Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Increase of sodium delivery stimulates the mitochondrial respiratory chain H2O2 production in rat renal medullary thick ascending limb

The mitochondria-rich epithelial cells of the renal medullary thick ascending limb (mTAL) reabsorb nearly 25% of filtered sodium (Na(+)) and are a major source of cellular reactive oxygen species. Although we have shown that delivery of Na(+) to the mTAL of rats increases superoxide (O(2)(·-)) production in mTAL, little is known about H(2)O(2) production, given the lack of robust and selective fluorescent indicators for determining changes within the whole cell, specifically in the mitochondria. The present study determined the effect of increased tubular flow and Na(+) delivery to mTAL on the production of mitochondrial H(2)O(2) in mTAL. H(2)O(2) responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H(2)O(2) indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H(2)O(2) indicator, peroxyfluor-6 acetoxymethyl ester. The results showed that mitochondrial H(2)O(2) and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ～10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na(+) transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H(2)O(2) production. Cytoplasmic H(2)O(2) (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H(2)O(2) (mitochondria peroxy yellow 1) and was partially attenuated (～65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H(2)O(2) is produced in the mitochondria in response to increased flow and delivery of Na(+) to the mTAL, and that whole cell H(2)O(2) levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H(2)O(2) may represent an important target for development of more effective antioxidant therapies.     

Role of 20-HETE in the antihypertensive effect of transfer of chromosome 5 from Brown Norway to Dahl salt-sensitive rats

This study examined whether substitution of chromosome 5 containing the CYP4A genes from Brown Norway rat onto the Dahl S salt-sensitive (SS) genetic background upregulates the renal production of 20-HETE and attenuates the development of hypertension. The expression of CYP4A protein and the production of 20-HETE were significantly higher in the renal cortex and outer medulla of SS.5(BN) (chromosome 5-substituted Brown Norway rat) consomic rats fed either a low-salt (LS) or high-salt (HS) diet than that seen in SS rats. The increase in the renal production of 20-HETE in SS.5(BN) rats was associated with elevated expression of CYP4A2 mRNA. MAP measured by telemetry rose from 117 ± 1 to 183 ± 5 mmHg in SS rats fed a HS diet for 21 days, but only increased to 151 ± 5 mmHg in SS.5(BN) rats. The pressure-natriuretic and diuretic responses were twofold higher in SS.5(BN) rats compared with SS rats. Protein excretion rose to 354 ± 17 mg/day in SS rats fed a HS diet for 21 days compared with 205 ± 13 mg/day in the SS.5(BN) rats, and the degree of glomerular injury was reduced. Baseline glomerular capillary pressure (Pgc) was similar in SS.5(BN) rats (43 ± 1 mmHg) and Dahl S (44 ± 2 mmHg) rats. However, Pgc increased to 59 ± 3 mmHg in SS rats fed a HS diet for 7 days, while it remained unaltered in SS.5(BN) rats (43 ± 2 mmHg). Chronic administration of an inhibitor of the synthesis of 20-HETE (HET0016, 10 mg·kg(-1)·day(-1) iv) reversed the antihypertensive phenotype seen in the SS.5(BN) rats. These findings indicate that the transfer of chromosome 5 from the BN rat onto the SS genetic background increases the renal expression of CYP4A protein and the production of 20-HETE and that 20-HETE contributes to the antihypertensive and renoprotective effects seen in the SS.5(BN) consomic strain.     

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Effect of lipid diet on mitochondrial palmitoyl-l-carnitine oxidation in kidney at postnatal development.

Oxygen consumption (VO2) and beta-hydroxyacyl-CoA dehydrogenase (beta OAC) activity were measured in isolated mitochondria of developing rat kidney from late fetal to adult age. In the presence of palmitoyl-L-carnitine, VO2 consumption was higher in suckling than in adult rats while beta OAC activity rose during the postnatal period and declined after weaning. During postnatal development, the high level of mitochondrial fatty acid oxidation was linked to the high level of fatty acid supply and any change in lipid diet altered mitochondrial fatty acid oxidation. By contrast at adult age, a high fat diet did not change either mitochondrial fatty acid oxidation or beta OAC activity measured in two nephron structures (PCT and mTAL). Dietary lipids seem to play an important role in the evolution of mitochondrial fatty acid oxidation in developing rat kidney.     

