Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

Spontaneous Ca²⁺ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 µM), but the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca²⁺ release events were also observed in 20% of cells treated with IP₃R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 µM; 0 Ca²⁺)-evoked Ca²⁺ transients but increased caffeine (10 mM; 0 Ca²⁺) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 µM; 0 Ca²⁺) were also enhanced by 2-APB. Ca²⁺ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 µM), which sensitizes the IP₃R to IP₃. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca²⁺ store content in smooth muscle is limited by tonic release of Ca²⁺ via an IP₃-dependent pathway. Blockade of IP₃Rs elevates sarcoplasmic reticulum store content, promoting Ca²⁺ sparks and STOC activity. calcium ion release; calcium ion transients; smooth muscle     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes

Localized Ca²⁺ transients inisolated murine colonic myocytes depend on Ca²⁺ releasefrom inositol 1,4,5-trisphosphate (IP₃) receptors.Localized Ca²⁺ transients couple to spontaneous transientoutward currents (STOCs) and mediate hyperpolarization responses inthese cells. We used confocal microscopy and whole cell patch-clamprecording to investigate how muscarinic stimulation, which causesformation of IP₃, can suppress Ca²⁺ transientsand STOCs that might override the excitatory nature of cholinergicresponses. ACh (10 µM) reduced localized Ca²⁺ transientsand STOCs, and these effects were associated with a rise in basalcytosolic Ca²⁺. These effects of ACh were mimicked bygeneralized rises in basal Ca²⁺ caused by ionomycin(250-500 nM) or elevated external Ca²⁺ (6 mM).Atropine (10 µM) abolished the effects of ACh. Pretreatment of cellswith nicardipine (1 µM), or Cd²⁺ (200 µM) had no effecton responses to ACh. An inhibitor of phospholipase C, U-73122, blockedCa²⁺ transients and STOCs but did not affect the increasein basal Ca²⁺ after ACh stimulation. Xestospongin C (Xe-C;5 µM), a membrane-permeable antagonist of IP₃ receptors,blocked spontaneous Ca²⁺ transients but did not prevent theincrease of basal Ca²⁺ in response to ACh. Gd³⁺(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca²⁺ after ACh and increased thefrequency and amplitude of Ca²⁺ transients and waves.Another inhibitor of receptor-mediated Ca²⁺ influxchannels, SKF-96365, also prevented the rise in basal Ca²⁺after ACh and increased Ca²⁺ transients and development ofCa²⁺ waves. FK-506, an inhibitor ofFKBP12/IP₃ receptor interactions, had no effect onthe rise in basal Ca²⁺ but blocked the inhibitory effectsof increased basal Ca²⁺ and ACh on Ca²⁺transients. These results suggest that the rise in basalCa²⁺ that accompanies muscarinic stimulation of colonicmuscles inhibits localized Ca²⁺ transients that couldcouple to activation of Ca²⁺-activated K⁺channels and reduce the excitatory effects of ACh.

     

IP(3) receptors, IP(3) transients, and nucleus-associated Ca(2+) signals in cultured skeletal muscle

Inositol1,4,5-trisphosphate (IP₃) receptors (IP₃R) andryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands (IP₃ andryanodine), and IP₃R were visualized in situ byfluorescence immunocytological techniques. Also explored was the effectof K⁺ depolarization on IP₃ mass andCa²⁺ transients studied using a radio-receptor displacementassay and fluorescence imaging of intracellular fluo 3. RyR werelocated in a microsomal fraction; IP₃R were preferentiallyfound in the nuclear fraction. Fluorescence associated withanti-IP₃R antibody was found in the region of the nuclearenvelope and in a striated pattern in the sarcoplasmic areas. Anincrease in external K⁺ affected membrane potential andproduced an IP₃ transient. Rat myotubes displayed afast-propagating Ca²⁺ signal, corresponding to theexcitation-contraction coupling transient and a much slowerCa²⁺ wave. Both signals were triggered by high externalK⁺ and were independent of external Ca²⁺. Slowwaves were associated with cell nuclei and were propagated leaving"glowing" nuclei behind. Different roles are proposed for atleast two types of Ca²⁺ release channels, each mediating anintracellular signal in cultured skeletal muscle.

