Insulin receptor signaling for the proliferation of pancreatic β-cells: Involvement of Ca2+ second messengers,IP3, NAADP and cADPR

Insulin has an autocrine/paracrine role through insulin receptors in pancreatic β-cells. Herein, we show the insulin receptor signaling pathway underlying CD38/ADPR-cyclase activation for NAADP/cADPR formation to induce Ca2+ rise, ultimately resulting in β-cell proliferation. Binding of insulin on insulin receptors leads to the activation of IRS/Akt/PI3K/PLC. Activation of PLC generates IP3 and DAG; the former induces Ca²⁺ release, resulting in activation of CD38/ADPR-cyclase for cADPR production via cGMP-dependent mechanism and the latter activates PKC, resulting in activation of ADPR-cyclase for NAADP synthesis. The NAADP-induced Ca²⁺ signal is required for IP₃-induced Ca²⁺ release from the ER. CD38 plays an important role in insulin receptor signaling in β-cells by reflecting a declined sustained Ca²⁺ signal, cADPR levels, and β-cell proliferation in response to insulin in CD38^-/- islets. However, evidence indicates that a hitherto-unidentified ADPR cyclase in addition to CD38 participates in insulin-induced signaling through cADPR and NAADP synthesis. In conclusion, insulin receptor signaling in β-cells employs three Ca²⁺ signaling messengers, IP₃, NAADP, and cADPR through a complex but concerted action of signaling molecules for Ca2+ signaling, which is involved in the proliferation of the islets.     

Involvement of actin filament in the generation of Ca2+ mobilizing messengers in glucose-induced Ca2+ signaling in pancreatic β-cells

Glucose is a metabolic regulator of insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca²⁺ signaling. We and others previously demonstrated that glucose activates CD38/ADP-ribosyl cyclase (ADPR-cyclase) to produce two Ca²⁺ second messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Although F-actin remodeling is known to be an important step in glucose stimulated insulin secretion, the role of actin cytoskeleton in regulating Ca²⁺ signaling in pancreatic β-cells remain to be solved. Here, we show that actin filaments are involved in the activation of CD38/ADPR-cyclase in pancreatic β-cells. Glucose induces a sequential formation of cADPR and NAADP. Pretreatment with jasplakinolide, an actin polymerizing agent, or a myosin heavy chain IIA (MHCIIA) blocker, blebbistatin, inhibited glucose-induced CD38 internalization, an essential step for cADPR formation. Blocking actin disassembly with jasplakinolide also abrogates glucose-induced cADPR and NAADP formation and sustained Ca²⁺ signals. These results indicate that actin filaments along with MHCIIA play an important role in CD38 internalization for the generation of Ca²⁺ mobilizing messengers for glucose-induced Ca²⁺ signaling in pancreatic β-cells.     

Remodeling of ryanodine receptor complex causes "leaky" channels: a molecular mechanism for decreased exercise capacity

During exercise, defects in calcium (Ca²⁺) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca²⁺ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca²⁺-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca²⁺ leak via calstabin1-depleted RyR1 channels leads to defective Ca²⁺ signaling, muscle damage, and impaired exercise capacity.     

Nicotinic acid adenine dinucleotide phosphate mediates Ca2+ signals and contraction in arterial smooth muscle via a two-pool mechanism

Previous studies of arterial smooth muscle have shown that inositol 1,4,5-trisphosphate (IP3) and cyclic ADP-ribose mobilize Ca2+ from the sarcoplasmic reticulum. In contrast, little is known about Ca2+ mobilization by nicotinic acid adenine dinucleotide phosphate, a pyridine nucleotide derived from beta-NADP+. We show here that intracellular dialysis of nicotinic acid adenine dinucleotide phosphate (NAADP) induces spatially restricted "bursts" of Ca2+ release that initiate a global Ca2+ wave and contraction in pulmonary artery smooth muscle cells. Depletion of sarcoplasmic reticulum Ca2+ stores with thapsigargin and inhibition of ryanodine receptors with ryanodine, respectively, block the global Ca2+ waves by NAADP. Under these conditions, however, localized Ca2+ bursts are still observed. In contrast, xestospongin C, an IP3 receptor antagonist, had no effect on Ca2+ signals by NAADP. We propose that NAADP mobilizes Ca2+ via a 2-pool mechanism, and that initial Ca2+ bursts are amplified by subsequent sarcoplasmic reticulum Ca2+ release via ryanodine receptors but not via IP3 receptors.     