Restoration of normal vascular relaxation mechanisms in cerebral arteries by chromosomal substitution in consomic SS.13BN rats

This study sought to identify the mechanisms of vascular relaxation that are rescued in middle cerebral arteries (MCA) of SS.13BN consomic rats by substituting chromosome 13 containing the renin gene from Brown Norway (BN) rats into the Dahl salt-sensitive (SS) genetic background. Isolated MCA from SS rats exhibited an indomethacin-sensitive constriction in response to acetylcholine (ACh) and hypoxia. ACh-induced dilation was NO dependent and hypoxic dilations were cyclooxygenase (COX) dependent in BN and SS.13BN rats. In SS rats, hypoxic dilation was restored by indomethacin and abolished by inhibiting cytochrome P-450 epoxygenases, suggesting a role for epoxyeicosatrienoic acids. MCA from SS and SS.13BN rats constricted and MCA from BN rats dilated in response to the stable prostacyclin analog iloprost. MCA from SS.13BN and BN rats (but not SS rats) dilated in response to the prostaglandin E2 receptor agonist butaprost. Hypoxia increased prostacyclin release in cerebral arteries from all the strains, whereas thromboxane A2 production was reduced in BN rat vessels only. These data suggest that SS rats may be less sensitive to vasodilator prostaglandins and that normalization of renin-angiotensin system regulation causes a switch from production of COX-derived vasoconstrictor metabolites (in SS rats) toward NO-dependent relaxation in response to ACh- and prostaglandin-dependent dilation in response to hypoxia in SS.13(BN) rats.     

Differential expression of cardiac mitochondrial proteins

Mitochondria were isolated from whole hearts of Dahl salt sensitive (SS) and chromosome 13 consomic control (SS.13BN/Mcwi) rats using a mechanical homogenization process followed by density centrifugation. The proteins present in the two mitochondria preparations were quantified; equal amounts of protein from each sample were taken and trypsinized in the presence of either 16O or 18O before pooling. Incorporation of one or two 18O atoms at the C-terminus of the peptide cleaved by trypsin allows the distinction between the two samples. The proteins were identified by automated MS/MS sequencing and relative amounts of each protein assessed by comparison of the intensities of the constituent peptides. Relative quantification was performed using the ZoomQuant (v1.24) software. Nine proteins were found to be differentially expressed. Electron transfer flavoprotein alpha (P13803, ETFA) protein expression was two-fold lower in the SS compared to the SS.13BN. This was confirmed by Western blot and 2-DE gel quantification. Potential functional implications of this differential expression include an impaired capacity of the heart to oxidize fatty acids in the SS strain compared to the control. Mathematical modeling of mitochondrial electron transport predicted that the observed change in ETFA expression may result in decreased activity of the electron transport chain.     

Lack of inhibition of vasopressin-stimulated NA+K+ATPase by atrial natriuretic factor in the rat renal medullary thick ascending limb of Henle's loop

The anti-diuretic hormone, arginine vasopressin (AVP) stimulates the activity of Na+K+ATPase in the rat renal medullary thick ascending limb of Henle's loop (mTAL). Atrial natriuretic factor (ANF) has been suggested to exert a tubular effect on the mammalian nephron, perhaps in part, by interacting with other hormones. In the present study, we investigated the effect of rat ANF with and without AVP upon mTAL Na+K+ATPase activity using cytochemical methods. ANF alone failed to inhibit or stimulate Na+K+ATPase activity in mTAL at any of the concentrations tested (10 nmol-0.1 pmol l-1). Unlike the rat hypothalamic digitalis-like factor, ANF (10 nmol-10 fmol l-1) did not inhibit Na+K+ATPase activity after stimulation with AVP (1 fmol l-1) for either 4 or 10 min. The results suggest that ANF does not exert an effect on mTAL, either alone or in conjunction with AVP.     

