ATP induces dephosphorylation of myosin light chain in endothelial cells

In cultured porcine aortic endothelial monolayers, theeffect of ATP on myosin light chain (MLC) phosphorylation, whichcontrols the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolicCa²⁺ concentration ([Ca²⁺]_i).Inhibition of the ATP-evoked [Ca²⁺]_i rise byxestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca²⁺ release from endoplasmicreticulum, did not affect the ATP-induced dephosphorylation of MLC. MLCdephosphorylation was prevented in the presence of calyculin A (10 nM),an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activatesMLC dephosphorylation in a Ca²⁺-independent manner. In thepresence of calyculin A, MLC phosphorylation was incremented afteraddition of ATP, an effect that could be abolished when cellswere loaded with the Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester (10 µM). Thus ATP also activates aCa²⁺-dependent kinase acting on MLC. In summary, ATPsimultaneously stimulates a functional antagonism toward bothphosphorylation and dephosphorylation of MLC in which thedephosphorylation prevails. In endothelial cells, ATP is the firstphysiological mediator identified to activate MLC dephosphorylation bya Ca²⁺-independent mechanism.

     

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 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.

     

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca²⁺-dependent MLC kinase or Ca²⁺-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 µM) increased ERK2 phosphorylation from basal 17 ± 3 to 53 ± 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 µM) or U0126 (10 µM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca²⁺ rise, because it was unaltered when this was suppressed by the Ca²⁺ chelator BAPTA (10 µM) or xestospongin C (3 µM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 µM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 µM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca²⁺-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase. mitogen-activated protein kinase; contractile machinery; myosin light chain kinase; myosin light chain phosphatase     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

Cyclopiazonic acid-induced changes in the contraction and Ca2+ transient of frog fast-twitch skeletal muscle

The effects ofcyclopiazonic acid (CPA) were investigated on isolated skeletal musclefibers of frog semitendinosus muscle. CPA (0.5-10 µM) enhancedisometric twitch but produced little change in resting tension. Athigher concentrations (10-50 µM), CPA depressed twitch andinduced sustained contracture without affecting resting and actionpotentials. In Triton-skinned fibers, CPA had no significant effect onmyofibrillar Ca²⁺ sensitivity butdecreased maximal activated force at concentrations >5 µM. Inintact cells loaded with the Ca²⁺fluorescence indicator indo 1, CPA (2 µM) induced an increase inCa²⁺-transient amplitude (10 ± 2.5%), which was associated with an increase in time to peak and inthe time constant of decay. Consequently, peak force was increased by35 ± 4%, and both time to peak and the time constant of relaxationwere prolonged. It is concluded that CPA effects, at a concentration ofup to 2 µM, were associated with specific inhibition of sarcoplasmicreticulumCa²⁺-adenosinetriphosphatase inintact skeletal muscle and that inhibition of the pump directlyaffected the handling of intracellularCa²⁺ and force production.

     

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.

     

Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+ signaling

High Ca²⁺ concentrationscan develop near Ca²⁺ sourcesduring intracellular signaling and might lead to localized regulationof Ca²⁺-dependent processes. Byshifting the amount of Ca²⁺ andother cations associated with ATP, local highCa²⁺ concentrations might alsoalter the substrate available for membrane-associated and cytoplasmicenzymes. To study this, simultaneous equations were solved over a rangeof Ca²⁺ andMg²⁺ concentrations to determinethe general effects of Ca²⁺ on theconcentrations of free and Ca²⁺-and Mg²⁺-bound forms of ATP. Toobtain a more specific picture of the changes that might occur insmooth muscle cells, mathematical models ofCa²⁺ diffusion and regulation wereused to predict the magnitude and time course of near-membraneCa²⁺ transients and their effectson the free and bound forms of ATP near the membrane. The results ofthis work indicate that changes in freeCa²⁺ concentration over the rangeof 50 nM-100 µM would result in significant changes in free ATPconcentration, MgATP concentration, and the CaATP-to-MgATPconcentration ratio.

     

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.

