Calmodulin and protein kinase C regulate gap junctional coupling in lens epithelial cells

The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca²⁺ (Ca_i²⁺) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Ca_i²⁺ (to micromolar concentrations) observed after the addition of ionophore plus Ca²⁺ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Ca_i²⁺, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Ca_i²⁺ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Ca_i²⁺ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Ca_i²⁺. calcium; connexin43; lens gap junctions     

Elementary events of agonist-induced Ca2+ release in vascular endothelial cells

The subcellular spatial and temporal organization ofagonist-induced Ca²⁺ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca²⁺ concentration([Ca²⁺]_i)in peripheral cytoplasmic processes from where activation propagated asa[Ca²⁺]_iwave toward the central regions of the cell. The average propagation velocity of the[Ca²⁺]_iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca²⁺]_idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca²⁺]_ideclined monotonically, whereas in the central region[Ca²⁺]_idecreased in an oscillatory fashion. Propagating[Ca²⁺]_iwaves were preceded by small, highly localized[Ca²⁺]_itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa²⁺ release was 23 nM, and theunderlying flux of Ca²⁺ amountedto ~1-2 × 10¹⁸mol/s or ~0.3 pA, consistent with aCa²⁺ flux through a single orsmall number of endoplasmic reticulum Ca²⁺-release channels.

     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Role of glycolytically generated ATP for CaMKII-mediated regulation of intracellular Ca2+ signaling in bovine vascular endothelial cells

The role of glycolytically generated ATP in Ca²⁺/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca²⁺ signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca²⁺ concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + -hydroxybutyrate (-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca²⁺]_i. The rise of [Ca²⁺]_i was characterized by a transient spike followed by a small sustained plateau of elevated [Ca²⁺]_i. In the absence of extracellular Ca²⁺ 2-DG caused an increase in [Ca²⁺]_i, suggesting that inhibition of glycolysis directly triggered release of Ca²⁺ from intracellular endoplasmic reticulum (ER) Ca²⁺ stores. The inositol-1,4,5-trisphosphate receptor (IP₃R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca²⁺ response. Ca²⁺ release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca²⁺]_i wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca²⁺]_i. Propagating [Ca²⁺]_i waves were preceded by [Ca²⁺]_i oscillations and small, highly localized elevations of [Ca²⁺]_i (Ca²⁺ puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca²⁺ response, and vice versa during inhibition of CaMKII 2-DG-induced Ca²⁺ release was attenuated. Similar results were obtained with pyr + -HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg²⁺ concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca²⁺ release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP₃R. Ca²⁺/calmodulin-dependent kinase II; glycolysis; calcium regulation     

The role of intracellular pH in cell growth arrest induced by ATP

In this study, we investigated ionic mechanisms involved in growth arrest induced by extracellular ATP in androgen-independent prostate cancer cells. Extracellular ATP reversibly induced a rapid and sustained intracellular pH (pH_i) decrease from 7.41 to 7.11. Inhibition of Ca²⁺ influx, lowering extracellular Ca²⁺, and buffering cytoplasmic Ca²⁺ inhibited ATP-induced acidification, thereby demonstrating that acidification is a consequence of Ca²⁺ entry. We show that ATP induced reuptake of Ca²⁺ by the mitochondria and a transient depolarization of the inner mitochondrial membrane. ATP-induced acidification was reduced after the dissipation of the mitochondrial proton gradient by rotenone and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, after inhibition of Ca²⁺ uptake into the mitochondria by ruthenium red, and after inhibition of the F₀F₁-ATPase with oligomycin. ATP-induced acidification was not induced by either stimulation of the Cl^–/HCO₃^– exchanger or inhibition of the Na⁺/H⁺ exchanger. In addition, intracellular acidification, induced by an ammonium prepulse method, reduced the amount of releasable Ca²⁺ from the endoplasmic reticulum, assessed by measuring change in cytosolic Ca²⁺ induced by thapsigargin or ATP in a Ca²⁺-free medium. This latter finding reveals cross talk between pH_i and Ca²⁺ homeostasis in which the Ca²⁺-induced intracellular acidification can in turn regulate the amount of Ca²⁺ that can be released from the endoplasmic reticulum. Furthermore, pH_i decrease was capable of reducing cell growth. Taken together, our results suggest that ATP-induced acidification in DU-145 cells results from specific effect of mitochondrial function and is one of the major mechanisms leading to growth arrest induced by ATP. prostate; cancer; acidification     