N-acetylcysteine with apocynin prevents hyperoxaluria-induced mitochondrial protein perturbations in nephrolithiasis

Diminished mitochondrial activities were deemed to play an imperative role in surged oxidative damage perceived in hyperoxaluric renal tissue. Proteomics is particularly valuable to delineate the damaging effects of oxidative stress on mitochondrial proteins. The present study was designed to apply large-scale proteomics to describe systematically how mitochondrial proteins/pathways govern the renal damage and calcium oxalate crystal adhesion in hyperoxaluria. Furthermore, the potential beneficial effects of combinatorial therapy with N-acetylcysteine (NAC) and apocynin were studied to establish its credibility in the modulation of hyperoxaluria-induced alterations in mitochondrial proteins. In an experimental setup with male Wistar rats, five groups were designed for 9?d. At the end of the experiment, 24-h urine was collected and rats were euthanized. Urinary samples were analyzed for kidney injury marker and creatinine clearance. Transmission electron microscopy revealed distorted renal mitochondria in hyperoxaluria but combinatorial therapy restored the normal mitochondrial architecture. Mitochondria were isolated from renal tissue of experimental rats, and mitochondrial membrane potential was analyzed. The two-dimensional electrophoresis (2-DE) based comparative proteomic analysis was performed on proteins isolated from renal mitochondria. The results revealed eight differentially expressed mitochondrial proteins in hyperoxaluric rats, which were identified by Matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) analysis. Identified proteins including those involved in important mitochondrial processes, e.g. antioxidant defense, energy metabolism, and electron transport chain. Therapeutic administration of NAC with apocynin significantly expunged hyperoxaluria-induced discrepancy in the renal mitochondrial proteins, bringing them closer to the controls. The results provide insights to further understand the underlying mechanisms in the development of hyperoxaluria-induced nephrolithiasis and the therapeutic relevance of the combinatorial therapy.     

Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K(+) channels in the TAL

We havepreviously demonstrated that the protein level of c-Src, anonreceptor type of protein tyrosine kinase (PTK), was higher in therenal medulla from rats on a K-deficient (KD) diet than that in rats ona high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000).We have now used the patch-clamp technique to investigate the role ofPTK in regulating the apical K channels in the medullary thickascending limb (mTAL) of the rat kidney. Inhibition of PTK withherbimycin A increased NP_o, a product of channelnumber (N) and open probability (P_o),of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats ona KD diet but had no significant effect in tubules from animals on a HKdiet. In contrast, herbimycin A did not affect the activity of the30-pS K channel in the mTAL from rats on a KD diet. Moreover, additionof N-methylsulfonyl-12,12-dibromododec-11-enamide, an agentthat inhibits the cytochrome P-450-dependent production of20-hydroxyeicosatetraenoic acid, further increasedNP_o of the 70-pS K channel in the presence ofherbimycin A. Furthermore, Western blot detected the presence ofPTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), inthe renal outer medulla. Inhibition of PTP with phenylarsine oxide(PAO) decreased NP_o of the 70-pS K channel inthe mTAL from rats on a HK diet. However, PAO did not inhibit theactivity of the 30-pS K channel in the mTAL. The effect of PAO on the70-pS K channel was due to indirectly stimulating PTK becausepretreatment of the mTAL with herbimycin A abolished the inhibitoryeffect of PAO. Finally, addition of exogenous c-Src reversibly blockedthe activity of the 70-pS K channel in inside-out patches. We concludethat PTK and PTP have no effect on the low-conductance K channels inthe mTAL and that PTK-induced tyrosine phosphorylation inhibits,whereas PTP-induced tyrosine dephosphorylation stimulates, the apical70-pS K channel in the mTAL.

