Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.

     

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.

     

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.

     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

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.     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

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.

     

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.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

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.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

Acidosis antagonizes intracellular calcium response to kappa -opioid receptor stimulation in the rat heart

To study the effects of -opioid receptor stimulation onintracellular Ca²⁺ concentration([Ca²⁺]_i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca²⁺]_iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca²⁺]_itransient, which results from the influx ofCa²⁺ and the subsequentmobilization of Ca²⁺ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca²⁺]_iand inhibited the[Ca²⁺]_itransient induced by caffeine, which mobilizesCa²⁺ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa²⁺ from its intracellular poolinto the cytoplasm. The Ca²⁺responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient were significantly attenuated when the extracellular pH(pH_e) was loweredto 6.8, which itself reduced intracellular pH(pH_i) and increased[Ca²⁺]_i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa⁺/H⁺exchange blocker, or 0.2 mM Ni²⁺,a putativeNa⁺/Ca²⁺exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa⁺/H⁺andNa⁺/Ca²⁺exchanges. When glucose at 50 mM, known to activate theNa⁺/H⁺exchange, was added, both the resting[Ca²⁺]_iand pH_i increased. Interestingly,the effects of U-50488H on [Ca²⁺]_iand the electrically induced[Ca²⁺]_itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca²⁺ (3 mM) solutionwas superfused, the resting[Ca²⁺]_iincreased; the increase was abolished by 0.2 mMNi²⁺, but thepH_i remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca²⁺]_iand electrically induced[Ca²⁺]_itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa²⁺ from SR. Activation of bothNa⁺/H⁺andNa⁺/Ca²⁺exchanges, leading to an elevation of[Ca²⁺]_i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

     

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     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

Regulation of cytoplasmic calcium levels by two nitric oxide receptors

We examined the effects of dissolved nitric oxide (NO) gas oncytoplasmic calcium levels ([Ca²⁺]_i) in C6glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca²⁺]_i was reached at 10 µM NO. Asecond application of NO was ineffective if the first was >0.5 µM.The NO donor diethylamine/NO mimicked the effects of NO. Acute exposureof the cells to low calcium levels was without effect on the NO-evokedresponse. Thapsigargin (TG) increased [Ca²⁺]_iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP waswithout effect on the NO-evoked response. If cells were pretreated withTG or exposed chronically to nominal amounts of calcium, NO decreased[Ca²⁺]_i. The results suggest that C6 gliomacells have two receptors for NO. One receptor (NO_A)elevates [Ca²⁺]_i and resides on theendoplasmic reticulum (ER). The other receptor (NO_B)decreases [Ca²⁺]_i and resides on theplasmalemma or the ER. The latter receptor dominates when the level ofcalcium within intracellular stores is diminished.

     

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca²⁺] ([Ca²⁺]_m) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca²⁺] ([Ca²⁺]_em) to >0.5 µM resulted in a [Ca²⁺]_em-dependent increase in the rate of mitochondrial Ca²⁺ accumulation ([Ca²⁺]_em resulting in half-maximal rate of Ca²⁺ accumulation = 4.4 µM) via Ca²⁺ uniporter. Ca²⁺ uptake was sensitive to the Ca²⁺ uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca²⁺]_m increase and recovery were dependent on the extramitochondrial [Na⁺] ([Na⁺]_em) due to Ca²⁺ extrusion via mitochondrial Na⁺/Ca²⁺ exchanger. The maximal rate of Ca²⁺ extrusion was observed with [Na⁺]_em in the range of 20–40 mM. Rapid switching (0.25–1 Hz) of [Ca²⁺]_em between 0 and 100 µM simulated rapid beat-to-beat changes in [Ca²⁺]_i (with [Ca²⁺]_i transient duration of 100–500 ms). No [Ca²⁺]_m oscillations were observed, either under conditions of maximal rate of Ca²⁺ uptake (100 µM [Ca²⁺]_em, 0 [Na⁺]_em) or with maximal rate of Ca²⁺ removal (0 [Ca²⁺]_em, 40 mM [Na⁺]_em). The slow frequency-dependent increase of [Ca²⁺]_m argues against a rapid transmission of Ca²⁺ signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca²⁺]_m changes elicited by continuous or pulsatile exposure to elevated [Ca²⁺]_em showed no difference in mitochondrial Ca²⁺ uptake. Thus in cardiac myocytes fast [Ca²⁺]_i transients are integrated by mitochondrial Ca²⁺ transport systems, resulting in a frequency-dependent net mitochondrial Ca²⁺ accumulation. mitochondrial Ca²⁺; excitation-contraction coupling; cardiomyocytes     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.