Quantitative targeted absolute proteomics of rat blood–cerebrospinal fluid barrier transporters: comparison with a human specimen

The purpose of this study was to determine absolute protein expression levels of transporters in rat choroid plexus, that is, the blood–cerebrospinal fluid barrier, and to compare them with the levels in the human choroid plexus. Plasma membrane fractions were prepared from pooled, freshly isolated choroid plexuses of 30 male Wistar rats and from frozen choroid plexus of one male human donor. Protein expression levels of 54 rat and 121 human molecules were measured, using a quantitative targeted absolute proteomics technique. In rat, oatp1a5 showed the most abundant protein expression (30.3 fmol/μg protein), and its expression level was 3.1‐, 4.5‐, 5.5‐, 8.4‐, 9.0‐, 9.9‐, 22‐, 91‐, and 95‐fold greater than those of glut1, oatp1c1, mrp1, mct1, oat3, pept2, mrp4, bcrp, and mdr1a, respectively. OATP1A2 (a possible homolog of rat oatp1a5), OATP1C1 and PEPT2 were not detected in human choroid plexus. MRP1, OAT3, and MRP4 showed 4.0‐, 1.8‐, and 1.7‐fold smaller expression levels in human than rat, respectively. MATE1 was detected in human, but not rat, and its expression level (8.61 fmol/μg protein) was the highest among the xenobiotic transporters examined in human choroid plexus. These findings should be useful for understanding rat blood–cerebrospinal fluid barrier function and its differences from that in human.

     

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive

Several studies suggest the involvement of Na⁺ and HCO₃^– transport in the formation of cerebrospinal fluid. Two Na⁺-dependent HCO₃^– transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601–C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pH_i) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH₂-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pH_i in choroidal cells increased from 7.03 ± 0.02 to 7.38 ± 0.02 (n = 41) after addition of CO₂/HCO₃^– into the bath solution. This increase was Na⁺ dependent and inhibited by the Cl^– and HCO₃^– transport inhibitor DIDS (200 µM). This suggests the presence of Na⁺-dependent, partially DIDS-sensitive HCO₃^– uptake. The pH_i recovery after acid loading revealed an initial Na⁺ and HCO₃^–-dependent net base flux of 0.828 ± 0.116 mM/s (n = 8). The initial flux in the presence of CO₂/HCO₃^– was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na⁺- and HCO₃^–-dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na⁺-driven Cl^– bicarbonate exchanger, and NBCe2 in this tissue. bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse     

Synthesis of transthyretin (pre-albumin) mRNA in choroid plexus epithelial cells, localized by in situ hybridization in rat brain

The sites of synthesis of transthyretin in the brain were investigated using in situ hybridization with [35S]-labeled recombinant cDNA probes specific for transthyretin mRNA. Autoradiography of hybridized coronal sections of rat brain revealed specific cellular localization of transthyretin mRNA in choroid plexus epithelial cells of the lateral, third, and fourth ventricles. Transferrin mRNA was also investigated and, in contrast to transthyretin mRNA, was localized mainly in the lateral ventricles. Our results indicate that substantial synthesis of transthyretin and transferrin mRNA may occur in the choroid plexus.     

Distribution of sodium transporters and aquaporin-1 in the human choroid plexus

The choroid plexus epithelium secretes electrolytes and fluid in the brain ventricular lumen at high rates. Several channels and ion carriers have been identified as likely mediators of this transport in rodent choroid plexus. This study aimed to map several of these proteins to the human choroid plexus. Immunoperoxidase-histochemistry was employed to determine the cellular and subcellular localization of the proteins. The water channel, aquaporin (AQP) 1, was predominantly situated in the apical plasma membrane domain, although distinct basolateral and endothelial immunoreactivity was also observed. The Na⁺-K⁺-ATPase ₁-subunit was exclusively localized apically in the human choroid plexus epithelial cells. Immunoreactivity for the Na⁺-K⁺-2Cl^– cotransporter, NKCC1, was likewise confined to the apical plasma membrane domain of the epithelium. The Cl^–/HCO₃^– exchanger, AE2, was localized basolaterally, as was the Na⁺-dependent Cl^–/HCO₃^– exchanger, NCBE, and the electroneutral Na⁺-HCO₃^– cotransporter, NBCn1. No immunoreactivity was found toward the Na⁺-dependent acid/base transporters NHE1 or NBCe2. Hence, the human choroid plexus epithelium displays an almost identical distribution pattern of water channels and Na⁺ transporters as the rat and mouse choroid plexus. This general cross species pattern suggests central roles for these transporters in choroid plexus functions such as cerebrospinal fluid production. immunohistochemistry; metabolism; cerebrospinal fluid secretion     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