Structural evidence for perinuclear calcium microdomains in cardiac myocytes

At each heartbeat, cardiac myocytes are activated by a cytoplasmic Ca²⁺ transient in great part due to Ca²⁺ release from the sarcoplasmic reticulum via ryanodine receptors (RyRs) clustered within calcium release units (peripheral couplings/dyads). A Ca²⁺ transient also occurs in the nucleoplasm, following the cytoplasmic transient with some delay. Under conditions where the InsP3 production is stimulated, these Ca²⁺ transients are regulated actively, presumably by an additional release of Ca²⁺ via InsP3 receptors (InsP3Rs). This raises the question whether InsP3Rs are appropriately located for this effect and whether sources of InsP3 and Ca²⁺ are available for their activation. We have defined the structural basis for InsP3R activity at the nucleus, using immunolabeling for confocal microscopy and freeze-drying/shadowing, T tubule “staining” and thin sectioning for electron microscopy. By these means we establish the presence of InsP3R at the outer nuclear envelope and show a close spatial relationship between the nuclear envelope, T tubules (a likely source of InsP3) and dyads (the known source of Ca²⁺). The frequency, distribution and distance from the nucleus of T tubules and dyads appropriately establish local perinuclear Ca²⁺ microdomains in cardiac myocytes.     

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2–dependent Ca2+ signaling

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca²⁺ signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca²⁺ mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca²⁺ stores, resulting in Ca²⁺ release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2^−/− mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca²⁺ release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca²⁺ release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2^−/− mice, but was unaffected in Tpcn1^−/− animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca²⁺ signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.In the adult the formation of new capillaries is an uncommon occurrence mostly restricted to pathological rather than physiological conditions, the majority of blood vessels remaining quiescent once organ growth is accomplished (1). Physiological neoangiogenesis is generally restricted to body sites undergoing regeneration or restructuring (e.g., tissue lesion repair and corpus luteum formation), whereas pathological neoangiogenesis takes place in different diseases ranging from macular degeneration to atherosclerosis, and is vital for the highly noxious development of solid tumors, thus representing a promising target for therapeutic strategies (2). Vascular endothelial growth factors (VEGF), and in particular the family member VEGF-A, are major regulators of angiogenesis and regulate ECs, mainly through the stimulation of VEGF receptor-2 (VEGFR2), a receptor tyrosine kinase, to induce cell proliferation, migration, and sprouting in the early stages of angiogenesis (3, 4). Antiangiogenic agents that target VEGF signaling have become an important component of therapies in multiple cancers, but their use is limited by acquisition of resistance to their therapeutic effects (5, 6). When overall VEGF receptor (VEGFR) signaling is experimentally impaired by the use of blocking antibodies or of specific tyrosine kinase inhibitors, alternative cellular and tissue strategies nullify the success of such interventions (5, 7, 8). Resistance to anti-VEGF therapies may occur through a variety of mechanisms, including evocation of alternative compensatory factors, selection of hypoxia-resistant tumor cells, action of proangiogenic circulating cells, and increased circulating nontumor proangiogenic factors. Moreover, cross-interactions (both cellular and humoral) between ECs and other environmental cues have to be taken into account for the ultimate aim of tailoring therapeutic interventions according to the specific pattern of the angiogenic microenvironment and EC conditions (5–7). The search for novel key downstream effectors is therefore of potential significance in the perspective of angiogenesis control in cancer progression.Autophosphorylation of VEGFR2 upon binding VEGF results in the activation of downstream signaling cascades through complex and manifold molecular interactions that transmit signals leading to angiogenic responses. Stimulation of different EC types via VEGFR2 results in increases in intracellular free calcium concentrations [Ca²⁺]_i (9, 10) and