Polyamines regulate beta-catenin tyrosine phosphorylation via Ca(2+) during intestinal epithelial cell migration

Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca²⁺concentration ([Ca²⁺]_cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca²⁺]_cyt induced by theCa²⁺ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca²⁺]_cyt.

     

Impaired G(s)alpha and adenylyl cyclase cause beta-adrenoceptor desensitization in chronically hypoxic rat hearts

The effectsof -adrenoceptor stimulation with isoproterenol on electricallyinduced contraction and intracellular calcium ([Ca²⁺]_i) transient, and cAMP inmyocytes from both hypertrophied right and nonhypertrophied leftventricles of rats exposed to 10% oxygen for 4 wk, were significantlyattenuated. The increased [Ca²⁺]_i transientin response to cholera toxin was abolished, whereas increased cAMPafter NaF significantly attenuated. The biologically activeisoform, G_s-small (45 kDa), was reduced while thebiologically inactive isoform, G_s-large (52 kDa),increased. The increased electrically induced[Ca²⁺]_i transient and cAMP with 10-100µM forskolin were significantly attenuated in chronically hypoxicrats. The content of G_i2, the predominantisoform of G_i protein in the heart, was unchanged. Resultsindicate that impaired functions of G_s protein and adenylyl cyclase cause -adrenoceptor desensitization. The impaired function of the G_s protein may be due to reducedG_s-small and/or increased G_s-large, whichdoes not result from changes in G_i protein. Responses toall treatments were the same for right and left ventricles, indicatingthat the impaired cardiac functions are not secondary to cardiac hypertrophy.

     

Arachidonic acid mediates dual effect of TNF-alpha on Ca2+ transients and contraction of adult rat cardiomyocytes

Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A₂ (PLA₂) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca²⁺ concentration([Ca²⁺]_i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca²⁺]_i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca²⁺]_i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca²⁺]_i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA₂.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA₂/AA pathway in mediating thecontractile effects of TNF-.

     

Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae

In the ratsphincter pupillae, as in other smooth muscles, the primary signaltransduction cascade for agonist activation is receptor  G protein phospholipase C  inositol trisphosphate  intracellularCa²⁺ concentration ([Ca²⁺]_i)  calmodulin  myosin light chain kinase  phosphorylated myosin  force development. Light stimulation of isolated sphincters pupillaecan be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMRideal for testing models of regulation of smooth muscle myosinphosphorylation. We measured force and[Ca²⁺]_i concurrently in sphincter pupillaefollowing stimulation by light flashes of varying duration andintensity. We sampled at unusually short (0.01-0.02 s) intervalsto adequately test a PMR model based on the myosin phosphorylationcascade. We found, surprisingly, contrary to the behavior of intestinalmuscle and predictions of the phosphorylation model, that during PMRsforce begins to decay while [Ca²⁺]_i is stillrising. We conclude that control of contraction in the sphincterpupillae probably involves an inhibitory process as well as activationby [Ca²⁺]_i.

     

Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria

The solubleCa²⁺-binding protein parvalbumin (PV) is expressed at highlevels in fast-twitch muscles of mice. Deficiency of PV in knockoutmice (PV /) slows down the speed of twitch relaxation, whilemaximum force generated during tetanic contraction is unaltered. Weobserved that PV-deficient fast-twitch muscles were significantly moreresistant to fatigue than were the wild type. Thus components involvedin Ca²⁺ homeostasis during the contraction-relaxation cyclewere analyzed. No upregulation of another cytosolicCa²⁺-binding protein was found. Mitochondria are thought toplay a physiological role during muscle relaxation and were thusanalyzed. The fractional volume of mitochondria in the fast-twitchmuscle extensor digitorum longus (EDL) was almost doubled in PV /mice, and this was reflected in an increase of cytochrome coxidase. A faster removal of intracellular Ca²⁺concentration ([Ca²⁺]_i) 200-700 ms afterfast-twitch muscle stimulation observed in PV / muscles supportsthe role for mitochondria in late [Ca²⁺]_iremoval. The present results also show a significant increase of thedensity of capillaries in EDL muscles of PV / mice. Thus alterations in the dynamics of Ca²⁺ transients detected infast-twitch muscles of PV / mice might be linked to the increase inmitochondria volume and capillary density, which contribute to thegreater fatigue resistance of these muscles.

