enhancing Ca(2+) sparks with caffeine (0.5 mM) caused membrane depolarization in PASMCs, in contrast to hyperpolarization in SASMCs. Norepinephrine and endothelin-1 both caused global elevations in cytosolic Ca(2+) concentration ([Ca(2+)]), but only endothelin-1 increased spark frequency. These results suggest that Ca(2+) sparks of PASMCs are similar to those of SASMCs, originate from ryanodine receptors, and are enhanced by Ca(2+) influx. However, they play a different modulatory role on membrane potential and are under agonist-specific regulation independent of global [Ca(2+)].     

Evidence for bile acid-evoked oscillations of Ca2(+)-dependent K+ permeability unrelated to a D-myo-inositol 1,4,5-trisphosphate effect in isolated guinea pig liver cells

In single liver cells, the D-myo-inositol 1,4,5-triphosphate (InsP3)-dependent agonists such as noradrenaline and angiotensin II evoke oscillations in intracellular calcium [Ca2+]i resulting mostly from the periodic release and reuptake of calcium from intracellular stores. In the present work, we have reexamined the effects of these agonists and investigated whether the natural bile acid taurolithocholic acid 3-sulfate (TLC-S), which permeabilizes the endoplasmic reticulum, could initiate oscillations of [Ca2+]i. Oscillations of [Ca2+]i were monitored with the Ca2(+)-dependent K+ permeability in whole-cell voltage-clamped guinea pig liver cells. Our results confirm the presence of two types of oscillations induced by hormones. They could be distinguished by their frequency periods. The fast (type I) had periods ranging from 5 to 12 s and the slow (type II) from 60 to 240 s. They have been respectively attributed to second messenger- and receptor-controlled oscillations, respectively. Our results also show that TLC-S, as noradrenaline and angiotensin II, induced the activation of this Ca(+)-dependent K+ current and was able to reproduce both types of oscillations. The bile acid effect was not blocked by intracellular perfusion of heparin known to inhibit both InsP3 binding and InsP3-evoked Ca2+ release in several tissues. In these conditions, TLC-S only evoked type I oscillations, suggesting that these fluctuations could originate from a mechanism that is independent of InsP3 and is an intrinsic property of internal Ca2+ stores.