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca²⁺, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca²⁺ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca²⁺ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X₃R > P2X_2b + X₄R > P2X_2bR > P2X_2a + X₄R > P2X₄R > P2X_2aR > P2X₇R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca²⁺ influx because of the coactivation of endogenously expressed Ca²⁺-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca²⁺ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca²⁺ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca²⁺ signals were not affected by voltage-gated Ca²⁺ influx and removal of extracellular sodium. Ca²⁺ signals reflected well the receptor-specific EC₅₀ values for ATP and the extracellular Zn²⁺ and pH sensitivities of P2XRs. These results indicate that intracellular Ca²⁺ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors. ATP-gated receptor channels; inward currents; intracellular calcium signals; desensitization-inactivation; voltage-gated calcium influx; localized and global calcium signals     

A P2X7 receptor stimulates plasma membrane trafficking in the FRTL rat thyrocyte cell line

Thyroid cells express a variety of P2Y and P2X purinergic receptor subtypes. G protein-coupled P2Y receptors influence a wide variety of thyrocyte-specific functions; however, functional P2X receptor-gated channels have not been observed. In this study, we used whole cell patch-clamp recording and fluorescence imaging of the plasma membrane marker FM1-43 to examine the effects of extracellular ATP on membrane permeability and trafficking in the Fisher rat thyroid cell line FRTL. We found a cation-selective current that was gated by ATP and 2',3'-O-(4-benzoylbenzoyl)-ATP but not by UTP. The ATP-evoked currents were inhibited by pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, adenosine 5'-triphosphate-2',3'-dialdehyde, 100 µM Zn²⁺, and 50 µM Cu²⁺. Fluorescence imaging revealed pronounced, temperature-sensitive stimulation of exocytosis and membrane internalization by ATP with the same pharmacological profile as observed for activation of current. The EC₅₀ for ATP stimulation of internalization was 440 µM in saline containing 2 mM Ca²⁺ and 2 mM Mg²⁺, and 33 µM in low-Mg²⁺, nominally Ca²⁺-free saline. Overall, the results are most consistent with activation of a P2X₇ receptor by ATP^4–. However, low permeability to N-methyl-D-glucamine⁺ and the propidium cation YO-PRO-1 indicates absence of the cytolytic pore that often accompanies P2X₇ receptor activation. ATP stimulation of internalization occurs in Na⁺-free, Ca²⁺-free, or low-Mg²⁺ saline and therefore does not depend on cation influx through the ATP-gated channel. We conclude that ATP activation of a P2X₇ receptor stimulates membrane internalization in FRTL cells via a transduction pathway that does not depend on cation influx. purinergic receptor; internalization; patch clamp     

Effect of Cytoplasmic Ca2+ on the Membrane Potential and Membrane Resistance of Chara Plasmalemma