the crucial role of this signaling element in the regulation of EC functions and angiogenesis is recognized (11, 12), and thought to be largely mediated by the phospholipase Cγ (PLCγ)/inositol 1,4,5 trisphosphate (IP₃) signaling pathway (10). It has been reported that IP₃ releases Ca²⁺ from intracellular stores in ECs, increasing [Ca²⁺]_i, and is augmented by store-operated Ca²⁺ influx (13). This signaling primes the endothelium for angiogenesis through the activation of downstream effectors such as endothelial nitric oxide synthase (eNOS), protein kinases C (PKC), and mitogen-activated protein kinases (MAPKs). Indeed, it has been reported that the interplay between IP₃-dependent Ca²⁺ mobilization and store-operated Ca²⁺ entry produces Ca²⁺ signals whose inhibition impairs the angiogenic effect of VEGF (14, 15). Given the complexity of both VEGF and Ca²⁺ signaling, and the crucial finding that VEGF evokes pro- and antiangiogenic responses, it is clear that the specificity of VEGF-evoked Ca²⁺ signatures deserves further investigation.Differences in Ca²⁺ signatures, which are key to determining specific Ca²⁺-dependent cellular responses, rely upon often complex spatiotemporal variations in [Ca²⁺]_i (16). A major determinant of these are based on functionally distinct intracellular Ca²⁺-mobilizing messengers, namely IP₃ and cyclic adenosine diphosphoribose (cADPR), which mobilize Ca²⁺ from the endoplasmic reticulum (ER) stores, and nicotinic acid adenine dinucleotide phosphate (NAADP), which triggers Ca²⁺ release from acidic organelles, such as lysosomes and endosomes (17, 18). NAADP likely targets a channel distinct from IP₃ and ryanodine receptors (RyRs), known as two-pore channels (TPCs) (19–25), and the resulting localized NAADP-evoked Ca²⁺ signals may in some cases be globalized via IP₃ and RyRs through Ca²⁺-induced Ca²⁺ release (26, 27). However, in a few cell types, direct activation of RyRs and Ca²⁺ influx channels by NAADP have also been proposed as alternative mechanisms (28, 29). It has been demonstrated that NAADP-sensitive Ca²⁺ stores are present in the endothelium, and that NAADP is capable of regulating vascular smooth muscle contractility and blood pressure by EC-dependent mechanisms (30). In addition, we have previously demonstrated that NAADP is a specific and essential intracellular mediator of ECs histamine H₁ receptors, evoking [Ca²⁺]_i release and secretion of von Willebrand factor, which requires the functional expression of TPCs (31).In the present work, we identify a novel pathway for VEGFR2 signal transduction whereby receptor activation leads to NAADP and TPC2-dependent Ca²⁺ release from acidic Ca²⁺ stores, which in turn controls angiogenic response in vitro and in vivo. These findings demonstrate, to our knowledge for the first time, the direct relationship between NAADP-mediated Ca²⁺ release and the signaling mechanisms underlying ECs angiogenesis mediated by VEGF.     

CD4+ T cell regulation of CD25 expression controls development of short-lived effector CD8+ T cells in primary and secondary responses

Both CD4⁺ T cell help and IL-2 have been postulated to “program” activated CD8⁺ T cells for memory cell development. However, the linkage between these two signals has not been well elucidated. Here we have studied effector and memory CD8⁺ T cell differentiation following infection with three pathogens (Listeria monocytogenes, vesicular stomatitis virus, and vaccinia virus) in the absence of both CD4⁺ T cells and IL-2 signaling. We found that expression of CD25 on antigen-specific CD8⁺ T cells peaked 3–4 days after initial priming and was dependent on CD4⁺ T cell help, likely through a CD28:CD80/86 mediated pathway. CD4⁺ T cell or CD25-deficiency led to normal early effector CD8⁺ T cell differentiation, but a subsequent lack of accumulation of CD8⁺ T cells resulting in overall decreased memory cell generation. Interestingly, in both primary and recall responses KLRG1^high CD127^low short-lived effector cells were drastically diminished in the absence of IL-2 signaling, although memory precursors remained intact. In contrast to previous reports, upon secondary antigen encounter CD25-deficient CD8⁺ T cells were capable of undergoing robust expansion, but short-lived effector development was again impaired. Thus, these results demonstrated that CD4⁺ T cell help and IL-2 signaling were linked via CD25 up-regulation, which controls the expansion and differentiation of antigen-specific effector CD8⁺ T cells, rather than “programming” memory cell traits.     

