Hepoxilin signaling in intact human neutrophils: biphasic elevation of intracellular calcium by unesterified hepoxilin A3

We have previously shown that the methyl ester of hepoxilin A3 causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A3 is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188+/-14 S.D., free acid = 135+/-11 S.D. nM, P < 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88+/-8 S.D., free acid = 107+/-15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calcium-free medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189+/-12; free acid, 145+/-6 S.D. nM, P<0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A3 demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.     

Mutual influence of plasma membrane structures and intracellular calcium stores on the formation of calcium signals in primary nociceptive neurons

Participation of different calcium-regulating mechanisms in the formation of intracellular calcium signals in rat primary sensory neurons was studied using two-wavelength fluorescent microscopy. Mitochondria were shown to be the most powerful intracellular calcium-regulating structures in the investigated neurons. These organelles were involved in the modulation of calcium signals induced either by Ca²⁺ entry from the extracellular medium or by Ca²⁺ release from endoplasmic reticulum (ER). Analysis of the mitochondrial calcium exchange showed that the efficiency of mitochondria depended on whether calcium entered the cytosol from ER or from the extracellular solution. Depletion of ER by activation of ryanodine-sensitive, inositol-3-phosphate-sensitive receptors of ER or by activation of the leak channels via the block of ATPases in ER activated the store-operated calcium entry from the extracellular medium to cytosol. The kinetics of the rising phase of these Ca²⁺ transients depended on the way of ER depletion. This allows suggesting the existence of different activation mechanisms for the studied signals. The block of the mitochondrial calcium uniporter resulted in a rapid recovery of the intracellular calcium concentration after the Ca²⁺ transient induced by store-operated calcium influx. We conclude that mitochondrial calcium uptake can prevent calcium-dependent inactivation of store-operated calcium channels.     

Chemical synthesis and actions of 11,12-thiirano-hepoxilin A3

A novel analog of hepoxilin A3 has been chemically synthesized in which the 11,12-epoxide group has been altered to a thiirano group. This has been accomplished through allylic rearrangement of unnatural (11R,12R)-hepoxilin B₃ under Mitsunobu conditions, first into unnatural (11R,12R)-hepoxilin A₃, followed by conversion of this compound with inversion of the epoxide centers into the thiirano-hepoxilin A₃ having the natural 11S,12S configuration. We also report herein evidence showing that thiirano-hepoxilin A₃ raises intracellular calcium concentrations in intact human neutrophils.     

Initiation of RVD response in human platelets: Mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A₃. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A₃ formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca²⁺-insensitive phospholipase A₂, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A₂ inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 m, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca²⁺ depletion by EGTA (up to 10 mm) does not affect RVD; (iii) intracellular Ca²⁺ depletion by BAPTAAM (100 m) inhibits RVD but not hepoxilin A₃ formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP S (1 m) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A₃; (v) NaF, 1 mm, induces hepoxilin A₃ formation, tested by the RVD reconstitution assay; and (vii) GDP S inhibits hepoxilin A₃ formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A₃ formation in human platelets.DeceasedThis work is dedicated to the memory of Prof. A.A. Livne. It was carried out at the Amelia (Mimi) Rose Laboratory for Cellular Signal Transduction at the Department of Life Sciences, Ben-Gurion University of the Negev. We thank A. Dannon for helpful discussion.     

Arachidonate mobilization is coupled to depletion of intracellular calcium stores and influx of extracellular calcium in differentiated U937 cells

We have previously reported that dimethylsulfoxide-differentiation of U937 cells induced significant A23187-stimulatable arachidonate mobilization, consistent with characteristics of cytosolic phospholipase A₂ (Rzigalinski, B.A. and Rosenthal, M.D. (1994) Biochim. Biophys. Acta 1223, 219–225). The present report demonstrates that differentiated cells attained higher elevations of intracellular free calcium in response to A23187 and thapsigargin, consistent with enhancement of the capacitative calcium influx pathway. Differentiation induced a significant increase in the size of the intracellular calcium stores, as well as in the capacity for store-activated calcium influx. Alterations in the capacitative calcium influx pathway were coupled to differentiation-induced activation of cPLA₂ and mobilization of arachidonate in response to thapsigargin and fMLP stimulation. Although cPLA₂ activity is often associated with influx of extracellular calcium, arachidonate mobilization in response to thapsigargin or fMLP was not simply a consequence of calcium influx. Assessment of intracellular free calcium elevations during thapsigargin or fMLP-induced stimulation suggest that a low level of arachidonic acid release was initiated upon release of intracellular store calcium. This initial release of arachidonate was unaffected by inhibition of calcium influx with nickel, EGTA, or SKF96365. Arachidonate release was observed when extracellular calcium was replaced with extracellular strontium, suggesting activation of the cytosolic PLA₂ rather than secretory PLA₂. Inhibition of PLA₂ with prostaglandin B oligomer prevented both thapsigargin and fMLP-stimulated influx of extracellular calcium. Furthermore, exogenous free arachidonate stimulated influx of extracellular calcium in differentiated U937 cells. These results suggest that cPLA₂-mediated release of free arachidonate may participate in the formation of a calcium influx factor which controls influx of extracellular calcium through store-controlled channels in the plasma membrane.     

