Calcium- and proton-dependent relocation of annexin A6 in Jurkat T cells stimulated for interleukin-2 secretion

Annexin A6 (AnxA6) is a Ca(2+)-dependent membrane-binding protein involved in vesicular traffic. The likely participation of AnxA6 in the response of lymphocytes to Ca(2+) signals has not been investigated yet. The present study focuses on intracellular relocation of AnxA6 in human Jurkat T lymphoblasts upon stimulation followed by transient increase of intracellular [Ca(2+)] and exocytosis of interleukin-2 (IL-2). Stimulation of the cells under different experimental conditions (by lowering pH and/or by rising extracellular [Ca(2+)] in the presence of ionomycin) induced time-dependent transients of intracellular [Ca(2+)] and concomitant changes in AnxA6 intracellular localization and in IL-2 secretion, with only minor effects on cell viability and apoptosis. In resting conditions (in the presence of EGTA or with no ionophore) AnxA6 was localized uniformly in the cytosol, whereas it translocated to vesicular structures beneath the plasma membrane within 5 min following stimulation of Jurkat T cells and rise of intracellular [Ca(2+)] at pH 7.4. Lowering the extracellular pH value from 7.4 to 6.0 significantly enhanced this process. AnxA6 changed its location from the cytosol to the secretory granules and early endosomes which seem to represent membranous targets for annexin. In conclusion, AnxA6 is sensitive to variations in intracellular [Ca(2+)] upon stimulation of Jurkat T cells, as manifested by a switch in its intracellular localization from the cytosol to vesicular structures located in close proximity to the plasma membrane, suggestive of participation of AnxA6 in calcium- and proton-dependent secretion of cytokines by lymphocytes.     

Potentiation by stannous chloride of calcium entry into osteoblastic MC3T3-E1 cells through voltage-dependent L-type calcium channels

The present study was undertaken to confirm that L-type Ca(2+) channels are involved in Ca(2+) entry into osteoblastic MC3T3-E1 cells and to examine the effect of SnCl2, a Ca(2+)]-channel activator, on the intracellular Ca(2+)concentration ([Ca(2+)]i). High K(+)concentration-dependently raised the [Ca(2+)]i. All of the L-type Ca(2+)channel blockers used here, such as nifedipine, nicardipine, verapamil, and diltiazem, and CdCl2 (a non-selective blocker) inhibited the high K(+)-induced [Ca(2+)]i rise, but v-conotoxin GVIA (an N-type blocker) and NiCl2(a T-type blocker) had no effect. Application of SnCl2 alone did not change the [Ca(2+)]i. However, in the presence of high K(+), SnCl2 enhanced the high K(+)-induced [Ca(2+)]i rise, which was inhibited by Ca(2+)]-free medium or nifedipine. In the case where high K(+)was applied prior to SnCl2, SnCl2 alone raised the [Ca(2+)]i by itself. In conclusion, MC3T3-E1 cells possess the voltage-dependent L-type Ca(2+)] channels and SnCl2 facilitates the Ca(2+) entry through the L-type ones under the condition of the membrane depolarization. There is the possibility that Ca(2+) release from intracellular Ca(2+) stores is involved in the action of SnCl2.     

Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca(2+)-ATPase pump

The isoform-specific interaction of plasma membrane Ca(2+)-ATPase (PMCA) pumps with partner proteins has been explored using a yeast two-hybrid technique. The 90 N-terminal residues of two pump isoforms (PMCA2 and PMCA4), which have a low degree of sequence homology, have been used as baits. Screening of 5 x 10(6) clones of a human brain cDNA library yielded approximately 100 LEU2- and galactoside-positive clones for both pumps. A clone obtained with the PMCA4 bait specified the epsilon-isoform of the 14-3-3 protein, whereas no 14-3-3epsilon clone was obtained with the PMCA2 bait. The 14-3-3epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca(2+) was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca(2+)] transients, higher post-transient plateaus of the cytosolic [Ca(2+)], and higher Ca(2+) levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3epsilon inhibited PMCA4. Silencing of the 14-3-3epsilon gene by RNA interference significantly reduced the expression of 14-3-3epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca(2+)] transient and of the post-transient cytosolic [Ca(2+)] plateau.     

Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling

Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.     

Calmodulin regulation and identification of calmodulin binding region of type-3 ryanodine receptor calcium release channel

Ryanodine receptors (RyRs) are a family of intracellular Ca(2+) channels that are regulated by calmodulin (CaM). At low Ca(2+) concentrations (<1 microM), CaM activates RyR1 and RyR3 and inhibits RyR2. At elevated Ca(2+) concentrations (>1 microM), CaM inhibits all three RyR isoforms. Here we report that the regulation of recombinant RyR3 by CaM is sensitive to redox regulation. RyR3 in the presence of reduced glutathione binds CaM with 10-15-fold higher affinity, at low and high Ca(2+) concentrations, compared to in the presence of oxidized glutathione. However, compared to RyR1 assayed at low Ca(2+) concentrations under both reducing and oxidizing conditions, CaM binds RyR3 with reduced affinity but activates RyR3 to a greater extent. Under reducing conditions, RyR1 and RyR3 activities are inhibited with a similar affinity at [Ca(2+)] > 1 microM. Mutagenesis studies demonstrate that RyR3 contains a single conserved CaM binding site. Corresponding amino acid substitutions in the CaM binding site differentially affect CaM binding and CaM regulation of RyR3 and those of the two other isoforms. The results support the suggestion that other isoform dependent regions have a major role in the regulation of RyRs by CaM [Yamaguchi et al. (2004) J. Biol. Chem. 279, 36433-36439].     

Lipid raft endocytosis and exosomal transport facilitate extracellular trafficking of annexin A2

Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2.     

Effect of nordihydroguaiaretic acid on intracellular Ca concentrations in C6 glioma cells

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in C6 glioma cells has been investigated. NDGA (5-100 microM) increased [Ca(2+)]i concentration-dependently. The [Ca(2+)]i increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced NDGA-induced [Ca(2+)]i signals by 52+/-2%. After incubation of cells with NDGA in Ca(2+)-free medium for 4 min, addition of 3 mM CaCl2 induced a concentration-dependent increase in [Ca(2+)]i. NDGA (100 microM)-induced [Ca(2+)]i increases in Ca(2+)-containing medium was not changed by pretreatment with 10 microM nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished 100 microM NDGA-induced [Ca(2+)]i increases. Inhibition of phospholipase C with 2 microM U73122 had little effect on 100 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)]i. Collectively, the results suggest that NDGA increased [Ca(2+)]i in glioma cells in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum in a manner independent of phospholipase C activity and by causing Ca(2+) influx.     

Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca2+ wave characteristics

The Ca(2+) dissociation constant (K(d)) of Fluo-3 was determined using confocal fluorescence microscopy in two different situations: (i) within the cytosol of a permeabilised cardiomyocyte; and (ii) in an intact cardiomyocyte after incubation with the acetoxymethyl ester form of Fluo-3 (AM). Measurements were made on isolated rabbit ventricular cardiomyocytes after permeabilisation by a brief treatment with beta-escin (0.1mg/ml) and equilibration with 10 microM Fluo-3. The K(d) of Fluo-3 within the cytosol was not significantly different from that in free solution (558 +/- 15 nM, n=6). Over a range of cytoplasmic [Ca(2+)], the minimum [Ca(2+)] values between Ca(2+) waves was relatively constant despite changes in wave frequency. After loading intact cardiomyocytes with Fluo-3 by incubation with the -AM, spontaneous Ca(2+) waves were produced by incubation with strophanthidin (10 microM). By assuming a common minimum [Ca(2+)] in permeabilised and intact cells, the intracellular K(d) of Fluo-3 in intact myocytes was estimated to be 898 +/-64 nM (n=6). Application of this K(d) to fluorescence records shows that Ca(2+) waves in intact cells have similar amplitudes to those in permeabilised cells. Stimulation of cardiac myocytes at 0.5 Hz in the absence of strophanthidin (room temperature) resulted in a Ca(2+) transient with a maximum and minimum [Ca(2+)] of 1190 +/- 200 and 158 +/- 30 nM (n=11), respectively.     