     

Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation

Westudied the interplay between matrix Ca²⁺ concentration([Ca²⁺]) and mitochondrial membrane potential() in regulation of the mitochondrial permeability transition(MPT) during anoxia and reoxygenation. Without Ca²⁺loading, anoxia caused near-synchronous dissipation,mitochondrial Ca²⁺ efflux, and matrix volume shrinkage whena critically low PO₂ was reached, which wasrapidly reversible upon reoxygenation. These changes were related toelectron transport inhibition, not MPT. Cyclosporin A-sensitive MPT didoccur when extramitochondrial [Ca²⁺] was increased topromote significant Ca²⁺ uptake during anoxia, depending onthe Ca²⁺ load size and ability to maintain . However,when [Ca²⁺] was increased after complete dissipation, MPT did not occur until reoxygenation, at which timereactivation of electron transport led to partial regeneration.In the setting of elevated extramitochondrial Ca²⁺, thisenhanced matrix Ca²⁺ uptake while promoting MPT because ofless than full recovery of . The interplay between andmatrix [Ca²⁺] in accelerating or inhibiting MPT duringanoxia/reoxygenation has implications for preventing reoxygenationinjury associated with MPT.

     

Impaired [Ca(2+)](i) and pH(i) responses to kappa-opioid receptor stimulation in the heart of chronically hypoxic rats

-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa²⁺ ([Ca²⁺]_i) transient andincreased the intracellular pH (pH_i) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na⁺/H⁺exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca²⁺]_i transient andpH_i were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca²⁺]_i and pH_i responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

     

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.

     

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.

     

Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia

Cellular sodium excess is cytotoxicbecause it increases both the intracellular osmotic load andintracellular calcium concentration ([Ca²⁺]_i). Because sodium levels rise duringhypoxia, it is thought to contribute to hypoxic injury. Thus thepresent study tested the hypothesis that taurine-linked reductions in[Na⁺]_i reduce hypoxia-induced cell injury.Taurine depletion was achieved by exposing isolated neonatalcardiomyocytes to medium containing the taurine analog -Alanine. Aspredicted, the -Alanine-treated cell exhibited less hypoxia-inducednecrosis and apoptosis than the control, as evidenced by lessswelling, shrinkage, TdT-mediated dUTP nick end labeling staining, andaccumulation of trypan blue. After 1 h of chemical hypoxia,[Na⁺]_i was 3.5-fold greater in the controlthan the taurine-deficient cell. Although more taurine was lost fromthe control cell than from the -Alanine-treated cell during hypoxia,the combined taurine and sodium osmotic load was lower in the-Alanine-treated cell. Taurine deficiency also reduced the degree ofhypoxia-induced calcium overload. Thus the observed resistance againsthypoxia-induced necrosis and apoptosis is probably related toan improvement in sodium and calcium handling.

     

Differences in Ca(2+) signaling underlie age-specific effects of secretagogues on colonic Cl(-) transport