Calcium influx and intracellular calcium release in anti-CD3 antibody-stimulated and thapsigargin-treated human T lymphoblasts

Summary Jurkat and MOLT-4 cultured T lymphoblasts were loaded with low concentrations (30–50 m) of indo-1 and with high concentrations (3.5–4.5mm) of quin-2, respectively, in order to follow the activation of calcium transport pathways after stimulation of the cells by a monoclonal antibody against the T cell antigen receptor (aCD3), or after the addition of thapsigargin, a presumed inhibitor of endoplasmic reticulum calcium pump. In the indo-1 loaded cells the dynamics of the intracellular calcium release and the calcium influx could be studied, while in the quin-2 overloaded cells the changes in cytoplasmic free calcium concentration ([Ca²⁺] _i ) were strongly buffered and the rate of calcium influx could be quantitatively determined. We found that in Jurkat lymphoblasts, in the absence of external calcium, both aCD3 and thapsigargin induced a rapid calcium release from internal stores, while upon the readdition of external calcium an increased rate of calcium influx could be observed in both cases, aCD3 and thapsigargin released calcium from the same intracellular pools. The calcium influx induced by either agent was of similar magnitude and had a nonadditive character if the two agents were applied simultaneously. As demonstrated in quin-2 overloaded cells, a significant initial rise in [Ca²⁺] _i or a pronounced depletion of internal calcium pools was not required to obtain a rapid calcium influx. The activation of protein kinase C by phorbol ester abolished the internal calcium release and the calcium influx induced by aCD3, while having only a small effect on these phenomena when evoked by thapsigargin. Membrane depolarization by gramicidin inhibited the rapid calcium influx in both aCD3- and thapsigargin-treated cells, although it did not affect the internal calcium release produced by either agent. In MOLT-4 cells, which have no functioning antigen receptors, aCD3 was ineffective in inducing a calcium signal, while thapsigargin produced similar internal calcium release and external calcium influx to those observed in Jurkat cells.     

Formation, Metabolism, and Action of Hepoxilin A3in the Rat Pineal Gland

Abstract: The present study was undertaken to investigate the possible formation of hepoxilin A₃ in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 μM) led to the appearance of hepoxilin A₃ (HxA₃) analyzed as its stable trihydroxy derivative, trioxilin A₃ by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA₃ is estimated to be 1.43 ± 0.66 ng//μg of protein. This amount is not modified when the tissue is boiled (2.07 ± 0.66 ng/μg of protein). However, the formation of this compound was stimulated to 21.26 ±5.82 ng/μg of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 /μg) resulted in a partial blockade of hepoxilin formation. Using [1-¹⁴C] H×A₃, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA₃ into trioxilin A₃. This hydrolysis was inhibited by 1 μmol/L of 3, 3, 3-trichloropropene-1, 2-oxide. In a separate study, HxA₃ in the presence of 3, 3, 3-trichloropropene-1, 2-oxide to block the hydrolysis of HxA₃ decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5′-N-ethylcarboxamidoadenosine, an A₁/A₂ adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (?88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA₃ in the rat pineal gland.     

Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V_max of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N’-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH^−/− mice; and iv) liver homogenates of sEH^−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone

The proton ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited antigen-stimulated secretion and calcium influx in rat basophilic leukemia cells. In a glucose-free solution the inhibitory effects of CCCP were due to a decrease in the intracellular ATP concentration; however, when glucose was present there was no decrease in ATP. Instead, we found that in a glucose-containing saline solution, CCCP inhibited antigen-stimulated calcium uptake because it depolarized the plasma membrane, which in rat basophilic leukemia cells inhibits antigen-stimulated calcium uptake. In the presence of glucose, relatively low concentrations of CCCP inhibited calcium uptake while higher concentrations were required to inhibit secretion. In contrast, the initial antigen-stimulated rise in cytoplasmic calcium, measured with the fluorescent calcium indicator quin2, was not inhibited by CCCP. This suggests that the release of calcium from intracellular stores might, in some cases, be sufficient to support antigen-stimulated secretion. In the presence of CCCP the pH gradient becomes important for regulating the membrane potential across the plasma membrane. When cells were depolarized with CCCP and the external pH was increased, the membrane potential returned to resting levels and antigen-stimulated calcium uptake was restored. Inhibition of antigen-stimulated secretion by higher concentrations of CCCP could also be reversed by increasing the external pH.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (R _LIS), phase I response, followed by an increase in tight junctional resistance (R _TJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC₅₀ of 6 μM). The objective of the study was to determine the degree to which the changes in [Ca²⁺]_i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La³⁺, and Mn²⁺ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease inR _LIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La³⁺ also inhibited the ATP-induced phase II response, but Mn²⁺ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase inR _TJ. KCl-induced membrane depolarization stimulated an acute decrease inR _LIS and a late increase inR _TJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca²⁺]_i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases inR _TJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic ATP-like changes inR _TE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease inR _LIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase inR _TJ.     

Hydrogen peroxide activates calcium influx in human neutrophils

The correlation between an increased production of reactive oxygen species (ROS) and an enhanced calcium entry in primed neutrophils stimulated with fMLP suggests that endogenous ROS could serve as an agonist to reinforce calcium signaling by positive feedback. This work shows that exogenous H₂O₂ produced a rapid influx of Mn²⁺ and an increase of intracellular calcium. The H₂O₂ was insufficient to produce significant changes in the absence of extracellular calcium but addition of Ca²⁺ to H₂O₂-treated cells suspended in a free Ca²⁺/EGTA buffer resulted in a great increase in [Ca²⁺]_i reflecting influx of Ca²⁺ across the cell membrane. The increase of intracellular calcium was inhibited by Ni²⁺, La³⁺, and hyperosmotic solutions of mannitol and other osmolytes. This raises the possibility that the secretion of H₂O₂ by activated neutrophils could act as an autocrine regulator of neutrophil function through the activation of calcium entry.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Correlation of prostaglandin-induced mitochondrial calcium release with contraction in bovine intrapulmonary vein

The effects of prostaglandins A₂, A₁, F_2α, E₂, E₁, F_1β and an analog of PGH₂ upon calcium release from mitochondria isolated from bovine intrapulmonary vein and contraction of helical strips of the same tissue were determined. The order of activity of the prostaglandins for calcium release was similar to that for contraction with the exception of the PGH₂ analog. It is suggested that prostaglandin A₂, F_2α, E₂ and A₁ induced release of mitochondrial calcium may influence the contractile state of bovine intrapulmonary vein. However, the PGH₂ analog has a subcellular mechanism other than or in addition to mitochondrial calcium release and is different from the other prostaglandins.     

Synergistically regulated spontaneous calcium signaling is attributed to cartilaginous extracellular matrix metabolism

Ca²⁺ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca ²⁺] _i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca ²⁺ signal of in-situ porcine chondrocytes was [Ca ²⁺] _o dependent, and mediated by [Ca ²⁺] _i store release. T-type voltage-dependent calcium channel (T-VDCC) mediated [Ca ²⁺] _o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5-trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca ²⁺ release and store-operated calcium entry significantly abolished spontaneous [Ca ²⁺] _i signaling of in-situ chondrocytes. Moreover, blocking ER Ca ²⁺ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca ²⁺] _i signaling of in-situ porcine chondrocytes was tightly regulated by [Ca ²⁺] _o influx, InsP3Rs mediated [Ca ²⁺] _i store release, and Orais mediated calcium release-activated calcium channels activation. Both T-VDCC mediated [Ca ²⁺] _o influx and InsP3Rs mediated ER Ca ²⁺ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.     

