Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca²⁺ accumulation resulting from functional coupling of Na⁺/Ca²⁺ exchange (NCE) with stimulated Na⁺/H⁺ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na⁺ and therefore Ca²⁺ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH_i), Na⁺ (Na), and calcium concentration ([Ca²⁺]_i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH_i, Na and [Ca²⁺]_i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH_i fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca²⁺]_i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca²⁺]_i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na⁺/H⁺ exchange; Na⁺/Ca²⁺ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation     

Quantitation of intracellular [Na+] in vivo by using TmDOTP5-asanNMRshiftreagentand extracellularmarker

A method ispresented to measure the absolute concentration of intracellularNa⁺([Na⁺]_i)in vivo by using interleaved ²³Na-and ³¹P-nuclear magnetic resonance(NMR) spectroscopy andTmDOTP⁵ as shift reagentand chemical marker of tissue extracellular space (ECS). The techniquewas used to determine[Na⁺]_iand relative ECS in livers of control rats (21 ± 3 and0.11 ± 0.02 mM, respectively) and in rats exposed to carbontetrachloride (103 ± 29 and 0.23 ± 0.03 mM, respectively). TheNMR measurements were confirmed independently on excised tissue samplesby using atomic absorption spectroscopy. The results confirm thatTmDOTP⁵ can be used as acombined cation shift reagent and ECS marker, thereby allowingquantitation of[Na⁺]_iin vivo by NMR.

     

Regulation of the epithelial Na+ channel by intracellular Na+

The hypothesis that the intracellularNa⁺ concentration([Na⁺]_i)is a regulator of the epithelialNa⁺ channel (ENaC) was tested withthe Xenopus oocyte expression systemby utilizing a dual-electrode voltage clamp.[Na⁺]_iaveraged 48.1 ± 2.2 meq (n = 27)and was estimated from the amiloride-sensitive reversal potential.[Na⁺]_iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 MNa₂SO₄.Within minutes of injection,[Na⁺]_istabilized and remained elevated at 97.8 ± 6.5 meq(n = 9) and 64.9 ± 4.4 (n = 5) meq 30 min after theinitial injection of 0.5 and 0.25 MNa₂SO₄,respectively. This increase of[Na⁺]_icaused a biphasic inhibition of ENaC currents. In oocytes injected with0.5 MNa₂SO₄(n = 9), a rapid decrease of inwardamiloride-sensitive slope conductance(g_Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slowerdecrease to 0.304 ± 0.043 of control at 30 min were observed.Similar but smaller inhibitions were also observed with the injectionof 0.25 MNa₂SO₄.Injection of isotonicK₂SO₄(70 mM) or isotonicK₂SO₄made hypertonic with sucrose (70 mMK₂SO₄-1.2M sucrose) was without effect. Injection of a 0.5 M concentration ofeitherK₂SO₄,N-methyl-D-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thusincreases of[Na⁺]_ihave multiple specific inhibitory effects on ENaC that can betemporally separated into a rapid phase that was complete within 2-3 min and a delayed slow phase that was observed between 5 and 30 min.

     

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

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

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Hypertonic perfusion inhibits intracellular Na and Ca accumulation in hypoxic myocardium

Muchevidence supports the view that hypoxic/ischemic injury is largely dueto increased intracellular Ca concentration([Ca]_i) resulting from 1) decreasedintracellular pH (pH_i), 2) stimulated Na/H exchangethat increases Na uptake and thus intracellular Na (Na_i),and 3) decreased Na gradient that decreases or reverses net Catransport via Na/Ca exchange. The Na/H exchanger (NHE) is alsostimulated by hypertonic solutions; however, hypertonic media mayinhibit NHE's response to changes in pH_i (Cala PM and Maldonado HM. J Gen Physiol 103: 1035-1054, 1994). Thus wetested the hypothesis that hypertonic perfusion attenuates acid-induced increases in Na_i in myocardium and, thereby, decreasesCa_i accumulation during hypoxia. Rabbit hearts wereLangendorff perfused with HEPES-buffered Krebs-Henseleit solutionequilibrated with 100% O₂ or 100% N₂. Hypertonic perfusion began 5 min before hypoxia or normoxicacidification (NH₄Cl washout). Na_i,[Ca]_i, pH_i, and high-energyphosphates were measured by NMR. Control solutions were 295 mosM, andhypertonic solutions were adjusted to 305, 325, or 345 mosM by additionof NaCl or sucrose. During 60 min of hypoxia (295 mosM),Na_i rose from 22 ± 1 to 100 ± 10 meq/kg dry wt while[Ca]_i rose from 347 ± 11 to 1,306 ± 89 nM.During hypertonic hypoxic perfusion (325 mosM), increases inNa_i and [Ca]_i were reduced by 65 and 60%, respectively (P < 0.05). Hypertonicperfusion also diminished Na uptake after normoxic acidification by87% (P < 0.05). The data are consistent with the hypothesisthat mild hypertonic perfusion diminishes acid-induced Na accumulationand, thereby, decreases Na/Ca exchange-mediated Ca_iaccumulation during hypoxia.