Taurodeoxycholic acid (TDC) stimulates Cl transport inadult (AD), but not weanling (WN) and newborn (NB), rabbit colonic epithelial cells (colonocytes). The present study demonstrates thatstimuli like neurotensin (NT) are also age specific and identifies theage-dependent signaling step. Bile acid actions are segment and bileacid specific. Thus although TDC and taurochenodeoxycholate stimulateCl transport in AD distal but not proximal colon,taurocholate has no effect in either segment. TDC increasesintracellular Ca²⁺ concentration([Ca²⁺]_i) in AD, but not in WN and NB,colonocytes. In AD cells, TDC (5 min) action on Cltransport needs intra- but not extracellular Ca²⁺. NT,histamine, and bethanechol increase Cl transport and[Ca²⁺]_i in AD, but not WN, distalcolonocytes. However, A-23187 increased [Ca²⁺]_i and Cl transport in allage groups, suggesting that Ca²⁺-sensitive Cltransport is present from birth. Study of the proximal steps inCa²⁺ signaling revealed that NT, but not TDC, activates aGTP-binding protein, G_q, in AD and WN cells. Inaddition, although WN and AD colonocytes had similar levels ofphosphatidylinositol 4,5-bisphosphate, NT and TDC increased1,4,5-inositol trisphosphate content only in AD cells.Nonresponsiveness of WN cells to Ca²⁺-dependent stimuli,therefore, is due to the absence of measurable phospholipase Cactivity. Thus delays in Ca²⁺ signaling afford a crucialprotective mechanism to meet the changing demands of the developing colon.

     

Suppression of cAMP by phosphoinositol/Ca2+ pathway in the cardiac kappa -opioid receptor

To determine whetherthe phosphoinositol/Ca²⁺ pathwayinteracts with the adenylate cyclase/adenosine 3',5'-cyclicmonophosphate (cAMP) pathway in the cardiac -receptor, the effectsof U-50488, a specific -receptor agonist, on the intracellularCa²⁺ concentration([Ca²⁺]_i)and forskolin-induced accumulation of cAMP in rat ventricular myocyteswere determined after interference of thephosphoinositol/Ca²⁺ pathway.U-50488 suppressed the forskolin-induced accumulation of cAMP andelevated[Ca²⁺]_i,which were blocked by norbinaltorphimine, a specific -receptor antagonist, and pertussis toxin. The effects of U-50488 werequalitatively similar to those of A-23187, aCa²⁺ ionophore, but opposite tothose of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a[Ca²⁺]_ichelator. Abolition of U-50488-induced elevation of[Ca²⁺]_iby BAPTA-AM also abolished the effect of U-50488 on forskolin-induced accumulation of cAMP. Inhibition of the phospholipase C by specific inhibitors, U-73122 and neomycin, abolished the effects of U-50488 onboth[Ca²⁺]_iand forskolin-induced accumulation of cAMP. The results showed for thefirst time that -receptor stimulation may suppress cAMP accumulationvia activation of thephosphoinositol/Ca²⁺ pathway inthe rat heart.

     

Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel

Protons regulateelectrogenic sodium absorption in a variety of epithelia, including thecortical collecting duct, frog skin, and urinary bladder. Recently,three subunits (, , ) coding for the epithelial sodium channel(ENaC) were cloned. However, it is not known whether pH regulatesNa⁺ channels directly byinteracting with one of the three ENaC subunits or indirectly byinteracting with a regulatory protein. As a first step to identifyingthe molecular mechanisms of proton-mediated regulation of apicalmembrane Na⁺ permeability inepithelia, we examined the effect of pH on the biophysical propertiesof ENaC. To this end, we expressed various combinations of -, -,and -subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clampand patch-clamp techniques. In addition, the effect of pH on the-ENaC subunit was examined in planar lipid bilayers. We report that ,,-ENaC currents were regulated by changes in intracellular pH(pH_i) but not by changes inextracellular pH (pH_o).Acidification reduced and alkalization increased channel activity by avoltage-independent mechanism. Moreover, a reduction ofpH_i reduced single-channel openprobability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited ,-ENaC, ,-ENaC, and -ENaC currents. We conclude thatpH_i but notpH_o regulates ENaC and that the-ENaC subunit is regulated directly bypH_i.     

Synergism between toxin-gamma from Brazilian scorpion Tityus serrulatus and veratridine in chromaffin cells

Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC₅₀ values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca²⁺concentration([Ca²⁺]_o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of⁴⁵Ca²⁺,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca²⁺ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca²⁺]_o.Veratridine induced oscillations of cytosolicCa²⁺ concentration([Ca²⁺]_i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca²⁺]_ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca²⁺]_iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa⁺ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na⁺channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na⁺ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca²⁺ channels, andCa²⁺ entry and secretion.