     

Possible role of brain salt-inducible kinase 1 in responses to central sodium in Dahl rats

In Dahl salt-sensitive (S) rats, Na(+) entry into the cerebrospinal fluid (CSF) and sympathoexcitatory and pressor responses to CSF Na(+) are enhanced. Salt-inducible kinase 1 (SIK1) increases Na(+)/K(+)-ATPase activity in kidney cells. We tested the possible role of SIK1 in regulation of CSF [Na(+)] and responses to Na(+) in the brain. SIK1 protein and activity were lower in hypothalamic tissue of Dahl S (SS/Mcw) compared with salt-resistant SS.BN13 rats. Intracerebroventricular infusion of the protein kinase inhibitor staurosporine at 25 ng/day, to inhibit SIK1 further increased mean arterial pressure (MAP) and HR but did not affect the increase in CSF [Na(+)] or hypothalamic aldosterone in Dahl S on a high-salt diet. Intracerebroventricular infusion of Na(+)-rich artificial CSF caused significantly larger increases in renal sympathetic nerve activity, MAP, and HR in Dahl S vs. SS.BN13 or Wistar rats on a normal-salt diet. Intracerebroventricular injection of 5 ng staurosporine enhanced these responses, but the enhancement in Dahl S rats was only one-third that in SS.BN13 and Wistar rats. Staurosporine had no effect on MAP and HR responses to intracerebroventricular ANG II or carbachol, whereas the specific protein kinase C inhibitor GF109203X inhibited pressor responses to intracerebroventricular Na(+)-rich artificial CSF or ANG II. These results suggest that the SIK1-Na(+)/K(+)-ATPase network in neurons acts to attenuate sympathoexcitatory and pressor responses to increases in brain [Na(+)]. The lower hypothalamic SIK1 activity and smaller effect of staurosporine in Dahl S rats suggest that impaired activation of neuronal SIK1 by Na(+) may contribute to their enhanced central responses to sodium.     

Introgression of chromosome 13 in Dahl salt-sensitive genetic background restores cerebral vascular relaxation

To evaluate the potential role of impaired renin-angiotensin system (RAS) function in contributing to reduced vascular relaxation in Dahl salt-sensitive (S) rats, responses to ACh (10(-6) mol/l) and hypoxia (Po(2) reduction to 40-45 mmHg) were determined in isolated middle cerebral arteries of Dahl S rats, Brown Norway (BN) rats, and consomic rats having chromosome 13 (containing the renin gene) or chromosome 16 of the BN rat substituted into the Dahl S genetic background (SS-13(BN) and SS-16(BN), respectively). Arteries of BN rats on a low-salt (LS) diet (0.4% NaCl) dilated in response to ACh and hypoxia, whereas dilation in response to these stimuli was absent in Dahl S rats on LS diet. Vasodilation to ACh and hypoxia was restored in SS-13(BN) rats on an LS diet but not in SS-16(BN) rats. High-salt diet (4% NaCl), to suppress ANG II, eliminated vasodilation to hypoxia and ACh in BN and in SS-13(BN) rats. Treatment of SS-13(BN) rats with the AT(1) receptor antagonist losartan also eliminated the restored vasodilation in response to ACh and hypoxia. These studies suggest that restoration of normal RAS regulation in SS-13(BN) consomic rats restores vascular relaxation mechanisms that are impaired in Dahl S rats.     

Exercise induces a cardiac mitochondrial phenotype that resists apoptotic stimuli

Ischemia-reperfusion-induced calcium overload and production of reactive oxygen species can trigger apoptosis by promoting the release of proapoptotic factors via the mitochondrial permeability transition pore. While it is clear that endurance exercise provides cardioprotection against ischemia-reperfusion-induced injury, it is unknown if exercise training directly alters mitochondria phenotype and confers protection against apoptotic stimuli in both subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. We hypothesized that exercise training increases expression of endogenous antioxidant enzymes and other antiapoptotic proteins, resulting in a SS and IMF mitochondrial phenotype that resists apoptotic stimuli. Mitochondria isolated from hearts of sedentary (n = 8) and exercised-trained (n = 8) adult male rats were studied. Endurance exercise increased the protein levels of primary antioxidant enzymes in both SS and IMF mitochondria. Furthermore, exercise increased the levels of antiapoptotic proteins in the heart, including the apoptosis repressor with a caspase recruitment domain and inducible heat shock protein 70. Importantly, our findings reveal that endurance exercise training attenuates reactive oxygen species-induced cytochrome c release from heart mitochondria. These changes are accompanied by a lower maximal rate of mitochondrial permeability transition pore opening (V(max)) and prolonged time to V(max) in both SS and IMF cardiac mitochondria. These novel findings reveal that endurance exercise promotes biochemical alterations in cardiac SS and IMF mitochondria, resulting in a phenotype that resists apoptotic stimuli. Furthermore, these results are consistent with the concept that exercise-induced mitochondrial adaptations contribute to exercise-induced cardioprotection.     