Transport of sulphate, thiosulphate and iodide by choroid plexus in vitro

—Isolated choroid plexuses of rabbits and cats were incubated in artificial cerebrospinal fluid medium containing [³⁵S]sulphate, [³⁵S]thiosulphate or [¹²⁵I]iodide and combinations thereof. After 1 hr incubation the mean ratio of tissue concentration to medium concentration was 2·46 for [³⁵S]sulphate, 2·39 for [³⁵S]thiosulphate, and 270 for [¹²⁵I]iodide. Uptake of all three anions was greatly reduced at 0° and by addition of dinitrophenol to the medium. Other inhibitors selectively reduced the uptake of particular anions; non-radioactive sulphate and thiosulphate reduced both [³⁵S]sulphate and [³⁵S]-thiosulphate uptake with much less effect on [¹²⁵I]iodide uptake, while non-radioactive iodide and thiocyanate greatly reduced [¹²⁵]iodide uptake with little or no effect on [³⁵S]sulphate or [³⁵S]thiosulphate uptake. It was concluded: (a) that sulphate and thiosulphate, like iodide, were accumulated by choroid plexus in vitro by active transport; (b) that sulphate and thiosulphate share and compete for a transport mechanism which is separate from the iodide transport mechanism; and (c) that the transport of sulphate out of cerebrospinal fluid demonstrated in vivo could occur at least in part in the choroid plexus.     

Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.     

Development of transport mechanisms for sulphate and iodide in immature choroid plexus

The time-course of development of sulphate and iodide transport mechanisms in choroid plexus was studied by measuring uptake of [³⁵S]sulphate and [¹²⁵I]iodide from an incubating medium by isolated choroid plexuses of foetal and newborn rabbits and cats. Sulphate uptake by choroid plexus was poorly developed in rabbit foetuses just before term, but highly developed in newborn animals. Iodide uptake was already well developed in the most immature foetuses studied.     

Choline uptake across the ventricular membrane of neonate rat choroid plexus

The uptake of[³H]choline from thecerebrospinal fluid (CSF) side of the rat neonatal choroid plexus wascharacterized in primary cultures of the choroidal epithelium grown onsolid supports. Cell-to-medium concentration ratios were ~5 at 1 minand as high as 70 at 30 min. Apical choline uptake was facilitated; theK_m was ~50µM. Several organic cations (e.g., hemicholinium-3 and N¹-methylnicotinamide)inhibited uptake. The reduction or removal of externalNa⁺ or the addition of 5 mM LiClhad no effect on uptake. However, increasing externalK⁺ concentration from 3 to 30 mMdepolarized ventricular membrane potential (70 to 15 mV)and reduced uptake to 45% of that for the control. Treatment with 1 mMouabain or 2 mM BaCl₂ reduced uptake 45%, and intracellular acidification reduced uptake to ~90%of that for controls. These data indicate that the uptake of choline from CSF across the ventricular membrane of the neonatal choroidal epithelium is not directly coupled toNa⁺ influx but is sensitive toplasma membrane electrical potential.

     

Bacterial biomass and production in a shallow bay

Bacterial biomass (BB, acridine orange) and bacterial secondaryproduction (BSP, [³H]thymidine incubations) were measured forthe first time in Concepción Bay (37°35'S, 73°01'W).BB ranged from 1.8x10⁶ to 22.5x10⁶ cells ml^–1 (the lattervalue observed near to an industrial effluent), BSP from 0.27x10⁶to 2.5x10⁶ cell ml^–1 day^–1 and heterotrophic turnoverrates between 0.15 and 1.0 doublings day^–1.     

Carbachol inhibits Na(+)-K(+)-ATPase activity in choroid plexus via stimulation of the NO/cGMP pathway

Secretion of cerebrospinal fluid by the choroid plexus canbe inhibited by its cholinergic innervation. We demonstrated that carbachol inhibits the Na⁺-K⁺-ATPase in bovinechoroid tissue slices and investigated the mechanism. Many of theactions of cholinergic agents are mediated by nitric oxide (NO), whichplays important roles in fluid homeostasis. The inhibition ofNa⁺-K⁺-ATPase was blocked by the NO synthaseinhibitor [N-nitro-L-argininemethyl ester] and was quantitatively mimicked by the NO agonistssodium nitroprusside (SNP) and diethylenetriamine NO. Inhibition by SNPcorrelated with an increase in tissue cGMP and was abolished by1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. Inhibition was mimicked bythe protein kinase G activator 8-bromo-cGMP and by okadaic acid, aninhibitor of protein phosphatases 1 and 2A. cGMP-dependent proteinkinase inhibitors Rp-8-pCPT-cGMP (0.5-5 µM) and KT-5823 (2.0 µM) did not block the effects of SNP, but higher concentrations ofthe more selective inhibitor (Rp-8-pCPT-cGMP) had a pharmacological inhibitory effect on Na⁺-K⁺-ATPase. The datasuggest that cholinergic regulation of theNa⁺-K⁺-ATPase is mediated by NO and involvesactivation of guanylate cyclase and elevation of cGMP.