Modeling Hermissenda: I. Differential contributions of I A and I C to type-B cell plasticity

We developed a multicompartmental Hodgkin-Huxley model of the Hermissenda type-B photoreceptor and used it to address the relative contributions of reductions of two K⁺ currents, I _a and I _C, to changes in cellular excitability and synaptic strength that occur in these cells after associative learning. We found that reductions of g_C, the peak conductance of I _C, substantially increased the firing frequency of the type-B cell during the plateau phase of a simulated light response, whereas reductions of g_A had only a modest contribution to the plateau frequency. This can be understood at least in part by the contributions of these currents to the light-induced (nonspiking) generator potential, the plateau of which was enhanced by g_C reductions, but not by g_A reductions. In contrast, however, reductions of g_A broadened the type-B cell action potential, increased Ca²⁺ influx, and increased the size of the postsynaptic potential produced in a type-A cell, whereas similar reductions of g_C had only negligible contributions to these measures. These results suggest that reductions of I _A and I _C play important but different roles in type-B cell plasticity.     

Receptor-mediated action of hepoxilin A3 releases diacylglycerol and arachidonic acid from human neutrophils

We have previously shown that hepoxilin A3 increases the intracellular concentration of Ca+2 in human neutrophils. Herein we address the initial events of hepoxilin action on the neutrophil which precede the rise in intracellular calcium. We show that hepoxilin A3 at 10-1000 nM concentrations releases from [1-14C]-arachidonic acid labeled neutrophils diacylglycerol and unesterified arachidonic acid in a time and concentration dependent fashion. The release of arachidonic acid and diacyglycerol are receptor-mediated events which are blocked by pertussis toxin. This data shows that hepoxilin A3 stimulates phospholipases C and A2 in the cell which may be involved in the rise in cytosolic calcium. Thus, hepoxilins may represent a hitherto unrecognised class of cellular mediators.     

Calcium transport in isolated bone cells. II. Calcium transport studies

Calcium transport was studied in bone cells isolated from fetal rat calvaria. ⁴⁵Ca uptake experiments revealed an active component of calcium exchange. Calcium uptake was inhibited by iodoacetamide, DNP, CCCP and oligomycin and appeared to be dependent on medium phosphate concentration. Initial influx values exhibited saturation kinetics from 0.6 mM to 1.5 mM extracellular calcium. Efflux of ⁴⁵Ca from loaded cells increased in the presence of iodoacetamide, DNP and CCCP. Incubation of the cells af 4° C inhibited both influx and efflux of calcium. Parathyroid hormone had no consistent effect on calcium uptake although characteristic increases in cyclic AMP levels were seen with the hormone. Calcitonin appeared to cause a transient increase in calcium uptake.     

Cytochalasin induces an increase in cytosolic free calcium in murine B lymphocytes

Cytochalasin promotes the progression of anti-immunoglobulin-treated B lymphocytes to S phase. However, the intracellular events induced by cytochalasin which may mediate signaling for progression have not been elucidated. In this study, the effect of cytochalasin on the level of intracellular free calcium in murine splenic B lymphocytes was assessed by using the fluorescent calcium indicator Indo-1. Cytochalasins A, B, D, and E induced a rapid and sustained elevation of intracellular free calcium. The calcium response to cytochalasin derived largely from the influx of extracellular calcium, although a small, transient elevation in intracellular calcium persisted when the suspension medium was made calcium-free with EGTA, implicating an intracellular source for a portion of the calcium response. Single cell fluorescence studies revealed that cytochalasin elicited a calcium response in most splenic B cells in suspension, indicating that this phenomenon is not restricted to a subpopulation of responding B cells. Phorbol esters inhibited the B cell calcium response to cytochalasin, and an established response to cytochalasin was rapidly and completely reversed by subsequently administered phorbol ester. T cells that lack the cytochalasin pathway showed a markedly diminished calcium response that was only apparent at higher cytochalasin concentration. However, B cells from xid-defective [CBA/N X DBA/2]F1 males, which fail to respond to anti-immunoglobulin plus cytochalasin, showed a calcium response to cytochalasin similar to that of phenotypically normal F1 females. These data, along with the finding that the rise in intracellular calcium occurred in naive B cells as well as B cells previously treated with anti-immunoglobulin, suggest that there is no clear association between the calcium response induced by cytochalasin and the ability of cytochalasin to stimulate progression to S phase. However, this effect of cytochalasin may suggest a connection between actin filaments and calcium influx in B cells.     

A Single Lys Residue on the First Intracellular Loop Modulates the Endoplasmic Reticulum Export and Cell-Surface Expression of α2A-Adrenergic Receptor

Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65also modulates the cell-surface transport of α_2A-adrenergic receptor (α_2A-AR). Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α_2A-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of α_2A-AR, mutation of Lys65 to Ala and Arg produced opposing effects on α_2A-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α_2A-AR mutant K65A displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell-surface expression of α_2A-AR. These data also suggest that the ICL1 may possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms.