Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus

This study investigated the effects of L-thyroxine-induced hyperthyroidism on Ca²⁺/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca²⁺ pump (Ca²⁺-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca²⁺-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30–50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) 1 (150%) Ca²⁺ pump isoform, unaltered levels of SERCA2 Ca²⁺ pump isoform, and lower levels of PLN (50%) and -, -, and -CaM kinase II (40 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities compared with that from euthyroid control. The V_max of Ca²⁺ uptake (in nmol Ca²⁺·mg SR protein^–1·min^–1: euthyroid, 818 ± 73; hyperthyroid, 1,649 ± 90) but not the apparent affinity of the Ca²⁺-ATPase for Ca²⁺ (euthyroid, 0.97 ± 0.02 µM, hyperthyroid, 1.09 ± 0.04 µM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca²⁺ uptake by soleus muscle SR was 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater (36%), and the time to peak force and relaxation time were significantly lower (30–40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca²⁺ pump. calmodulin kinase II; phospholamban ; calcium ion-adenosinetriphosphatase; sarcoplasmic reticulum     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by miconazole

The inhibition of sarcoplasmic reticulumCa²⁺-ATPase activity by miconazole was dependent on theconcentration of ATP and membrane protein. Half-maximal inhibition wasobserved at 12 µM miconazole when the ATP concentration was 50 µMand the membrane protein was 0.05 mg/ml. When ATP was 1 mM, a lowmicromolar concentration of miconazole activated the enzyme, whereashigher concentrations inhibited it. A qualitatively similar responsewas observed when Ca²⁺ transport was measured. Likewise,the half-maximal inhibition value was higher when the membraneconcentration was raised. Phosphorylation studies carried out aftersample preequilibration in different experimental settings shed lighton key partial reactions such as Ca²⁺ binding and ATPphosphorylation. The miconazole effect on Ca²⁺-ATPaseactivity can be attributed to stabilization of theCa²⁺-free enzyme conformation giving rise to a decrease inthe rate of the Ca²⁺ binding transition. The phosphoryltransfer reaction was not affected by miconazole.

     

Isoproterenol potentiates alpha -adrenergic and muscarinic receptor-mediated Ca2+ response in rat parotid cells

The effects of the cAMP pathway on theCa²⁺ response elicited byphospholipase C-coupled receptor stimulations were studied in ratparotid cells. Although 1 µM isoproterenol (Iso) itself had no effect on the cytosolicCa²⁺ concentration, thepretreatment with Iso potentiatedCa²⁺ responses evoked byphenylephrine. The potentiating effect of Iso was attributed to ashifting of the concentration-response curves of phenylephrine to theleft and an increase in the maximal response. Half-maximal potentiationoccurred at 3 nM Iso. Iso also potentiated theCa²⁺ response elicited bycarbachol. The potentiating effect of Iso was mimicked by forskolin (10 µM) and dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) and was blocked by 10 µM H-89. Iso potentiated thephenylephrine-induced Ca²⁺response in the absence of extracellularCa²⁺, but Iso did not increase theinositol trisphosphate (IP₃)production induced by phenylephrine. These results suggest that thepotentiation of the Ca²⁺ responsecan be attributed to a sensitization ofIP₃ receptors by cAMP-dependentprotein kinase.

     

Peroxide resistance of ER Ca2+ pump in endothelium: implications to coronary artery function

We examined the effects of peroxide on the sarco(endo)plasmicreticulum Ca²⁺ (SERCA) pump in pigcoronary artery endothelium and smooth muscle at three organizationallevels: Ca²⁺ transport inpermeabilized cells, cytosolicCa²⁺ concentration in intactcells, and contractile function of artery rings. We monitored theATP-dependent, azide-insensitive, oxalate-stimulated ⁴⁵Ca²⁺uptake by saponin-permeabilized cultured cells. Low concentrations ofperoxide inhibited the uptake less effectively in endothelium than insmooth muscle whether we added the peroxide directly to theCa²⁺ uptake solution or treatedintact cells with peroxide and washed them before the permeabilization.An acylphosphate formation assay confirmed the greater resistance ofthe SERCA pump in endothelial cells than in smooth muscle cells.Pretreating smooth muscle cells with 300 µM peroxide inhibited (by 77 ± 2%) the cyclopiazonic acid (CPA)-induced increase in cytosolicCa²⁺ concentration in aCa²⁺-free solution, but it did notaffect the endothelial cells. Peroxide pretreatment inhibited theCPA-induced contraction in deendothelialized arteries with a 50%inhibitory concentration of 97 ± 13 µM, but up to 500 µMperoxide did not affect the endothelium-dependent, CPA-inducedrelaxation. Similarly, 500 µM peroxide inhibited the angiotensin-induced contractions in deendothelialized arteries by 93 ± 2%, but it inhibited the bradykinin-induced,endothelium-dependent relaxation by only 40 ± 13%. The greaterresistance of the endothelium to reactive oxygen may be importantduring ischemia-reperfusion or in the postinfection immune response.

     

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.

     

Role of aspartate residues in Ca(2+) affinity and permeation of the distal ECaC1

The Ca²⁺ affinity andpermeation of the epithelial Ca²⁺ channel (ECaC1) wereinvestigated after expression in Xenopus oocytes. ECaC1displayed anomalous mole-fraction effects. Extracellular Ca²⁺ and Mg²⁺ reversibly inhibited ECaC1 wholecell Li⁺ currents: IC₅₀ = 2.2 ± 0.4 µM (n = 9) and 235 ± 35 µM (n = 10), respectively. These values compare well with theCa²⁺ affinity of the L-type voltage-gated Ca²⁺(Ca_V1.2) channel measured under the same conditions,suggesting that high-affinity Ca²⁺ binding is awell-conserved feature of epithelial and voltage-gated Ca²⁺channels. Neutralization of D550 and E535 in the pore region had nosignificant effect on Ca²⁺ and Mg²⁺ affinities.In contrast, neutralization of D542 significantly decreasedCa²⁺ affinity (IC₅₀ = 1.1 ± 0.2 mM,n = 6) and Mg²⁺ affinity(IC₅₀ > 25 ± 3 mM, n = 4).Despite a 1,000-fold decrease in Ca²⁺ affinity in D542N,Ca²⁺ permeation properties and theCa²⁺-to-Ba²⁺ conductance ratio remainedcomparable to values for wild-type ECaC1. Together, our observationssuggest that D542 plays a critical role in Ca²⁺ affinitybut not in Ca²⁺ permeation in ECaC1.

     

Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes

Depletion of Ca²⁺ stores inthe sarcoplasmic reticulum (SR) activates extracellularCa²⁺ influx via capacitativeCa²⁺ entry (CCE). Here, CCE levelsin proliferating and growth-arrested human pulmonary artery smoothmuscle cells (PASMCs) were compared by digital imaging fluorescencemicroscopy. Resting cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)in proliferating PASMCs was twofold higher than that in growth-arrestedcells. Cyclopiazonic acid (CPA; 10 µM), which inhibits SRCa²⁺-ATPase and depletes inositol1,4,5-trisphosphate-sensitiveCa²⁺ stores, transiently increased[Ca²⁺]_cytin the absence of extracellularCa²⁺. The addition of 1.8 mMCa²⁺ to the extracellular solutionin the presence of CPA induced large increases in[Ca²⁺]_cyt,indicative of CCE. The CPA-induced SRCa²⁺ release in proliferatingPASMCs was twofold higher than that in growth-arrested cells, whereasthe transient rise of[Ca²⁺]_cytdue to CCE was fivefold greater in proliferating cells. CCE wasinsensitive to nifedipine but was significantly inhibited by 50 mMK⁺, which reduces the drivingforce for Ca²⁺ influx, and by 0.5 mM Ni²⁺, a putative blocker ofstore-operated Ca²⁺ channels.These data show that augmented CCE is associated with proliferation ofhuman PASMCs and may be involved in stimulating and maintaining cell growth.