     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the synthesis and release of catecholamine in the rat carotid body in vitro

The aim of this work was to determinecontents and turnover rates for dopamine (DA) andnorepinephrine (NE) and to identify the catecholamine (CA) releasedduring stimulation of the rat carotid body (CB). Turnover rates and therelease of CA were measured in an in vitro preparation using acombination of HPLC and radioisotopic methods. Mean rat CB levels of DAand NE were 209 and 45 pmol/mg tissue, respectively. With[³H]tyrosine asprecursor, rat CB synthesized[³H]CA in a time- andconcentration-dependent manner; calculated turnover times for DA and NEwere 5.77 and 11.4 h, respectively. Hypoxia and dibutyryl adenosine3',5'-cyclic monophosphate significantly increased[³H]CA synthesis. Innormoxia, rat CB released[³H]DA and[³H]NE in a ratio of5:1, comparable to that of the endogenous tissue CA. Hypoxia and highK⁺ preferentially released[³H]DA, nicotinepreferentially released[³H]NE, and acidicstimuli released both amines in proportion to tissue content. Releaseof [³H]CA induced byhypoxia and high K⁺ was nearlyfully dependent on extracellularCa²⁺, whereas basal normoxicrelease was not altered by removal of Ca²⁺ from the incubating solution.We conclude that the rat CB is an organ with higher levels of DA thanNE that preferentially releases DA or NE in a stimulus-specific manner.

     

Protein secretion by choroid plexus: isolated apical fragments synthesize protein in vitro

Protein synthesis was studied in the isolated rat choroid plexus. When the choroid plexus was studied by transmission electron microscopy, membrane-bound structures were often observed in the ventricular space. These structures appear to bud from the apical surface of the epithelial cells. In the present study, we attempted to isolate these membrane-bound cellular fragments from the choroid plexus and to determine their ability to synthesize proteins. The apical fragments (aposomes) were isolated from the choroid plexus by allowing tissue explants to incubate in media (37 degrees C) for 1 h. The tissue was removed and the media, now containing aposomes, was incubated with [S35]methionine (100 microCi). The media was collected and analysed by SDS-PAGE followed by fluorography. Parallel [S35]methionine incubations were done with whole tissue explants. The SDS-PAGE protein derived from the aposomes was similar to the profile derived from the tissue. In addition, proteins detected in CSF had relative molecular weights comparable to the products synthesized by aposomes. These observations suggest that aposomes provide an additional route of entry for proteins into CSF.     

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse

Tg737^orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737^orpk mutants is associated with defects in ion transport, we compared the steady-state pH_i and Na⁺-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737^orpk mutant and wild-type mice. The data indicate that Tg737^orpk mutant choroid plexus epithelium have lower pH_i and higher Na⁺-dependent HCO₃^– transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na⁺-dependent HCO₃^– transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737^orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH_i and ion transport activity. cAMP; ion transport     

Nucleoside Triphosphate(NTP)-Binding Proteins and Endogenous ADP-ribosyl Transferase in Neurospora crassa

Nucleoside triphosphate(NTP)-binding proteins were detectedin the crude extract of mycelia of Neurospora crassa, whichwas treated with 1% Lubrol PX and fractionated by gel filtration.Protein fractions showing the capacity to bind [³⁵S]ATPS or[³⁵S]GTPS were designated as AGN1 to 6. The binding of [³⁵S]ATPSor [³⁵S]GTPS was prevented in the presence of 0.1 mM ATP orGTP except that in fractions AGN1 and 2, the presence of GTPstimulated the binding of [³⁵S] ATPS to ATP(NTP)-binding proteins.ATP or GTP was 1 to 2 orders of magnitude more effective thanCTP or UTP in preventing the binding of [³⁵S]GTPS in AGN1, 2and 5. Among these fractions AGN1, 2, 5 and 6 showed activityto hydrolyze 1 nM [–³²P]ATP or [–³²P]GTP. NTP-bindingproteins bound with [³⁵S]ATPS or [³⁵S]GTPS had lower apparentmolecular weights than the same proteins without bound nucleotide.Proteins bound with [³⁵S]ATPS or [³⁵S]GTPS and those [³²P]ADP-ribosylatedby endogenous ADP-ribosyl transferase in each fraction wereanalyzed by SDS-PAGE. About 20 species of ATP or ATP-GTP-bindingproteins were detected, several of which were ADP-ribosylated.The binding of [³⁵S]ATPS or [³⁵S]GTPS to NTP-binding proteinswas confirmed by the comparison of non-boiled and boiled samplesimmediately before loading to SDS-PAGE. ATP, GTP, CTP or UTPat the concentration of 0.1 mM effectively removed [³³S]ATPSor [³⁵S]GTPS bound to NTP-binding proteins. (Received December 10, 1990; Accepted April 18, 1991)     