     

Developmental changes in ryanodine- and IP3-sensitive Ca2+ pools in ovine basilar artery

To explore thehypothesis that cerebrovascular maturation alters ryanodine- andinositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pool sizes, we measured total intracellularCa²⁺ with ⁴⁵Ca and the fractions ofintracellular Ca²⁺ released by IP₃ and/orcaffeine in furaptra-loaded permeabilized basilar arteries fromnonpregnant adult and term fetal (139-141 days) sheep.Ca²⁺ mass (nmol/mg dry weight) was similar in adult(1.60 ± 0.18) and fetal (1.71 ± 0.16) arteries in the poolsensitive to IP₃ alone but was significantly lower foradult (0.11 ± 0.01) than for fetal (1.22 ± 0.11) arteriesin the pool sensitive to ryanodine alone. The pool sensitive to bothryanodine and IP₃ was also smaller in adult (0.14 ± 0.01) than in fetal (0.85 ± 0.08) arteries. Because theCa²⁺ fraction in the ryanodine-IP₃ pool wassmall in both adult (5 ± 1%) and fetal (7 ± 4%) arteries,the IP₃ and ryanodine pools appear to be separate in thesearteries. However, the pool sensitive to neither IP₃ norryanodine was 10-fold smaller in adult (0.87 ± 0.10) than infetal (8.78 ± 0.81) arteries, where it accounted for 72% oftotal intracellular membrane-bound Ca²⁺. Thus, duringbasilar artery maturation, intracellular Ca²⁺ mass plummetsin noncontractile pools, decreases modestly in ryanodine-sensitivepools, and remains constant in IP₃-sensitive pools. Inaddition, age-related increases in IP₃ efficacy must involve factors other than IP₃ pool size alone.

     

Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release. intracellular calcium stores; oscillations; pulmonary hypertension     

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

To investigatethe Ca²⁺-dependent plasticity ofsarcoplasmic reticulum (SR) function in vascular smooth muscle,transient responses to agents releasing intracellularCa²⁺ by either ryanodine(caffeine) orD-myo-inositol1,4,5-trisphosphate [IP₃;produced in response to norepinephrine (NE),5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptorsin rat tail arterial rings were evaluated after 4 days of organculture. Force transients induced by all agents were increased comparedwith those induced in fresh rings. Stimulation by 10% FCSduring culture further potentiated the force andCa²⁺ responses to caffeine (20 mM)but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effectwas persistent, and SR capacity was not altered after reversibledepletion of stores with cyclopiazonic acid. The effects of serum couldbe mimicked by culture in depolarizing medium (30 mMK⁺) and blocked by the additionof verapamil (1 µM) or EGTA (1 mM) to the medium, loweringintracellular Ca²⁺ concentration([Ca²⁺]_i)during culture. These results show that modulation of SR function canoccur in vitro by a mechanism dependent on long-term levels of basal[Ca²⁺]_iand involving ryanodine- but notIP₃ receptor-mediatedCa²⁺release.     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.

     

Cell cycle-dependent calcium oscillations in mouse embryonic stem cells

During cell cycle progression, somatic cells exhibit different patterns of intracellular Ca²⁺ signals during the G₀ phase, the transition from G₁ to S, and from G₂ to M. Because pluripotent embryonic stem (ES) cells progress through cell cycle without the gap phases G₁ and G₂, we aimed to determine whether mouse ES (mES) cells still exhibit characteristic changes of intracellular Ca²⁺ concentration during cell cycle progression. With confocal imaging of the Ca²⁺-sensitive dye fluo-4 AM, we identified that undifferentiated mES cells exhibit spontaneous Ca²⁺ oscillations. In control cultures where 50.4% of the cells reside in the S phase of the cell cycle, oscillations appeared in 36% of the cells within a colony. Oscillations were not initiated by Ca²⁺ influx but depended on inositol 1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ release and the refilling of intracellular stores by a store-operated Ca²⁺ influx (SOC) mechanism. Using cell cycle synchronization, we determined that Ca²⁺ oscillations were confined to the G₁/S phase (70% oscillating cells vs. G₂/M with 15% oscillating cells) of the cell cycle. ATP induced Ca²⁺ oscillations, and activation of SOC could be induced in G₁/S and G₂/M synchronized cells. Intracellular Ca²⁺ stores were not depleted, and all three IP₃ receptor isoforms were present throughout the cell cycle. Cell cycle analysis after EGTA, BAPTA-AM, 2-aminoethoxydiphenyl borate, thapsigargin, or U-73122 treatment emphasized that IP₃-mediated Ca²⁺ release is necessary for cell cycle progression through G₁/S. Because the IP₃ receptor sensitizer thimerosal induced Ca²⁺ oscillations only in G₁/S, we propose that changes in IP₃ receptor sensitivity or basal levels of IP₃ could be the basis for the G₁/S-confined Ca²⁺ oscillations. pluripotent; IP₃; store operated Ca entry; IP₃ receptor     

Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells

The regulationof intracellular Ca²⁺ signals in smooth muscle cells andarterial diameter by intravascular pressure was investigated in ratcerebral arteries (~150 µm) using a laser scanning confocal microscope and the fluorescent Ca²⁺ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca²⁺spark frequency 2.6-fold, Ca²⁺ wave frequency 1.9-fold, andglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) 1.4-fold in smooth muscle cells,and constricted arteries. Ryanodine (10 µM), an inhibitor ofryanodine-sensitive Ca²⁺ release channels, or thapsigargin(100 nM), an inhibitor of the sarcoplasmic reticulumCa²⁺-ATPase, abolished sparks and waves, elevated global[Ca²⁺]_i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 µM), a voltage-dependentCa²⁺ channel (VDCC) blocker, significantly reduced sparks,waves, and global [Ca²⁺]_i, and dilatedpressurized (60 mmHg) arteries. Steady membrane depolarization elevatedCa²⁺ signaling similar to pressure and increased transientCa²⁺-sensitive K⁺ channel current frequencye-fold for ~7 mV, and these effects were prevented by VDCCblockers. Data are consistent with the hypothesis that pressure inducesa steady membrane depolarization that activates VDCCs, leading to anelevation of spark frequency, wave frequency, and global[Ca²⁺]_i. In addition, pressure inducescontraction via an elevation of global[Ca²⁺]_i, whereas the net effect of sparks andwaves, which do not significantly contribute to global[Ca²⁺]_i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.

     

Imaging of intracellular calcium stores in single permeabilized lens cells

Intracellular Ca²⁺ storesin permeabilized sheep lens cells were imaged with mag-fura 2 tocharacterize their distribution and sensitivity toCa²⁺-releasing agents. Inositol1,4,5-trisphosphate (IP₃) orcyclic ADP-ribose (cADPR) releasedCa²⁺ from intracellularCa²⁺ stores that were maintainedby an ATP-dependent Ca²⁺ pump. TheIP₃ antagonist heparin inhibitedIP₃- but not cADPR-mediated Ca²⁺ release, whereas the cADPRantagonist 8-amino-cADPR inhibited cADPR- but notIP₃-mediatedCa²⁺ release, indicating thatIP₃ and cADPR were operatingthrough separate mechanisms. ACa²⁺ store sensitive toIP₃, cADPR, and thapsigarginappeared to be distributed throughout all intracellular regions. Insome cells a Ca²⁺ storeinsensitive to IP₃, cADPR,thapsigargin, and 2,4-dinitrophenol, but not ionomycin, was present ina juxtanuclear region. We conclude that lens cells containintracellular Ca²⁺ stores that aresensitive to IP₃, cADPR, andthapsigargin, as well as a Ca²⁺store that appears insensitive to all these agents.     