     

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)     

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.

     

Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum

Favero, Terence G., David Colter, Paul F. Hooper, andJonathan J. Abramson. Hypochlorous acid inhibitsCa²⁺-ATPase from skeletal musclesarcoplasmic reticulum. J. Appl. Physiol. 84(2): 425-430, 1998.Hypochlorous acid(HOCl) is produced by polymorphonuclear leukocytes that migrate andadhere to endothelial cells as part of the inflammatory response totissue injury. HOCl is an extremely toxic oxidant that can react with avariety of cellular components, and concentrations reaching 200 µMhave been reported in some tissues. In this study, we show that HOClinteracts with the skeletal sarcoplasmic reticulumCa²⁺-adenosinetriphosphatase(ATPase), inhibiting transport function. HOCl inhibits sarcoplasmicreticulum Ca²⁺-ATPase activity ina concentration-dependent manner with a concentration required toinhibit ATPase activity by 50% of 170 µM and with completeinhibition of activity at 3 mM. A concomitant reduction infree sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluoresceinisothiocyanate to the ATPase protein, indicating some structural damagemay have occurred. These findings suggest that the reactive oxygenspecies HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygenspecies disrupt the normalCa²⁺-handling kinetics in musclecells.

     

Effects of estradiol on phenylephrine contractility associated with intracellular calcium release in rat aorta

The ability of estradiol to affect phenylephrine-induced contraction and the subsequent increase in resting tone, associated with capacitative Ca²⁺ entry across the plasma membrane, was evaluated in rat aortic rings incubated in Ca²⁺-free solution. The incubation with estradiol (1–100 nM, 5 min) inhibited both the phenylephrine-induced contraction and the IRT. Neither cycloheximide (1 µM; inhibitor of protein synthesis) nor tamoxifen (1 µM; blocker of estrogenic receptors) modified the effects of estradiol. Estradiol (100 µM) also blocked the contractile response to serotonin (10 µM) but not to caffeine (10 mM). In addition, estradiol (100 µM) inhibited the contractile responses to cyclopiazonic acid (1 µM; selective Ca²⁺-ATPase inhibitor) associated with capacitative Ca²⁺ influx through non-L-type Ca²⁺ channels. Finally, estradiol inhibited the Ca²⁺-induced increases in intracellular free Ca²⁺ (after pretreatment with phenylephrine) in cultured rat aorta smooth muscle cells incubated in Ca²⁺-free solution. In conclusion, estradiol interfered in a concentration-dependent manner with Ca²⁺-dependent contractile effects mediated by the stimuli of ₁-adrenergic and serotonergic receptors and inhibited the capacitative Ca²⁺ influx through both L-type and non-L-type Ca²⁺ channels. Such effects are in essence nongenomic and not mediated by the intracellular estrogenic receptor. estrogen; ₁-adrenergic agonists     

Cross-bridge blocker BTS permits direct measurement of SR Ca2+ pump ATP utilization in toadfish swimbladder muscle fibers

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca²⁺ pumps. Specific and potent SR Ca²⁺ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl-p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca²⁺ pump ATP utilization in saponin-skinned fibers. At 25 µM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca²⁺ concentration ([Ca²⁺]) relationship, we measured SR Ca²⁺ pump ATP utilization in the presence and absence of BTS. At 25 µM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca²⁺]at 15°C. Curve fits to SR Ca²⁺ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior. muscle energetics; skinned muscle fibers; sarcoplasmic reticulum calcium ion pumps; cross bridges     

Differences in regulation of Ca2+-activated Cl- channels in colonic and parotid secretory cells