     

Regulation of arterial tone by smooth muscle myosin type II

Theinitiation of contractile force in arterial smooth muscle (SM) isbelieved to be regulated by the intracellular Ca²⁺concentration and SM myosin type II phosphorylation. We tested thehypothesis that SM myosin type II operates as a molecular motor proteinin electromechanical, but not in protein kinase C (PKC)-induced,contraction of small resistance-sized cerebral arteries. We utilized aSM type II myosin heavy chain (MHC) knockout mouse model and measuredarterial wall Ca²⁺ concentration([Ca²⁺]_i) and the diameter of pressurizedcerebral arteries (30-100 µm) by means of digital fluorescencevideo imaging. Intravasal pressure elevation caused a graded[Ca²⁺]_i increase and constricted cerebralarteries of neonatal wild-type mice by 20-30%. In contrast,intravasal pressure elevation caused a graded increase of[Ca²⁺]_i without constriction in (/)MHC-deficient arteries. KCl (60 mM) induced a further[Ca²⁺]_i increase but failed to inducevasoconstriction of (/) MHC-deficient cerebral arteries. Activationof PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM)induced a strong, sustained constriction of (/) MHC-deficientcerebral arteries without changing [Ca²⁺]_i.These results demonstrate a major role for SM type II myosin in thedevelopment of myogenic tone and Ca²⁺-dependentconstriction of resistance-sized cerebral arteries. In contrast, thesustained contractile response did not depend on myosin andintracellular Ca²⁺ but instead depended on PKC. We suggestthat SM myosin type II operates as a molecular motor protein in thedevelopment of myogenic tone but not in pharmacomechanical coupling byPKC in cerebral arteries. Thus PKC-dependent phosphorylation ofcytoskeletal proteins may be responsible for sustained contraction invascular SM.

     

Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio

We proposed andtested the use of nontraditional excitation wavelengths(₁ and ₂) and an emission wavelength(_em) to define conditions under which free calciumconcentration and a fluorescence ratio are linearly related.Fluorescence spectra were determined for aqueous solutions thatcontained 25 µM fura 2, 125 mM K⁺, and either 0 mM or 0.1 mM Ca²⁺. Effectively linear relationships between[Ca²⁺] and a fluorescence ratio, i.e., <5% bias when[Ca²⁺]  5 × dissociation constant, were apparentwhen ₁  400 nm, ₂  370 nm, and_em  510 nm. Combinations with longer ₁and _em and/or with shorter ₂ reduced thisbias further. Although the method described does not obviate thecomplications that surround the correction for fluorescence background,choosing a nontraditional combination of excitation and emissionwavelengths offers several practical advantages over more traditionalfura 2 fluorescence methodologies in a variety of experimental settings.

     

Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice

Thecalcium-binding protein parvalbumin (PV) occurs at high concentrationsin fast-contracting vertebrate muscle fibers. Its putative role infacilitating the rapid relaxation of mammalian fast-twitch musclefibers by acting as a temporary buffer for Ca²⁺ is still controversial. Wegenerated knockout mice for PV (PV /) and compared theCa²⁺ transients and the dynamicsof contraction of their muscles with those from heterozygous (PV+/) and wild-type (WT) mice. In the muscles of PV-deficientmice, the decay of intracellularCa²⁺ concentration([Ca²⁺]_i)after 20-ms stimulation was slower compared with WT mice and led to aprolongation of the time required to attain peak twitch tension and toan extension of the half-relaxation time. The integral [Ca²⁺]_iin muscle fibers of PV / mice was higher and consequently the force generated during a single twitch was ~40% greater than inPV +/ and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in theperformance of rapid, phasic movements.     

Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of G(i-1) and G(i-2)

This study examined the ability of protein kinase C (PKC) toinduce heterologous desensitization by targeting specific G proteinsand limiting their ability to transduce signals in smooth muscle.Activation of PKC by pretreatment of intestinal smooth muscle cellswith phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, orthe phosphatase 1 and phosphatase 2A inhibitor, calyculin A,selectively phosphorylated G_i-1 and G_i-2,but not G_i-3 or G_o, and blockedinhibition of adenylyl cyclase mediated by somatostatin receptorscoupled to G_i-1 and opioid receptors coupled toG_i-2, but not by muscarinic M₂ and adenosineA₁ receptors coupled to G_i-3. Phosphorylationof G_i-1 and G_i-2 and blockade of cyclaseinhibition were reversed by calphostin C and bisindolylmaleimide, andadditively by selective inhibitors of PKC and PKC. Blockade ofinhibition was prevented by downregulation of PKC. Phosphorylation ofG-subunits by PKC also affected responses mediated by-subunits. Pretreatment of muscle cells withcANP-(4-23), a selective agonist of the natriureticpeptide clearance receptor, NPR-C, which activates phospholipase C(PLC)-3 via the -subunits of G_i-1 andG_i-2, inhibited the PLC- response to somatostatin and[D-Pen^2,5]enkephalin. The inhibition waspartly reversed by calphostin C. Short-term activation of PKC had noeffect on receptor binding or effector enzyme (adenylyl cyclase orPLC-) activity. We conclude that selective phosphorylation ofG_i-1 and G_i-2 by PKC partly accounts forheterologous desensitization of responses mediated by the - and-subunits of both G proteins. The desensitization reflects adecrease in reassociation and thus availability of heterotrimeric G proteins.

     

Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts

The effects ofhuman cytomegalovirus (HCMV) infection onCl/HCO₃exchanger activity in human lung fibroblasts (MRC-5 cells) were studiedusing fluorescent, ion-sensitive dyes. The intracellular pH(pH_i) of mock- and HCMV-infectedcells bathed in a solution containing 5%CO₂-25 mMHCO₃ were nearly the same. However,replacement of external Clwith gluconate caused anH₂DIDS-inhibitable (100 µM)increase in the pH_i ofHCMV-infected cells but not in mock-infected cells. Continuous exposureto hyperosmotic external media containing CO₂/HCO₃caused the pH_i of both cell typesto increase. The pH_i remainedelevated in mock-infected cells. However, in HCMV-infected cells, thepH_i peaked and then recoveredtoward control values. This pH_irecovery phase was completely blocked by 100 µMH₂DIDS. In the presence ofCO₂/HCO₃, there was an H₂DIDS-sensitivecomponent of net Cl efflux(external Cl wassubstituted with gluconate) that was less in mock- than in HCMV-infected cells. When nitrate was substituted for external Cl (in the nominal absenceofCO₂/HCO₃),the H₂DIDS-sensitive netCl efflux was much greaterfrom HCMV- than from mock-infected cells. In mock-infected cells,H₂DIDS-sensitive, netCl efflux decreased aspH_i increased, whereas forHCMV-infected cells, efflux increased aspH_i increased. All these resultsare consistent with an HCMV-induced enhancement ofCl/HCO₃exchanger activity.

     

Mechanical stress-induced Ca2+ entry and Cl- current in cultured human aortic endothelial cells

A fluid streamthrough a microtube was applied to cultured human aortic endothelialcells to investigate the endothelial responses of both the ioniccurrents and intracellular Ca²⁺concentration([Ca²⁺]_i)to mechanical stimulation. The fluid stream induced an increase in[Ca²⁺]_ithat was dependent on both the flow rate and the extracellular Ca²⁺ concentration.Gd³⁺ and niflumic acid inhibitedthe fluid stream-induced increase in[Ca²⁺]_i,whereas Ba²⁺ andtetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca²⁺]_iincrease was accompanied by the activation of an inward current at52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the externalCl concentration wasreduced but was not affected by variation of the externalNa⁺ concentration. During theexposure to the fluid stream,[Ca²⁺]_iwas voltage dependent, i.e., depolarization decreased[Ca²⁺]_i.We therefore conclude that the fluid stream-induced current is largelycarried by Cl and that theCl current may thus play arole in modulating the Ca²⁺ influxby altering the membrane potential of endothelial cells.