     

Extracellular ATP stimulates volume decrease in Necturus red blood cells

This study examined whether extracellular ATP stimulatesregulatory volume decrease (RVD) in Necturusmaculosus (mudpuppy) red blood cells (RBCs). Thehemolytic index (a measure of osmotic fragility) decreased withextracellular ATP (50 µM). In contrast, the ATP scavenger hexokinase(2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, theATP-dependent K⁺ channelantagonist glibenclamide (100 µM) increased the hemolytic index, andthis inhibition was reversed with ATP (50 µM). We also measured cellvolume recovery in response to hypotonic shock electronically with aCoulter counter. Extracellular ATP (50 µM) enhanced cell volumedecrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml)inhibited cell volume recovery. The inhibitory effect of hexokinase wasreversed with the Ca²⁺ ionophoreA-23187 (1 µM); it also was reversed with the cationophore gramicidin(5 µM in a choline-Ringer solution), indicating that ATP was linkedto K⁺ efflux. In addition,glibenclamide (100 µM) and gadolinium (10 µM) inhibited cell volumedecrease, and the effect of these agents was reversed with ATP (50 µM) and A-23187 (1 µM). Using the whole cell patch-clamp technique,we found that ATP (50 µM) stimulated a whole cell current underisosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide(100 µM), and gadolinium (10 µM) inhibited whole cell currents thatwere activated during hypotonic swelling. The inhibitory effect ofapyrase was reversed with the nonhydrolyzable analog adenosine5'-O-(3-thiotriphosphate) (50 µM), and the effect of glibenclamide or gadolinium was reversed withATP (50 µM). Finally, anionic whole cell currents were activated withhypotonic swelling when ATP was the only significant charge carrier,suggesting that increases in cell volume led to ATP efflux through aconductive pathway. Taken together, these results indicate thatextracellular ATP stimulated cell volume decrease via aCa²⁺-dependent step that led toK⁺ efflux.

     

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.

     

Model of beta -cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables

In the preceding article[Am. J. Physiol. 274 (Cell Physiol. 43):C1158-C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca²⁺ handlingand electrical activity in the pancreatic -cell. Here we describefurther results of those simulations, focusing on mitochondrialvariables, the rate of respiration, and fluxes of metabolicintermediates as a function of D-glucose concentration. Oursimulations predict relatively smooth increases of O₂consumption, adenine nucleotide transport, oxidative phosphorylation,and ATP production by the tricarboxylic acid cycle asD-glucose concentrations are increased from basal to 20 mM.On the other hand, we find that the active fraction of pyruvatedehydrogenase saturates, due to increases in matrix Ca²⁺,near the onset of bursting electrical activity and that the NADH/NAD⁺ ratio in the mitochondria increases by roughly anorder of magnitude as glucose concentrations are increased. Themitochondrial ATP/ADP ratio increases by factor of <2 between theD-glucose threshold for bursting and continuousspiking. According to our simulations, relatively small changes inmitochondrial membrane potential (~1 mV) caused by uptake ofCa²⁺ are sufficient to alter the cytoplasmic ATP/ADP ratioand influence ATP-sensitive K⁺ channels in the plasmamembrane. In the simulations, these cyclic changes in the mitochondrialmembrane potential are due to synchronization of futile cycle ofCa²⁺ from the cytoplasm through mitochondria viaCa²⁺ uniporters and Na⁺/Ca²⁺exchange. Our simulations predict steady mitochondrial Ca²⁺concentrations on the order of 0.1 µM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 µMduring bursting. Abrupt increases in mitochondrial Ca²⁺concentration >5 µM may occur during continuous electricalactivity.

     

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.