We investigatedthe regulation of Ca²⁺-activatedCl channels in cells fromthe human colonic cell line T84 and acinar cells from rat parotidglands. The participation of multifunctional Ca²⁺- and calmodulin-dependentprotein kinase (CaM kinase) II in the activation of these channels wasstudied using selective inhibitors of calmodulin and CaM kinase II.Ca²⁺-dependentCl currents were recordedusing the whole cell patch-clamp technique. Direct inhibition of CaMkinase II by 40 µM peptide 281-302 or by 10 µM KN-62, anotherCaM kinase inhibitor, did not block the Cl current in parotidacinar cells, whereas in T84 cells KN-62 markedly inhibited theCa²⁺-dependentCl current. We also usedthe calmodulin-binding domain peptide 290-309 (0.5 µM), whichcompetitively inhibits the activation of CaM kinase II. This peptidereduced the Cl current inT84 cells by ~70% but was without effect on the channels in parotidacinar cells. We conclude that theCa²⁺-dependentCl channels in T84 cellsare activated by CaM kinase II but that the channels in parotid acinarcells must be regulated by a fundamentally differentCa²⁺-dependent mechanism that doesnot utilize CaM kinase II or any calmodulin-dependent process.

     

Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase

Ca²⁺ has been postulated as a cytosolicsecond messenger in the regulation of cardiac oxidativephosphorylation. This hypothesis draws support from the well-knowneffects of Ca²⁺ on muscle activity, which is stimulated inparallel with the Ca²⁺-sensitive dehydrogenases (CaDH). Theeffects of Ca²⁺ on oxidative phosphorylation were furtherinvestigated in isolated porcine heart mitochondria at the level ofmetabolic driving force (NADH or ) and ATPproduction rates (flow). The resulting force-flow (F-F) relationshipspermitted the analysis of Ca²⁺ effects on several putativecontrol points within oxidative phosphorylation, simultaneously. TheF-F relationships resulting from additions of carbon substrates aloneprovided a model of pure CaDH activation. Comparing this curve withvariable Ca²⁺ concentration([Ca²⁺]) effects revealed an approximatetwofold higher ATP production rate than could be explained by a simpleincrease in NADH or via CaDH activation. The half-maximal effectof Ca²⁺ at state 3 was 157 nM and was completely inhibitedby ruthenium red (1 µM), indicating matrix dependence of theCa²⁺ effect. Arsenate was used as a probe to differentiatebetween F₀/F₁-ATPase and adenylate translocaseactivity by a futile recycling of ADP-arsenate within the matrix,catalyzed by the F₀/F₁-ATPase. Ca²⁺increased the ADP arsenylation rate more than twofold, suggesting adirect effect on the F₀/F₁-ATPase. Theseresults suggest that Ca²⁺ activates cardiac aerobicrespiration at the level of both the CaDH andF₀/F₁-ATPase. This type of parallel control ofboth intermediary metabolism and ATP synthesis may provide a mechanismof altering ATP production rates with minimal changes in thehigh-energy intermediates as observed in vivo.

     

Spontaneous acetylcholine release in mammalian neuromuscular junctions

Spontaneous secretion of the neurotransmitter acetylcholine inmammalian neuromuscular synapsis depends on theCa²⁺ content of nerve terminals.The Ca²⁺ electrochemical gradientfavors the entry of this cation. We investigated the possibleinvolvement of three voltage-dependent Ca²⁺ channels (VDCC) (L-, N-, andP/Q-types) on spontaneous transmitter release at the rat neuromuscularjunction. Miniature end-plate potential (MEPP) frequency was clearlyreduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to alesser extent by the N-type VDCC blocker, -conotoxin GVIA (-CgTx,5 µM). On the other hand, nifedipine and -CgTx had no effect onK⁺-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, preventsacetylcholine release induced byK⁺ depolarization but failed toaffect MEPP frequency in basal conditions. These results suggest thatin the mammalian neuromuscular junction Ca²⁺ enters nerve terminalsthrough at least three different channels, two of them (L- and N-types)mainly related to spontaneous acetylcholine release and the other(P/Q-type) mostly involved in depolarization-induced neurotransmitterrelease. Ca²⁺-bindingmolecule-related spontaneous release apparently binds Ca²⁺ very rapidly and wouldprobably be located very close toCa²⁺ channels, since the fastCa²⁺ chelator (BAPTA-AM)significantly reduced MEPP frequency, whereas EGTA-AM, exhibitingslower kinetics, had a lower effect. The increase in MEPP frequencyinduced by exposing the preparation to hypertonic solutions wasaffected by neither external Ca²⁺concentration nor L-, N-, and P/Q-type VDCC blockers, indicating thatextracellular Ca²⁺ is notnecessary to produce hyperosmotic neurosecretion. On the other hand,MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the sameextent, supporting the view that hypertonic response is promoted by"bulk" intracellular Ca²⁺concentration increases.