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.     

Fast biphasic regulation of type 3 inositol trisphosphate receptors by cytosolic calcium

In cytosol-like medium (CLM) with a free [Ca(2+)] of 200 nm, a supramaximal concentration of inositol 1,4,5-trisphosphate (IP(3)) (30 microm) evoked (45)Ca(2+) release from type 3 IP(3) receptors only after a latency of 48 +/- 6 ms; this latency could not be reduced by increasing the IP(3) concentration. In CLM containing a low free [Ca(2+)] ( approximately 4 nm), 300 microm IP(3) evoked (45)Ca(2+) release after a latency of 66 +/- 11 ms; this was reduced to 14 +/- 3 ms when the [Ca(2+)] was 1 mm. Preincubation with CLM containing 100 microm Ca(2+) caused a rapid (half-time = 33 +/- 9 ms), complete, and fully reversible inhibition that could not be overcome by a high concentration of IP(3) (300 microm). Hepatic (type 2) IP(3) receptors were not inhibited by Ca(2+) once they had bound IP(3), but 100 microm Ca(2+) rapidly inhibited type 3 IP(3) receptors whether it was delivered before addition of IP(3) or at any stage during a response to IP(3). Ca(2+) increases the affinity of IP(3) for hepatic receptors by slowing IP(3) dissociation, but Ca(2+) had no effect on IP(3) binding to type 3 receptors. The rate of inhibition of type 3 IP(3) receptors by Ca(2+) was faster than the rate of IP(3) dissociation, and occurred at similar rates whether receptors had bound a high (adenophostin) or low affinity (3-deoxy-3-fluoro-IP(3)) agonist. Dissociation of agonist is not therefore required for Ca(2+) to inhibit type 3 IP(3) receptors. We conclude that type 2 and 3 IP(3) receptors are each biphasically regulated by Ca(2+), but by different mechanisms. For both, IP(3) binding causes a stimulatory Ca(2+)-binding site to be exposed allowing Ca(2+) to bind and open the channel. IP(3) binding protects type 2 receptors from Ca(2+) inhibition, but type 3 receptors are inhibited by Ca(2+) whether or not they have IP(3) bound. Increases in cytosolic [Ca(2+)] will immediately inhibit type 3 receptors, but inhibit type 2 receptors only after IP(3) has dissociated.     

Annexin A6-Linking Ca(2+) signaling with cholesterol transport

Annexin A6 (AnxA6) belongs to a conserved family of Ca(2+)-dependent membrane-binding proteins. Like other annexins, the function of AnxA6 is linked to its ability to bind phospholipids in cellular membranes in a dynamic and reversible fashion, in particular during the regulation of endocytic and exocytic pathways. High amounts of AnxA6 sequester cholesterol in late endosomes, thereby lowering the levels of cholesterol in the Golgi and the plasma membrane. These AnxA6-dependent redistributions of cellular cholesterol pools give rise to reduced cytoplasmic phospholipase A2 (cPLA(2)) activity, retention of caveolin in the Golgi apparatus and a reduced number of caveolae at the cell surface. In addition to regulating cholesterol and caveolin distribution, AnxA6 acts as a scaffold/targeting protein for several signaling proteins, the best characterized being the Ca(2+)-dependent membrane targeting of p120GAP to downregulate Ras activity. AnxA6 also stimulates the Ca(2+)-inducible involvement of PKC in the regulation of HRas and possibly EGFR signal transduction pathways. The ability of AnxA6 to recruit regulators of the EGFR/Ras pathway is likely potentiated by AnxA6-induced actin remodeling. Accordingly, AnxA6 may function as an organizer of membrane domains (i) to modulate intracellular cholesterol homeostasis, (ii) to create a scaffold for the formation of multifactorial signaling complexes, and (iii) to regulate transient membrane-actin interactions during endocytic and exocytic transport. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Unitary Ca(2+) current through recombinant type 3 InsP(3) receptor channels under physiological ionic conditions