Oxidation of lactate and acetate in rat skeletal muscle: analysis by 13C-nuclear magnetic resonance spectroscopy

Bertocci, Loren A., John G. Jones, Craig R. Malloy, RonaldG. Victor, and Gail D. Thomas. Oxidation of lactateand acetate in rat skeletal muscle: analysis by¹³C-nuclear magnetic resonancespectroscopy. J. Appl. Physiol. 83(1): 32-39, 1997.The balance between carbohydrate and fatty acidutilization in skeletal muscle previously has been studied in vivo byusing a variety of methods such as arteriovenous concentrationdifferences and radioactive isotope tracer techniques. However, thesemethodologies provide only indirect estimates of substrate oxidation.We used ¹³C-nuclear magneticresonance (NMR) spectroscopy and non-steady-state isotopomer analysisto directly quantify the relative oxidation of two competing exogenoussubstrates in rat skeletal muscles. We infused[1,2-¹³C]acetate and[3-¹³C]lactateintravenously in anesthetized rats during the final 30 min of 35 (n = 10) or 95 (n = 10) min of intense, unilateral, rhythmic hindlimb contractions.¹³C-NMR spectroscopy andisotopomer analysis were performed on extracts of gastrocnemius andsoleus muscles from both the contracting and contralateralresting hindlimbs. We found that1)[¹³C]lactate and[¹³C]acetate were taken up and oxidized by both restingand contracting skeletal muscles; and2) high-intensity musclecontractions altered the pattern of substrate utilization such that therelative oxidation of acetate decreased while that of lactate remainedunchanged or increased. Based on these findings, we propose that¹³C-NMR spectroscopy incombination with isotopomer analysis can be used to study the generaldynamics of substrate competition between carbohydrates and fats in ratskeletal muscle.

     

Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells

The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K⁺ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K⁺ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K⁺ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 µM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min (P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT_2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl-sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT_2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K⁺ efflux at the apical membrane of the choroid plexus. delayed-rectifying potassium channel; serotonin     

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa²⁺ release channel (RyR1) in the presence of nanomolarCa²⁺ concentrations. However, the role of CaM activation inthe mechanisms that control Ca²⁺ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca²⁺, the rate of⁴⁵Ca²⁺ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle⁴⁵Ca²⁺ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca²⁺ sensitivityof [³H]ryanodine binding to skeletal muscle SR, whereasthe Ca²⁺ sensitivity of cardiac SR[³H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [³⁵S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca²⁺. In nanomolar Ca²⁺, however,the affinity and stoichiometry of RyR2 [³⁵S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca²⁺ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca²⁺ dependence of channelactivation in skeletal and cardiac muscle.

     

Limited myogenic response to a single bout of weight-lifting exercise in old rats

The purpose of the present study was to compare themyogenic response of hindlimb muscles in young (14-20 wk of age)and old (>120 wk of age) rats with a single exhaustive bout of heavyresistance weight lifting. [³H]thymidine and[¹⁴C]leucine labeling were monitored for up to2 wk after the exercise bout to estimate serial changes in mitoticactivity and the level of amino acid uptake and myosin synthesis.Histological, histochemical, and immunohistochemical[anti-5-bromo-2'-deoxyuridine and myogenic determinationgenes (MyoD)] analyses of whole muscles and analysis ofmuscle-specific gene expression (MyoD) using Western blotting andRT-PCR were performed. Old rats showed significant muscle atrophy and alower exercise capacity than young rats. Exercise-induced muscledamage, as assessed in histological sections, and increases in serumcreatine kinase activity were evident in both young and old exercisedgroups. Mitotic activity was increased in young, but not old, rats 2 days after exercise. There was a biphasic increase in[¹⁴C]leucine uptake during the 14 dayspostexercise (peaks at 1-4 and 10 days) in young rats: only thefirst peak was observed in old rats. There was a lower uptake of[¹⁴C]leucine in the myosin fraction and animpaired expression of MyoD at the protein (immunohistochemistry andWestern blotting) and mRNA (RT-PCR) levels in old rats throughout thepostexercise period. These results demonstrate a reduced reparativecapability of muscle in response to a single bout of exercise in oldcompared with young rats.