Crystal structure of type I ryanodine receptor amino-terminal β-trefoil domain reveals a disease-associated mutation “hot spot” loop

Muscle contraction and relaxation is regulated by transient elevations of myoplasmic Ca²⁺. Ca²⁺ is released from stores in the lumen of the sarco(endo)plasmic reticulum (SER) to initiate formation of the Ca²⁺ transient by activation of a class of Ca²⁺ release channels referred to as ryanodine receptors (RyRs) and is pumped back into the SER lumen by Ca²⁺-ATPases (SERCAs) to terminate the Ca²⁺ transient. Mutations in the type 1 ryanodine receptor gene, RYR1, are associated with 2 skeletal muscle disorders, malignant hyperthermia (MH), and central core disease (CCD). The evaluation of proposed mechanisms by which RyR1 mutations cause MH and CCD is hindered by the lack of high-resolution structural information. Here, we report the crystal structure of the N-terminal 210 residues of RyR1 (RyR_NTD) at 2.5 Å. The RyR_NTD structure is similar to that of the suppressor domain of type 1 inositol 1,4,5-trisphosphate receptor (IP₃Rsup), but lacks most of the long helix-turn-helix segment of the “arm” domain in IP₃Rsup. The N-terminal β-trefoil fold, found in both RyR and IP₃R, is likely to play a critical role in regulatory mechanisms in this channel family. A disease-associated mutation “hot spot” loop was identified between strands 8 and 9 in a highly basic region of RyR1. Biophysical studies showed that 3 MH-associated mutations (C36R, R164C, and R178C) do not adversely affect the global stability or fold of RyR_NTD, supporting previously described mechanisms whereby mutations perturb protein–protein interactions.     

Leaky RyR2 trigger ventricular arrhythmias in Duchenne muscular dystrophy

Patients with Duchenne muscular dystrophy (DMD) have a progressive dilated cardiomyopathy associated with fatal cardiac arrhythmias. Electrical and functional abnormalities have been attributed to cardiac fibrosis; however, electrical abnormalities may occur in the absence of overt cardiac histopathology. Here we show that structural and functional remodeling of the cardiac sarcoplasmic reticulum (SR) Ca²⁺ release channel/ryanodine receptor (RyR2) occurs in the mdx mouse model of DMD. RyR2 from mdx hearts were S-nitrosylated and depleted of calstabin2 (FKBP12.6), resulting in “leaky” RyR2 channels and a diastolic SR Ca²⁺ leak. Inhibiting the depletion of calstabin2 from the RyR2 complex with the Ca²⁺ channel stabilizer S107 (“rycal”) inhibited the SR Ca²⁺ leak, inhibited aberrant depolarization in isolated cardiomyocytes, and prevented arrhythmias in vivo. This suggests that diastolic SR Ca²⁺ leak via RyR2 due to S-nitrosylation of the channel and calstabin2 depletion from the channel complex likely triggers cardiac arrhythmias. Normalization of the RyR2-mediated diastolic SR Ca²⁺ leak prevents fatal sudden cardiac arrhythmias in DMD.     

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca²⁺ channels (VDCCs) inducing Ca²⁺ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca²⁺ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca²⁺ release, leading to a significant increase in the activity of Ca²⁺ sparks and contraction in airway SMCs. The increased Ca²⁺ sparks are independent of VDCCs and the associated extracellular Ca²⁺ influx. This format of local Ca²⁺ release results from a direct activation of G protein-coupled, M₃ muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP₃), inducing initial Ca²⁺ release through IP₃ receptors and then further Ca²⁺ release via RyR2 due to a local Ca²⁺-induced Ca²⁺ release process. These findings demonstrate an important mechanism for Ca²⁺ signaling and attendant physiological function in SMCs.     