The ubiquitous inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) channel, localized primarily in the endoplasmic reticulum (ER) membrane, releases Ca(2+) into the cytoplasm upon binding InsP(3), generating and modulating intracellular Ca(2+) signals that regulate numerous physiological processes. Together with the number of channels activated and the open probability of the active channels, the size of the unitary Ca(2+) current (i(Ca)) passing through an open InsP(3)R channel determines the amount of Ca(2+) released from the ER store, and thus the amplitude and the spatial and temporal nature of Ca(2+) signals generated in response to extracellular stimuli. Despite its significance, i(Ca) for InsP(3)R channels in physiological ionic conditions has not been directly measured. Here, we report the first measurement of i(Ca) through an InsP(3)R channel in its native membrane environment under physiological ionic conditions. Nuclear patch clamp electrophysiology with rapid perfusion solution exchanges was used to study the conductance properties of recombinant homotetrameric rat type 3 InsP(3)R channels. Within physiological ranges of free Ca(2+) concentrations in the ER lumen ([Ca(2+)](ER)), free cytoplasmic [Ca(2+)] ([Ca(2+)](i)), and symmetric free [Mg(2+)] ([Mg(2+)](f)), the i(Ca)-[Ca(2+)](ER) relation was linear, with no detectable dependence on [Mg(2+)](f). i(Ca) was 0.15 +/- 0.01 pA for a filled ER store with 500 microM [Ca(2+)](ER). The i(Ca)-[Ca(2+)](ER) relation suggests that Ca(2+) released by an InsP(3)R channel raises [Ca(2+)](i) near the open channel to approximately 13-70 microM, depending on [Ca(2+)](ER). These measurements have implications for the activities of nearby InsP(3)-liganded InsP(3)R channels, and they confirm that Ca(2+) released by an open InsP(3)R channel is sufficient to activate neighboring channels at appropriate distances away, promoting Ca(2+)-induced Ca(2+) release.     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Maitotoxin-induced calcium entry in human lymphocytes: modulation by yessotoxin, Ca(2+) channel blockers and kinases

We have studied the effect of the ciguatera-related toxin maitotoxin (MTX) on the cytosolic free calcium concentration ([Ca(2+)]i) of human peripheral blood lymphocytes loaded with the fluorescent probe Fura2 and the regulation of MTX action by different drugs known to interfere in cellular Ca(2+) signalling mechanisms and by the marine phycotoxin yessotoxin (YTX). MTX produced a concentration-dependent elevation of [Ca(2+)]i in a Ca(2+)-containing medium. This effect was stimulated by pretreatment with YTX 1 microM and NiCl(2) 15 microM. The voltage-independent Ca(2+) channel antagonist 1-[beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenyl]-1H-imidazole hydrochloride (SKF96365) blocked the MTX-induced [Ca(2+)]i elevation, while the L-type channel blocker nifedipine had no effect. Pretreatment with NiCl(2) or nifedipine did not modify YTX-induced potentiation of MTX effect, and SKF96365-induced inhibition was reduced in the presence of YTX, which suggest different pathways to act on [Ca(2+)]i. Preincubation with N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H-89) or genistein (10 microM) also had no effect on the MTX-induced [Ca(2+)]i increment. In contrast, the PKC inhibitor bisindolilmaleimide I (GF109203X 1 microM) potentiated the MTX effect, whereas phosphatidylinositol (PI) 3-kinase inhibition with wortmannin (10 nM) reduced the MTX-elicited Ca(2+) entry. In summary, MTX produced Ca(2+) influx into human lymphocytes through a SKF96365-sensitive, nifedipine-insensitive pathway. The MTX-induced [Ca(2+)]i elevation was stimulated by the marine toxin YTX through a mechanism insensitive to SKF96365, nifedipine or NiCl(2). It was also stimulated by the divalent cation Ni(2+) and PKC inhibition and was partially inhibited by PI 3-kinase inhibition.     