     

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.

     

Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle

The role of Na⁺/Ca²⁺ exchange inregulating intracellular Ca²⁺ concentration([Ca²⁺]_i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na⁺ concentration resulted in agraded rise of [Ca²⁺]_i; 50-100 µMstrophanthidin also increased [Ca²⁺]_i. Asmall outward current accompanied the rise of[Ca²⁺]_i in low-Na⁺ solutions(17.1 ± 1.8 pA in 29.4 mM Na⁺). The quantity ofCa²⁺ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca²⁺]_i,after correction was made for intracellular Ca²⁺ buffering.Ca²⁺ influx through the exchanger was able to loadintracellular Ca²⁺ stores. It is concluded that the levelof resting [Ca²⁺]_i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na⁺ concentration would besufficient to generate significant net Ca²⁺ influx.

     

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

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

     

NHE3-dependent cytoplasmic alkalinization is triggered by Na(+)-glucose cotransport in intestinal epithelia

Cytoplasmic pH (pH_i) was evaluated duringNa⁺-glucose cotransport in Caco-2 intestinal epithelialcell monolayers. The pH_i increased by 0.069 ± 0.002 within 150 s after initiation of Na⁺-glucosecotransport. This increase occurred in parallel with glucose uptake andrequired expression of the intestinal Na⁺-glucosecotransporter SGLT1. S-3226, a preferential inhibitor ofNa⁺/H⁺ exchanger (NHE) isoform 3 (NHE3),prevented cytoplasmic alkalinization after initiation ofNa⁺-glucose cotransport with an ED₅₀ of 0.35 µM, consistent with inhibition of NHE3, but not NHE1 or NHE2. Incontrast, HOE-694, a poor NHE3 inhibitor, failed to significantlyinhibit pH_i increases at <500 µM.Na⁺-glucose cotransport was also associated with activationof p38 mitogen-activated protein (MAP) kinase, and the p38 MAP kinase inhibitors PD-169316 and SB-202190 prevented pH_i increasesby 100 ± 0.1 and 86 ± 0.1%, respectively. Conversely,activation of p38 MAP kinase with anisomycin induced NHE3-dependentcytoplasmic alkalinization in the absence of Na⁺-glucosecotransport. These data show that NHE3-dependent cytoplasmic alkalinization occurs after initiation of SGLT1-mediatedNa⁺-glucose cotransport and that the mechanism of this NHE3activation requires p38 MAP kinase activity. This coordinatedregulation of glucose (SGLT1) and Na⁺ (NHE3) absorptiveprocesses may represent a functional activation of absorptiveenterocytes by luminal nutrients.

     

Sodium gradient-dependent transport of magnesium in rat ventricular myocytes

Cytoplasmic concentration of Mg²⁺([Mg²⁺]_i) was measured with a fluorescentindicator furaptra in ventricular myocytes enzymatically dissociatedfrom rat hearts (25°C). To study Mg²⁺ transport acrossthe cell membrane, cells were treated with ionomycin inCa²⁺-free (0.1 mM EGTA) and high-Mg²⁺ (10 mM)conditions to facilitate passive Mg²⁺ influx. Rate of riseof [Mg²⁺]_i due to the net Mg²⁺influx was significantly smaller in the presence of 130 mMextracellular Na⁺ than in its absence. We also tested theextracellular Na⁺ dependence of the net Mg²⁺efflux from cells loaded with Mg²⁺. After[Mg²⁺]_i was raised by ionomycin and highMg²⁺ to the level 0.5-0.6 mM above the basal value(~0.7 mM), washout of ionomycin and lowering extracellular[Mg²⁺] to 1.2 mM caused rapid decline of[Mg²⁺]_i in the presence of 140 mMNa⁺. This net efflux of Mg²⁺ was completelyinhibited by withdrawal of extracellular Na⁺ and waslargely attenuated by imipramine, a known inhibitor of Na⁺/Mg²⁺ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg²⁺ efflux andextracellular Na⁺ concentration([Na⁺]_o) had a Hill coefficient of 2 and[Na⁺]_o at half-maximal rate of 82 mM. Theseresults demonstrate the presence of Na⁺ gradient-dependentMg²⁺ transport, which is consistent withNa⁺/Mg²⁺ exchange, in cardiac myocytes.