Sarcoplasmic reticulum Ca2+ release in neonatal rat cardiac myocytes

In the neonatal mammalian heart, the role of ryanodine receptor (= Ca²⁺ release channel)-mediated sarcoplasmic reticulum (SR) Ca²⁺ release for excitation–contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes. Measurement of Ca²⁺ transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca²⁺ content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca²⁺ storage but unchanged Ca²⁺ signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation–contraction coupling in the developing mammalian heart.     

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca²⁺ imaging and two-photon Na⁺ imaging. GABA evoked Ca²⁺ transients in astrocytes that persisted in the presence of GABA_A and GABA_B receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca²⁺ blocked GABA-induced Ca²⁺ transients, and depletion of Ca²⁺ stores with cyclopiazonic acid reduced Ca²⁺ transients by approximately 90%. This indicates that the Ca²⁺ transients depend on external Ca²⁺, but are mainly mediated by intracellular Ca²⁺ release, conforming with Ca²⁺-induced Ca²⁺ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca²⁺ transients, whereas the InsP₃ receptor blocker 2-APB inhibited the Ca²⁺ transients. GABA also induced Na⁺ increases in astrocytes, potentially reducing Na⁺/Ca²⁺ exchange. To test whether reduction of Na⁺/Ca²⁺ exchange induces Ca²⁺ signaling, we inhibited Na⁺/Ca²⁺ exchange with KB-R7943, which mimicked GABA-induced Ca²⁺ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca²⁺ transients in astrocytes. The significance of GABAergic Ca²⁺ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na⁺ rises that reduce Na⁺/Ca²⁺ exchange, thereby leading to a Ca²⁺ increase sufficient to trigger Ca²⁺-induced Ca²⁺ release via InsP₃ receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.     

An emerging role for NAADP-mediated Ca2+ signaling in the pancreatic β-cell

Several recent reports, including one in this journal, have reignited the debate about whether the calcium-mobilizing messenger, nicotinic adenine nucleotide diphosphate (NAADP) plays a central role in the regulation of calcium signalling in pancreatic β-cell.^1-5 These studies have highlighted a role for NAADP-induced Ca²⁺ mobilization not only in mediating the effects of the incretin, GLP-1 and the autocrine proliferative effects of insulin, but also possibly a fundamental role in glucose-mediated insulin secretion in the pancreatic β-cell.     

Blockade of receptor-operated calcium channels by mibefradil (Ro 40-5967): Effects on intracellular calcium and platelet aggregation

Summary The goal of the present study was to evaluate if mibefradil, a novel nondihydropyridine Ca²⁺ antagonist, could block receptor-operated calcium channels (ROCC) present in human platelets and to determine the functional consequences of this blockade. Therefore, the effect of mibefradil on increases in intracellular Ca²⁺ concentrations and aggregation of human platelets induced by platelet activating factor (PAF) was examined. In order to differentiate effects on Ca²⁺ mobilization from intracellular stores from those on Ca²⁺ influx through ROCC, intracellular Ca²⁺ concentrations were measured either in fura-2-loaded platelets or in cells loaded with both BAPTA and fura-2. Mibefradil totally and dose dependently inhibited PAF-induced Ca²⁺ influx with a maximal effective concentration of 10 µM, but at this concentration only reduced Ca²⁺ mobilization from intracellular stores. A similar effect was observed when platelets were stimulated with ADP, suggesting that mibefradil was indeed interfering with ROCC and not specifically with PAF receptors. In the same range of concentrations, mibefradil inhibited Ca²⁺-dependent platelet aggregation induced by PAF. This effect was most likely due to the inhibition of ROCC, as Ca²⁺-independent aggregation induced by phorbolmyristyl-acetate (PMA) was insensitive to mibefradil. We conclude that mibefradil, which has previously been described to be an antagonist for L- and T-Type Ca²⁺ channels, also blocks receptor-operated Ca²⁺ channels. This blockade seems to be functionally relevant for platelet aggregation.     

Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai in Drosophila cells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca²⁺ concentration near the interface, indicating Ca²⁺ influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca²⁺ signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca²⁺ influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca²⁺ signaling during subsequent antigen encounter.     

If and SR Ca release both contribute to pacemaker activity in canine sinoatrial node cells

Increasing evidence suggests that cardiac pacemaking is the result of two sinoatrial node (SAN) cell mechanisms: a ‘voltage clock’ and a Ca²⁺ dependent process, or ‘Ca²⁺ clock.’ The voltage clock initiates action potentials (APs) by SAN cell membrane potential depolarization from inward currents, of which the pacemaker current (I_f) is thought to be particularly important. A Ca²⁺ dependent process triggers APs when sarcoplasmic reticulum (SR) Ca²⁺ release activates inward current carried by the forward mode of the electrogenic Na⁺/Ca²⁺ exchanger (NCX). However, these mechanisms have mostly been defined in rodents or rabbits, but are unexplored in single SAN cells from larger animals. Here, we used patch-clamp and confocal microscope techniques to explore the roles of the voltage and Ca²⁺ clock mechanisms in canine SAN pacemaker cells. We found that ZD7288, a selective I_f antagonist, significantly reduced basal automaticity and induced irregular, arrhythmia-like activity in canine SAN cells. In addition, ZD7288 impaired but did not eliminate the SAN cell rate acceleration by isoproterenol. In contrast, ryanodine significantly reduced the SAN cell acceleration by isoproterenol, while ryanodine reduction of basal automaticity was modest (∼ 14%) and did not reach statistical significance. Importantly, pretreatment with ryanodine eliminated SR Ca²⁺ release, but did not affect basal or isoproterenol-enhanced I_f. Taken together, these results indicate that voltage and Ca²⁺ dependent automaticity mechanisms coexist in canine SAN cells, and suggest that I_f and SR Ca²⁺ release cooperate to determine baseline and catecholamine-dependent automaticity in isolated dog SAN cells.     

Presence of tubular and reticular structures in the nucleus of human vascular smooth muscle cells

In recent decades, studies addressing nuclear calcium (Ca²⁺) homeostasis and signaling contributed to redefining the role of the nucleus. Yet many aspects of nuclear Ca²⁺ signaling and homeostasis are only modestly understood. The present study aimed at investigating the presence of nuclear structures which could contribute to the regulation of nuclear Ca²⁺ homeostasis. Using real 3D confocal microscopy, coupled to utilization of appropriate organelle probes and specific antibodies, we identified two entities in the nuclei of intact human vascular smooth muscle cells (hVSMCs) as well as in isolated hVSMCs nuclei. Our results demonstrate the presence of an ER-like nuclear reticular structure in nuclei of intact hVSMCs and in isolated nuclei. Similar to the ER/SR, this structure possesses thapsigargin binding sites, IP₃Rs and RyRs, thus it was named nucleoplasmic reticulum (NR). Furthermore, nuclear tubular structures were also detected. The latter, similar to the nuclear envelope membranes, possess nuclear pores, thapsigargin binding sites, Angiotensin II receptor AT₂, and are associated with Lamin A/C. However, unlike the NR and the nuclear envelope membranes, these tubular structures disappeared when the nuclei were isolated from the cells. The nuclear tubular structures were called Nuclear T-Tubules (NTTs). Our calcium studies in isolated nuclei utilizing IP₃ and Ryanodine suggest that the NR may participate in nuclear Ca²⁺ signaling. On the other hand, presence of nuclear pores on the NTTs suggests that these structures can play a role in cytosol-nucleus exchange. In conclusion, two distinct structures are present in the nucleus of hVSMCs and might play an important role in nuclear Ca²⁺ homeostasis.