     

Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel

Calcium entry in nonexcitable cells occurs throughCa²⁺-selective channels activatedsecondarily to store depletion and/or through receptor- orsecond messenger-operated channels. In amphibian liver, hormones thatstimulate the production of adenosine 3',5'-cyclic monophosphate (cAMP) also regulate the opening of an ion gate in theplasma membrane, which allows a noncapacitative inflow ofCa²⁺. To characterize thisCa²⁺ channel, we studied theeffects of inhibitors of voltage-dependent Ca²⁺ channels and of nonselectivecation channels on 8-bromoadenosine 3',5'-cyclicmonophosphate (8-BrcAMP)-dependentCa²⁺ entry in single axolotlhepatocytes. Ca²⁺ entry provokedby 8-BrcAMP in the presence of physiologicalCa²⁺ followed first-order kinetics(apparent Michaelis constant = 43 µM at the cellsurface). Maximal values of cytosolicCa²⁺ (increment ~300%) werereached within 15 s, and the effect was transient (half time of 56 s).We report a strong inhibition of cAMP-dependentCa²⁺ entry by nifedipine[half-maximal inhibitory concentration(IC₅₀) = 0.8 µM], byverapamil (IC₅₀ = 22 µM), andby SK&F-96365 (IC₅₀ = 1.8 µM).Depolarizing concentrations of K⁺were without effect. Gadolinium and the anti-inflammatory compound niflumate, both inhibitors of nonselective cation channels, suppressed Ca²⁺ influx. This "profile"indicates a novel mechanism ofCa²⁺ entry in nonexcitable cells.

     

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.

     

State-dependent modification of ATP-sensitive K+ channels by phosphatidylinositol 4,5-bisphosphate

With inside-out patchrecordings in ventricular myocytes from the hearts of guinea pigs, westudied ATP-sensitive K⁺ (K_ATP) channelsactivated by phosphatidylinositol 4,5-bisphosphate (PIP₂)with respect to sensitivity to ATP when in either a rundown state (RS)or a non-rundown state (NRS). Rundown of K_ATP channels wasinduced by exposure either to ATP-free solution or to ATP-free solutioncontaining 19 µM Ca²⁺. Exposure of membrane patches to 10 µM PIP₂ reactivated channels with both types of rundown.The reactivation by PIP₂ did not require ATP in the bath.The IC₅₀ of channels recovered from RS and before therundown was 37.1 and 31.1 µM, respectively. PIP₂irreversibly increased the mean current when the channel was in theNRS. This was associated with a shift of IC₅₀ to 250.6 µMafter PIP₂ exposure. PIP₂ activates NRSK_ATP channels by decreasing their sensitivity to ATP,whereas PIP₂ reactivates RS-K_ATP channelsindependently of ATP without changing ATP sensitivity.

     

Protein kinase C mediates erythrocyte "programmed cell death" following glucose depletion

Glucose depletion of erythrocytes leads to activation of Ca²⁺-permeable cation channels, Ca²⁺ entry, activation of a Ca²⁺-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca²⁺ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 µM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 µM) and the phosphatase inhibitor okadaic acid (1–10 µM) mimicked the effect of glucose depletion and similarly led to translocation of PKC and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 µM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca²⁺ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine     

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.

     

NAD Kinase in Corn: Regulation by Far Red Light is Mediated by Ca2+ and Calmodulin

Far red light irradiation of intact corn seedlings (Zea maysL.) has neither an effect on the cellular distribution nor onthe Ca²⁺, calmodulin-dependence of the NAD kinase (EC 2.7.1.23 [EC] ).The enzyme is located in the outer mitochondrial membrane andits activity is totally dependent on the presence of both Ca²⁺and calmodulin, independently of the illumination. In intactmitochondria and the presence of calmodulin the enzyme activityincreases linearly from 100 nM to 1 mM. At 100 µM Ca²⁺halfmaximal activation occurs at about 10 nM calmodulin. After solubilizationand purification by calmodulin-Sepharose chromatography theCa²⁺dependence of the enzyme changes. The activation reachesa plateau at about 100 µM Ca²⁺ and half maximal activationoccurs at about 6 µM Ca²⁺. On the other hand irradiationof intact corn seedlings as well as an increase of the cellularCa²⁺ concentration leads to an increase of NADP and a correspondingdecrease of NAD. Based on these data we suggest that the lighteffect on the NAD kinase activity is mediated by Ca²⁺ and calmodulin. (Received May 31, 1986; Accepted July 14, 1986)     

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.