     

Na+/Ca2+ exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function

The role of theNa⁺/Ca²⁺exchanger in intracellular Ca²⁺regulation was investigated in freshly dissociated catfish retinalhorizontal cells (HC).Ca²⁺-permeable glutamate receptorsand L-type Ca²⁺ channels as wellas inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitiveintracellular Ca²⁺ stores regulateintracellular Ca²⁺ in these cells.We used the Ca²⁺-sensitive dyefluo 3 to measure changes in intracellularCa²⁺ concentration([Ca²⁺]_i)under conditions in whichNa⁺/Ca²⁺exchange was altered. In addition, the role of theNa⁺/Ca²⁺exchanger in the refilling of the caffeine-sensitiveCa²⁺ store followingcaffeine-stimulated Ca²⁺ releasewas assessed. Brief applications of caffeine (1-10 s) producedrapid and transient changes in[Ca²⁺]_i.Repeated applications of caffeine produced smallerCa²⁺ transients until no furtherCa²⁺ was released. Store refillingoccurred within 1-2 min and required extracellularCa²⁺. Ouabain-induced increases inintracellular Na⁺ concentration([Na⁺]_i)increased both basal free[Ca²⁺]_iand caffeine-stimulated Ca²⁺release. Reduction of external Na⁺concentration([Na⁺]_o)further and reversibly increased[Ca²⁺]_iin ouabain-treated HC. This effect was not abolished by the Ca²⁺ channel blocker nifedipine,suggesting that increases in[Na⁺]_ipromote net extracellular Ca²⁺influx through aNa⁺/Ca²⁺exchanger. Moreover, when[Na⁺]_owas replaced by Li⁺, caffeine didnot stimulate release of Ca²⁺ fromthe caffeine-sensitive store afterCa²⁺ depletion. TheNa⁺/Ca²⁺exchanger inhibitor 2',4'-dimethylbenzamil significantlyreduced the caffeine-evoked Ca²⁺response 1 and 2 min after store depletion.

     

Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat

The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca²⁺]_i andCa²⁺ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca²⁺), the resting length and[Ca²⁺]_i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 10⁵ M) caused transient increasein [Ca²⁺]_i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca²⁺]_i transient was notsignificantly different, but the maintained [Ca²⁺]_i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa²⁺-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca²⁺]_i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca²⁺]_i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca²⁺]_i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca²⁺]_i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca²⁺]_i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca²⁺]_i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (10⁸ M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca²⁺]_i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca²⁺]_i and contraction of vascularsmooth muscle triggered by Ca²⁺ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca²⁺]_i due to Ca²⁺release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca²⁺]_i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

     

Intracellular pH homeostasis in cultured human placental syncytiotrophoblast cells: recovery from acidification

Resting or basal intracellular pH (pH_i) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO₃^–) or 7.24 ± 0.03 (with HCO₃^–). Ion substitution and inhibitor experiments were performed to determine whether common H⁺-transporting species were operating to maintain basal pH_i. Removal of extracellular Na⁺ or Cl^– or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H₂DIDS) had no effect. Acidification with the K⁺/H⁺ exchanger nigericin reduced pH_i to 6.25 ± 0.15 (without HCO₃^–) or 6.53 ± 0.10 (with HCO₃^–). In the presence of extracellular Na⁺, recovery to basal pH_i was prompt and occurred at similar rates in the absence and presence of HCO₃^–. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pH_i after acidification. Recovery was inhibited by removal of Na⁺ or addition of amiloride, whereas removal of Cl^– and addition of H₂DIDS were ineffective. Addition of the Na⁺/H⁺ exchanger monensin to cells that had returned to basal pH_i elicited a further increase in pH_i to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pH_i approached the basal level, despite the continued presence of a driving force for H⁺ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pH_i is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na⁺/H⁺ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein     

LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na(+) transport in A6 epithelia

Blocker-inducednoise analysis of epithelial Na⁺ channels (ENaCs) was usedto investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na⁺transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na⁺ transport rates(I_Na) of 4.0 ± 0.1 (unstimulated) and9.1 ± 0.9 µA/cm² (aldosterone), 10 µM LY-294002caused, following a relatively small initial increase of transport, acompletely reversible inhibition of transport within 90 min to 33 ± 6% and 38 ± 2% of respective baseline values. Initialincreases of transport could be attributed to increases of channel openprobability (P_o) within 5 min to 143 ± 17% (unstimulated) and 142 ± 10% of control (aldosterone) frombaseline P_o averaging near 0.5. Inhibition oftransport was due to much slower decreases of functional channeldensities (N_T) to 28 ± 4% (unstimulated)and 35 ± 3% (aldosterone) of control at 90 min. LY-294002 (50 µM) caused larger but completely reversible increases ofP_o (215 ± 38% of control at 5 min) andmore rapid but only slightly larger decreases ofN_T. Basolateral exposure to LY-294002 induced nodetectable effect on transport, P_o or N_T. We conclude that an LY-294002-sensitive PI3-kinase plays an important role in regulation of transport bymodulating N_T and P_o ofENaCs, but only when presented to apical surfaces of the cells.

     

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.

     

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

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

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts

The ability to image calciumsignals at subcellular levels within the intact depolarizing heartcould provide valuable information toward a more integratedunderstanding of cardiac function. Accordingly, a system combiningtwo-photon excitation with laser-scanning microscopy was developed tomonitor electrically evoked [Ca²⁺]_itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca²⁺]_itransients were recorded at depths 100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked[Ca²⁺]_i transients were highly synchronizedamong neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50%(t_90-50%) and from 50 to 10%(t_50-10%) of the peak[Ca²⁺]_i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and94 ± 6 ms (n = 19, P < 0.05 vs.1 Hz) in rhod-2-loaded cardiomyocytes.[Ca²⁺]_i decay was markedly slower infura-2-loaded hearts (t_90-50% at 1 Hz,128 ± 9 ms and at 2 Hz, 88 ± 5 ms;t_50-10% at 1 Hz, 214 ± 18 ms and at2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of[Ca²⁺]_i decline resulted from increasedcytosolic Ca²⁺ buffering, because the kinetics of rhod-2decay resembled those obtained with fura-2 after incorporation of theCa²⁺ chelator BAPTA. Propagating calcium waves and[Ca²⁺]_i amplitude alternans were readilydetected in paced hearts. This approach should be of general utility tomonitor the consequences of genetic and/or functional heterogeneity incellular calcium signaling within whole mouse hearts at tissue depthsthat have been inaccessible to single-photon imaging.

     

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

The general phosphate need in mammalian cells is accommodated by members of the P_i transport (PiT) family (SLC20), which use either Na⁺ or H⁺ to mediate inorganic phosphate (P_i) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na⁺-dependent P_i (NaP_i) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with ³²P_i as a traceable P_i source. For PiT1, the Michaelis-Menten constant for P_i was determined as 322.5 ± 124.5 µM. PiT2 was analyzed for the first time and showed positive cooperativity in P_i uptake with a half-maximal activity constant for P_i of 163.5 ± 39.8 µM. PiT1- and PiT2-mediated Na⁺-dependent P_i uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na⁺ dependency patterns. However, only PiT2 was capable of Na⁺-independent P_i transport at acidic pH. Study of the impact of divalent cations Ca²⁺ and Mg²⁺ revealed that Ca²⁺ was important, but not critical, for NaP_i transport function of PiT proteins. To gain insight into the NaP_i cotransport function, we analyzed PiT2 and a PiT2 P_i transport knockout mutant using ²²Na⁺ as a traceable Na⁺ source. Na⁺ was transported by PiT2 even without P_i in the uptake medium and also when P_i transport function was knocked out. This is the first time decoupling of P_i from Na⁺ transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E⁵⁵ and E⁵⁷⁵ are responsible for linking P_i import to Na⁺ transport in PiT2. inorganic phosphate transport; retroviral receptor; SLC20     

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

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