Effects of cytoplasmic Ca²⁺ on the electrical properties ofthe plasma membrane were investigated in tonoplast-free cellsof Chara australis that had been internally perfused with media,containing either 1 mM ATP to fuel the electrogenic pump orhexokinase and glucose to deplete the ATP and stop the pump. In the presence of ATP, cytoplasmic Ca²⁺ up to 2.5?10^–5M did not affect the membrane potential (about -190 mV), butmembrane resistance decreased uniformly with increasing [Ca²⁺]_i.In the absence of ATP, the membrane potential, which was onlyabout -110 mV, was depolarized further by raising [Ca²⁺]_i from1.4?10^–6 to 2.5?10^–5 M. Membrane resistance, whichwas nearly the twofold that of ATP-provided cells, decreasedmarkedly with an increase in [Ca²⁺]_i from zero to 1.38?10^–6M, but showed no change for further increases. Internodal cellsof Nitellopsis obtusa were more sensitive to intracellular Ca²⁺with respect to membrane potential than were those of Charaaustralis, reconfirming the results obtained by Mimura and Tazawa(1983). The effect of cytoplasmic Ca²⁺ on the ATP-dependent H⁺ effluxwas measured. No marked difference in H⁺ effluxes was detectedbetween zero and 2.5?10^–5 M [Ca²⁺]_i; but, at 10^–4M the ATP-dependent H⁺ efflux was almost zero. Ca²⁺ efflux experimentswere done to investigate dependencies on [Ca²⁺]_i and [ATP]_i.The efflux was about 1 pmol cm^–2 s^–1 at all [Ca²⁺]_iconcentrations tested (1.38?10^–6, 2.5?10^–5, 10^–4M).This value is much higher than the influx reported by Hayamaet al. (1979), and this efflux was independent of [ATP]_i. Thepossibility of a Ca²⁺-extruding pump is discussed. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, 5300 Bonn, F.R.G. (Received September 22, 1984; Accepted February 19, 1985)     

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.

     

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.

     

Regulation of exocytosis by purinergic receptors in pancreatic duct epithelial cells

In epithelial cells, several intracellular signals regulate the secretion of large molecules such as mucin via exocytosis and the transport of ions through channels and transporters. Using carbon fiber amperometry, we previously reported that exocytosis of secretory granules in dog pancreatic duct epithelial cells (PDEC) can be stimulated by pharmacological activation of cAMP-dependent protein kinase (PKA) or protein kinase C (PKC), as well as by an increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). In this study, we examined whether exocytosis in these cells is modulated by activation of endogenous P2Y receptors, which increase cAMP and [Ca²⁺]_i. Low concentrations of ATP (<10 µM) induced intracellular Ca²⁺ oscillation but no significant exocytosis. In contrast, 100 µM ATP induced a sustained [Ca²⁺]_i rise and increased the exocytosis rate sevenfold. The contribution of Ca²⁺ or cAMP pathways to exocytosis was tested by using the Ca²⁺ chelator BAPTA or the PKA inhibitors H-89 or Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate. Removal of [Ca²⁺]_i rise or inhibition of PKA each partially reduced exocytosis; when combined, they abolished exocytosis. In conclusion, ATP at concentrations >10 µM stimulates exocytosis from PDEC through both Ca²⁺ and cAMP pathways. secretion; amperometry; photometry; calcium, adenosine 3',5'-cyclic monophosphate     

Regulation of KCa current by store-operated Ca2+ influx depends on internal Ca2+ release in HSG cells

This study examines theCa²⁺ influx-dependent regulationof the Ca²⁺-activatedK⁺ channel(K_Ca) in human submandibulargland (HSG) cells. Carbachol (CCh) induced sustained increases in theK_Ca current and cytosolic Ca²⁺ concentration([Ca²⁺]_i),which were prevented by loading cells with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Removal of extracellularCa²⁺ and addition ofLa³⁺ orGd³⁺, but notZn²⁺, inhibited the increases inK_Ca current and[Ca²⁺]_i.Ca²⁺ influx during refill (i.e.,addition of Ca²⁺ to cells treatedwith CCh and then atropine inCa²⁺-free medium) failed to evokeincreases in the K_Ca current but achieved internal Ca²⁺ storerefill. When refill was prevented by thapsigargin,Ca²⁺ readdition induced rapidactivation of K_Ca. These dataprovide further evidence that intracellularCa²⁺ accumulation provides tightbuffering of[Ca²⁺]_iat the site of Ca²⁺ influx (H. Mogami, K. Nakano, A. V. Tepikin, and O. H. Petersen. Cell 88: 49-55, 1997). We suggestthat the Ca²⁺ influx-dependentregulation of the sustained K_Cacurrent in CCh-stimulated HSG cells is mediated by the uptake ofCa²⁺ into the internalCa²⁺ store and release via theinositol 1,4,5-trisphosphate-sensitive channel.