Ca2+ signaling in mouse mesenteric small arteries: myogenic tone and adrenergic vasoconstriction

Arteries that have developed myogenic tone (MT) are in a markedly different physiological state compared with those that have not, with higher cytosolic [Ca(2+)] and altered activity of several signal transduction pathways. In this study, we sought to determine whether alpha(1)-adrenoceptor-induced Ca(2+) signaling is different in pressurized arteries that have spontaneously developed MT (the presumptive physiological state) compared with those that have not (a common experimental state). At 32 degrees C and intraluminal pressure of 70 mmHg, cytoplasmic [Ca(2+)] was steady in most smooth muscle cells (SMCs). In a minority of cells (34%), however, at least one propagating Ca(2+) wave occurred. alpha(1)-Adrenoceptor activation (phenylephrine, PE; 0.1-10.0 microM) caused strong vasoconstriction and markedly increased the frequency of Ca(2+) waves (in virtually all cells). However, when cytosolic [Ca(2+)] was elevated experimentally in these arteries ([K(+)] 20 mM), PE failed to elicit Ca(2+) waves, although it did elevate [Ca(2+)] (F/F(0)) further and caused further vasoconstriction. During development of MT, the cytosolic [Ca(2+)] (F/F(0)) in individual SMCs increased, Ca(2+) waves disappeared (from SMCs that had them), and small Ca(2+) ripples (frequency approximately 0.05 Hz) appeared in approximately 13% of cells. PE elicited only spatially uniform increases in [Ca(2+)] and a smaller change in diameter (than in the absence of MT). Nevertheless, when cytosolic [Ca(2+)] and MT were decreased by nifedipine (1 microM), PE did elicit Ca(2+) waves. Thus alpha(1)-adrenoceptor-mediated Ca(2+) signaling is markedly different in arteries with and without MT, perhaps due to the elevated [Ca(2+)], and may have a different molecular basis. alpha(1)-Adrenoceptor-induced vasoconstriction may be supported either by Ca(2+) waves or by steady elevation of cytoplasmic [Ca(2+)], depending on the amount of MT.     

Effects of mitochondrial uncoupling on adipocyte intracellular Ca(2+) and lipid metabolism

Previous data from this laboratory demonstrate that increased intracellular Ca(2+) ([Ca(2+)]i) coordinately regulates human and murine adipocyte lipid metabolism by stimulating lipogenesis and inhibiting lipolysis. However, recent data demonstrate metabolic uncoupling increases [Ca(2+)]i but inhibits lipogenesis by suppressing fatty acid synthase (FAS) activity. Accordingly, we have evaluated the interaction between mitochondrial uncoupling, adipocyte [Ca(2+)]i, and adipocyte lipid metabolism. Pretreatment of 3T3-L1 cells with mitochondrial uncouplers (DNP or FCCP) amplified the [Ca(2+)]i response to depolarization with KCl by 2-4 fold (p <0.001), while this increase was prevented by [Ca(2+)]i channel antagonism with lanthanum. Mitochondrial uncouplers caused rapid (within 4hr) dose-dependent inhibition of FAS activity (p <0.001), while lanthanum caused a further additive inhibition. The suppression of FAS activity induced by uncoupling was reversed by addition of ATP. Mitochondrial uncouplers increased FAS expression significantly while [Ca(2+)]i antagonism with lanthanum decreased FAS expression (P <0.001). In contrast, mitochondrial uncouplers independently inhibited basal and isoproterenol-stimulated lipolysis (20-40%, p <0.001), while this inhibition was fully reversed by lanthanum. Thus, mitochondrial uncoupling exerted short-term regulatory effects on adipocyte [Ca(2+)]i and lipogenic and lipolytic systems, serving to suppress lipolysis via a Ca(2+) -dependent mechanism and FAS activity via a Ca(2+)-independent mechanism.     

cGMP signals mainly through cAMP kinase in permeabilized murine aorta

GMP affects vascular tone by multiple mechanisms, including inhibition of the Rho/Rho kinase-mediated Ca(2+) sensitization, a process identified as Ca(2+) desensitization. Ca(2+) desensitization is mediated probably by both cGMP- and cAMP-dependent protein kinases (cGKI and PKA). We investigate to which extent Ca(2+) desensitization is initiated by cGKI and PKA. cGMP/cAMP-induced relaxation was studied at constant [Ca(2+)] in permeabilized aortas from wild-type and cGKI-deficient mice. [Ca(2+)] increased aortic tone in the absence and presence of 50 microM GTPgammaS with EC(50) values of 160 and 30 nM, respectively. In the absence of GTPgammaS, the EC(50) for [Ca(2+)] was shifted rightward from 0.16 microM to 0.43 and 0.82 microM by 1 and 300 microM 8-bromo-cGMP (8-Br-cGMP), and to 8 microM by 10 microM Y-27632. Contractions induced by 300 nM [Ca(2+)] were relaxed by 8-Br-cGMP with an EC(50) of 2.6 microM. Surprisingly, [Ca(2+)]-induced contractions were also relaxed by 8-Br-cGMP in aortas from cGKI(-/-) mice (EC(50) of 19 microM). Western blot analysis of the vasodilator-stimulated phosphoprotein indicated "cross"-activation of PKA by 1 mM 8-Br-cGMP in aortic smooth muscle cells from cGKI(-/-) mice. Indeed, the PKA inhibitor peptide (PKI 5-24) completely abolished the relaxant effect of 8-Br-cGMP in muscles from cGKI(-/-) mice and to 65% in wild-type aortas. The thromboxane analogue U-46619 induced contraction at constant [Ca(2+)], which was only partially relaxed by 8-Br-cGMP but completely relaxed by Y-27632. The effect of 8-Br-cGMP on U-46619-induced contraction was attenuated by PKI 5-24. These results show that cGKI has only a small inhibitory effect on Ca(2+) sensitization in murine aortas.     

Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia and mitochondria inhibitors on the capacitative Ca(2+) entry (CCE) in cultured smooth muscle cells from rabbit small pulmonary arteries. Cyclopiazonic acid (CPA) depleted Ca(2+) from sarcoplasmic reticulum (SR) in Ca(2+)-free medium and subsequent addition of Ca(2+) led to the nifedipine-insensitive, La(3+)-sensitive Ca(2+) influx. The presence of CCE was further verified by the measurement of unidirectional Mn(2+) influx. During the decay phase of the CCE-induced [Ca(2+)]c transients, hypoxia (P(O2) < 50 mmHg) and the mitochondria inhibitor FCCP reversibly increased [Ca(2+)]c, that is La(3+)-sensitive. Once SR is depleted by CPA, subsequent treatment of FCCP slowed the decay of CCE-induced [Ca(2+)]c transients but it did not attenuate Mn(2+) influx. Mitochondrial uptake of incoming Ca(2+) through CCE was demonstrated by additional increase in [Ca(2+)]c with Ca(2+) ionophore after terminating CCE. Together, it is suggested that the augmentation of CCE-induced [Ca(2+)]c transients by hypoxia and FCCP reflects a net gain of [Ca(2+)]c by the inhibition of mitochondrial Ca(2+) uptake.