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells

Cl^– is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is Cl^– dependent. After incubating the cells at different extracellular Cl^– concentration ([Cl^–]_e) for 40 min, the ANG II-induced Ca²⁺ transients at 120 meq/l Cl^– were more than twice those at either 80 or 20 meq/l Cl^–. Replacing Cl^– with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca²⁺, ANG II-induced as well as platelet-derived growth factor-induced Ca²⁺ release exhibited Cl^– dependency. The difference of Ca²⁺ release with high vs. low [Cl^–]_e was not affected by acutely altering [Cl^–]_e 1 min before administration of ANG II when [Cl^–]_i was yet to be equilibrated with [Cl^–]_e. Pretreatment of a Cl^– channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca²⁺ release and entry at 20 meq/l Cl^– but did not alter those at 120 meq/l Cl^–. However, after equilibration, a reduced [Cl^–]_e did not affect thapsigargin-induced Ca²⁺ release, suggesting that Cl^– may not affect the size of intracellular Ca²⁺ stores. Nevertheless, at high [Cl^–], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] induced by ANG II was approximately sixfold that at low [Cl^–]. Thus the Cl^–-dependent effects of ANG II on Ca²⁺ transients may be mediated, at least in part, by a Cl^–-dependent Ins(1,4,5)P₃ accumulation in VSMC. anion; inositol 1,4,5-trisphosphate; Ca²⁺ release     

Mitochondrial transport in processes of cortical neurons is independent of intracellular calcium

Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca²⁺] ([Ca²⁺]_i) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 µM. The average velocity was 0.52 µm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca²⁺]_i associated with spontaneous firing of action potentials. Stimulation of Ca²⁺ transients with forskolin (10 µM) or bicuculline (10 µM), or sustained elevations of [Ca²⁺]_i evoked by glutamate (10 µM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca²⁺, depletion of intracellular Ca²⁺ stores with thapsigargin, or inhibition of synaptic activity with TTX (1 µM) or a cocktail of CNQX (10 µM) and MK801 (10 µM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca²⁺]_i associated with action potential firing, synaptic activity, or release of Ca²⁺ from intracellular stores. calcium transient; dendrites     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Upregulation of Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation     

Thrombin-, bradykinin-, and arachidonic acid-induced Ca2+ signaling in Ehrlich ascites tumor cells

Stimulation ofsingle Ehrlich ascites tumor cells with agonists (bradykinin, thrombin)and with arachidonic acid (AA) induces increases in the freeintracellular Ca²⁺ concentration([Ca²⁺]_i)in the presence and absence of extracellularCa²⁺, measured using theCa²⁺-sensitive probe fura 2. Sequential stimulation with two agonists elicits sequential increasesin[Ca²⁺]_i,unlike addition of the same agonist twice. Bradykinin and thrombin haveadditive effects on[Ca²⁺]_iin Ca²⁺-free medium. Thephosphoinositidase C inhibitor U-73122 inhibits the agonist-inducedincreases in[Ca²⁺]_i,whereas ryanodine has no effect. Pretreatment of cells in Ca²⁺-free medium with thapsigarginabolishes the bradykinin-induced increase in[Ca²⁺]_ibut not the response to thrombin. The AA-induced response is notinhibited by U-73122 and cannot be mimicked by the inactive structuralanalog trifluoromethylarachidonyl ketone. Pretreatment of the cellswith 50 µM AA (but not with 10 µM AA) abolishes the agonist-inducedincrease in[Ca²⁺]_i.Thus bradykinin, thrombin, and AA induce increases in[Ca²⁺]_iin Ehrlich cells due to Ca²⁺ entryand release from intracellular stores. Thrombin causes release ofCa²⁺ from an intracellular storethat is insensitive to bradykinin and is not depleted by thapsigarginbut is depleted by AA.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Modulation of intracellular Ca2+ release and capacitative Ca2+ entry by CaMKII inhibitors in bovine vascular endothelial cells

The effects of inhibitors of CaMKII on intracellular Ca²⁺ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca²⁺]_i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca²⁺ resulted in a dose-dependent increase of [Ca²⁺]_i consisting of a rapid and transient Ca²⁺ spike followed by a small sustained plateau phase of elevated [Ca²⁺]_i. Exposure to KN-93 in the absence of extracellular Ca²⁺ caused a transient rise of [Ca²⁺]_i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca²⁺ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP₃) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca²⁺ response, suggesting that exposure to KN-93 affects Ca²⁺ release from an IP₃-sensitive store. Depletion of Ca²⁺ stores by exposure to ATP or to the ER Ca²⁺ pump inhibitor thapsigargin triggered robust capacitative Ca²⁺ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca²⁺ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca²⁺ from intracellular stores and activation of CCE. Ca²⁺/calmodulin-dependent kinase II; calcium regulation; capacitative calcium entry