     

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     

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.

     

Tunicamycin increases intracellular calcium levels in bovine aortic endothelial cells

Tunicamycin is anucleoside antibiotic that inhibits protein glycosylation andpalmitoylation. The therapeutic use of tunicamycin is limited inanimals because of its toxic effects, particularly in cerebralvasculature. Tunicamycin decreases palmitoylation of the endothelialisoform of nitric oxide synthase, stimulates nitric oxide synthesis,and increases the concentration of intracellular calcium([Ca²⁺]_i)in bovine aortic endothelial cells (B. J. Buckley and A. R. Whorton.FASEB J. 11: A110, 1997). In the present study,we investigated the mechanism by which tunicamycin alters[Ca²⁺]_iusing the Ca²⁺-sensitive dye fura2. We found that tunicamycin increased[Ca²⁺]_iwithout increasing levels of inositol phosphates. When cells wereincubated in the absence of extracellularCa²⁺,[Ca²⁺]_irapidly rose in response to tunicamycin, although a full response wasnot achieved. The pool of intracellularCa²⁺ mobilized by tunicamycinoverlapped with that mobilized by thapsigargin. Extracellular nickelblocked a full response to tunicamycin when cells were incubated in thepresence of extracellular Ca²⁺.The effects of tunicamycin on[Ca²⁺]_iwere partially reversed by washing out the drug, and the remainder ofthe response was inhibited by removing extracellularCa²⁺. These results indicate thattunicamycin mobilizes Ca²⁺ fromintracellular stores in a manner independent of phospholipase Cactivation and increases the influx ofCa²⁺ across the plasma membrane.

     

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.

     

RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry

A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca²⁺-dependent Cl^– channels via stimulation of Ca²⁺ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca²⁺ in human airway epithelial cells that translates into stimulation of sustained secretory Cl^– transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca²⁺ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X₄, P2X₅, and P2X₆ subtypes in non-CF (16HBE14o^–) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca²⁺ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X₄ and P2X₆ (but not P2X₅) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca²⁺ entry markedly in fura-2 Ca²⁺ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca²⁺ entry channel subtypes are expressed in airway epithelia. P2X₄ and P2X₆ may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy     

P2 purinoceptor of the globular substance in the otoconial membrane of the guinea pig inner ear

The biological characteristics of the globular substance, aprecursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca²⁺ concentration([Ca²⁺]_i) changes of the globular substanceand the ATP distribution in the vestibular organ were investigatedusing a Ca²⁺ indicator, fluo 3, and an adeninenucleotide-specific fluorochrome, quinacrine, by means of confocallaser scanning microscopy. [Ca²⁺]_i showed arapid and dose-dependent increase in response to ATP with a 50%effective concentration (EC₅₀) of 16.7 µM. Thisreaction was independent of external Ca²⁺, indicating thepresence of an internal Ca²⁺ reservoir. Neither adenosine,,-methylene-ATP, 3'-O-(4-benzoylbenzoyl)-ATP, ADP, norUTP evoked this reaction, whereas 2-methylthio-ATP induced an increaseof [Ca²⁺]_i with an EC₅₀ of 14.4 µM. Moreover, P2 antagonists, reactive blue 2 and suramin, and aphospholipase C inhibitor, U-73122, inhibited the ATP-induced[Ca²⁺]_i increase. These findings indicate thepresence of a P2Y purinoceptor on the globular substance. In addition,granular fluorescence was observed in the quinacrine-stained macularsensory epithelium, indicating the presence of ATP-containing granulesin this tissue. These results suggest that a paracrine mechanisminvolving ATP may exist in the macula and that this mechanism regulatesthe biological behavior of the globular substance.

     

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.