     

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells

Two H⁺-K⁺-ATPase isoforms are presentin kidney: the gastric, highly sensitive to Sch-28080, and the colonic,partially sensitive to ouabain. Upregulation of Sch-28080-sensitiveH⁺-K⁺-ATPase, or "gastric"H⁺-K⁺-ATPase, has been demonstrated inhypokalemic rat inner medullary collecting duct cells (IMCDs).Nevertheless, only colonic H⁺-K⁺-ATPase mRNAand protein abundance increase in this condition. This study wasdesigned to determine whether Sch-28080 inhibits transporters otherthan the gastric H⁺-K⁺-ATPase. In the presenceof bumetanide, Sch-28080 (200 µM) and ouabain (2 mM) inhibited⁸⁶Rb⁺ uptake (>90%). That⁸⁶Rb⁺ uptake was almost completely abolished bySch-28080 indicates an effect of this agent on theNa⁺-K⁺-ATPase. ATPase assays in membranes, orlysed cells, demonstrated sensitivity to ouabain but not Sch-28080.Thus the inhibitory effect of Sch-28080 was dependent on cellintegrity. ⁸⁶Rb⁺-uptake studies withoutbumetanide demonstrated that ouabain inhibited activity by only50%. Addition of Sch-28080 (200 µM) blocked all residualactivity. Intracellular ATP declined after Sch-28080 (200 µM) butrecovered after removal of this agent. In conclusion, highconcentrations of Sch-28080 inhibit K⁺-ATPase activity inmouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

     

Comparative effects of a low-carbohydrate diet and exercise plus a low-carbohydrate diet on muscle sarcoplasmic reticulum responses in males

We employed a glycogen-depleting session of exercise followed by a low-carbohydrate (CHO) diet to investigate modifications that occur in muscle sarcoplasmic reticulum (SR) Ca²⁺-cycling properties compared with low-CHO diet alone. SR properties were assessed in nine untrained males [peak aerobic power (O_{2 peak}) = 43.6 ± 2.6 (SE) ml·kg^–1·min^–1] during prolonged cycle exercise to fatigue performed at 58% O_{2 peak} after 4 days of low-CHO diet (Lo CHO) and after glycogen-depleting exercise plus 4 days of low-CHO (Ex+Lo CHO). Compared with Lo CHO, Ex+Lo CHO resulted in 12% lower (P < 0.05) resting maximal Ca²⁺-ATPase activity (V_max = 174 ± 12 vs. 153 ± 10 µmol·g protein^–1·min^–1) and smaller reduction in V_max induced during exercise. A similar effect was observed for Ca²⁺ uptake. The Hill coefficient, defined as slope of the relationship between cytosolic free Ca²⁺ concentration and Ca²⁺-ATPase activity, was higher (P < 0.05) at rest (2.07 ± 0.15 vs. 1.90 ± 0.10) with Ex+Lo CHO, an effect that persisted throughout the exercise. The coupling ratio, defined as the ratio of Ca²⁺ uptake to V_max, was 23–30% elevated (P < 0.05) at rest and during the first 60 min of exercise with Ex+Lo CHO. The 27 and 34% reductions (P < 0.05) in phase 1 and phase 2 Ca²⁺ release, respectively, observed during exercise with Lo CHO were not altered by Ex+Lo CHO. These results indicate that when prolonged exercise precedes a short-term Lo CHO diet, Ca²⁺ sequestration properties and efficiency are improved compared with those during Lo CHO alone. calcium cycling; vastus lateralis; contractile activity; glycogen; phosphorylation potential     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium     

Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

In the present study, we used laser scanning confocal microscopy in combination with fluorescent indicator dyes to investigate the effects of nitric oxide (NO) produced endogenously by stimulation of the mitochondria-specific NO synthase (mtNOS) or applied exogenously through a NO donor, on mitochondrial Ca²⁺ uptake, membrane potential, and gating of mitochondrial permeability transition pore (PTP) in permeabilized cultured calf pulmonary artery endothelial (CPAE) cells. Higher concentrations (100–500 µM) of the NO donor spermine NONOate (Sper/NO) significantly reduced mitochondrial Ca²⁺ uptake and Ca²⁺ extrusion rates, whereas low concentrations of Sper/NO (<100 µM) had no effect on mitochondrial Ca²⁺ levels ([Ca²⁺]_mt). Stimulation of mitochondrial NO production by incubating cells with 1 mM L-arginine also decreased mitochondrial Ca²⁺ uptake, whereas inhibition of mtNOS with 10 µM L-N⁵-(1-iminoethyl)ornithine resulted in a significant increase of [Ca²⁺]_mt. Sper/NO application caused a dose-dependent sustained mitochondrial depolarization as revealed with the voltage-sensitive dye tetramethylrhodamine ethyl ester (TMRE). Blocking mtNOS hyperpolarized basal mitochondrial membrane potential and partially prevented Ca²⁺-induced decrease in TMRE fluorescence. Higher concentrations of Sper/NO (100–500 µM) induced PTP opening, whereas lower concentrations (<100 µM) had no effect. The data demonstrate that in calf pulmonary artery endothelial cells, stimulation of mitochondrial Ca²⁺ uptake can activate NO production in mitochondria that in turn can modulate mitochondrial Ca²⁺ uptake and efflux, demonstrating a negative feedback regulation. This mechanism may be particularly important to protect against mitochondrial Ca²⁺ overload under pathological conditions where cellular [NO] can reach very high levels. nitric oxide synthase; permeability transition pore; endothelium     

Chronic intermittent hypoxia alters Ca2+ handling in rat cardiomyocytes by augmented Na+/Ca2+ exchange and ryanodine receptor activities in ischemia-reperfusion

This study examined Ca²⁺ handling mechanisms involved in cardioprotection induced by chronic intermittent hypoxia (CIH) against ischemia-reperfusion (I/R) injury. Adult male Sprague-Dawley rats were exposed to 10% inspired O₂ continuously for 6 h daily from 3, 7, and 14 days. In isolated perfused hearts subjected to I/R, CIH-induced cardioprotection was most significant in the 7-day group with less infarct size and lactate dehydrogenase release, compared with the normoxic group. The I/R-induced alterations in diastolic Ca²⁺ level, amplitude, time-to-peak, and the decay time of both electrically and caffeine-induced Ca²⁺ transients measured by spectrofluorometry in isolated ventricular myocytes of the 7-day CIH group were less than that of the normoxic group, suggesting an involvement of altered Ca²⁺ handling of the sarcoplasmic reticulum (SR) and sarcolemma. We further determined the protein expression and activity of ⁴⁵Ca²⁺ flux of SR-Ca²⁺-ATPase, ryanodine receptor (RyR) and sarcolemmal Na⁺/Ca²⁺ exchange (NCX) in ventricular myocytes from the CIH and normoxic groups before and during I/R. There were no changes in expression levels of the Ca²⁺-handling proteins but significant increases in the RyR and NCX activities were remarkable during I/R in the CIH but not the normoxic group. The augmented RyR and NCX activities were abolished, respectively, by PKA inhibitor (0.5 µM KT5720 or 0.5 µM PKI_14-22) and PKC inhibitor (5 µM chelerythrine chloride or 0.2 µM calphostin C) but not by Ca²⁺/calmodulin-dependent protein kinase II inhibitor KN-93 (1 µM). Thus, CIH confers cardioprotection against I/R injury in rat cardiomyocytes by altered Ca²⁺ handling with augmented RyR and NCX activities via protein kinase activation. cardioprotection; intracellular calcium