Biogenesis of mammalian mitochondria in the renoprival kidney. I. Amino acid incorporation and respiratory activity

Changes in the yield of mitochondrial protein, in the incorporation of leucine into mitochondrial proteins, and in the respiratory activity of isolated mitochondria were determined in the remaining kidney (renoprival kidney) of the rat during the first 72 hr postmononephrectomy. At 24, 48, and 72 hr the yield of mitochondrial protein isolated from the renoprival kidney increased 13, 23, and 34%, respectively, whereas renal mass increased 9, 14, and 19%. Incorporation of [³H]-leucine in vivo into total mitochondrial protein was increased 96 and 130% over control at 12 and 24 hr, respectively. Incorporation of leucine in vitro by mitochondria was increased 27% over control at 24 hr; chloroamphenicol, but not cycloheximide, inhibited the in vitro incorporation.     

Renal medullary endothelin-1 is decreased in Dahl salt-sensitive rats

Although it is well established that the renal endothelin (ET-1) system plays an important role in regulating sodium excretion and blood pressure through activation of renal medullary ET(B) receptors, the role of this system in Dahl salt-sensitive (DS) hypertension is unclear. The purpose of this study was to determine whether the DS rat has abnormalities in the renal medullary endothelin system when maintained on a high sodium intake. The data indicate that Dahl salt-resistant rats (DR) on a high-salt diet had a six-fold higher urinary endothelin excretion than in the DR rats with low Na(+) intake (17.8 ± 4 pg/day vs. 112 ± 44 pg/day). In sharp contrast, urinary endothelin levels increased only twofold in DS rats in response to a high Na(+) intake (13 ± 2 pg/day vs. 29.8 ± 5.5 pg/day). Medullary endothelin concentration in DS rats on a high-Na(+) diet was also significantly lower than DR rats on a high-Na(+) diet (31 ± 2.8 pg/mg vs. 70.9 ± 5 pg/mg). Furthermore, DS rats had a significant reduction in medullary ET(B) receptor expression compared with DR rats while on a high-Na(+) diet. Finally, chronic infusion of ET-1 directly into the renal medulla blunted Dahl salt-sensitive hypertension. These data indicate that a decrease in medullary production of ET-1 in the DS rat could play an important role in the development of salt-sensitive hypertension observed in the DS rat.     

Increased expression of NAD(P)H oxidase subunit p67(phox) in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension

NAD(P)H oxidase has been shown to be important in?the development of salt-sensitive hypertension. Here, we show that the expression of a subunit of NAD(P)H oxidase, p67(phox), was increased in response to a high-salt diet in the outer renal medulla of the Dahl salt-sensitive (SS) rat, an animal model for human salt-sensitive hypertension. The higher expression of p67(phox), not the other subunits observed, was associated with higher NAD(P)H oxidase activity and salt sensitivity in SS rats compared with a salt-resistant strain. Genetic mutations of the SS allele of p67(phox) were found in the promoter region and contributed to higher promoter activity than that of the salt-resistant strain. To verify the importance of p67(phox), we disrupted p67(phox) in SS rats using zinc-finger nucleases. These rats exhibited a significant reduction of salt-sensitive hypertension and renal medullary oxidative stress and injury. p67(phox) could represent a target for salt-sensitive hypertension therapy.     

Genetic determinants on rat chromosome 6 modulate variation in the hypercapnic ventilatory response using consomic strains.

To understand the genetic basis of pathways involved in the control of breathing, a large scale, high-throughput study using chromosomal substitution strains of rats is underway. Eight new consomic rat stains (SS-2(BN), SS-4(BN), SS-6(BN), SS-7(BN), SS-8(BN), SS-11(BN), SS-12(BN), SS-14(BN), SS-Y(BN)), containing one homozygous BN/NHsdMcwi (BN) chromosome on a background of SS/JrHsdMcwi (SS), were created by PhysGen (http://pga.mcw.edu) Program for Genomic Applications. Male and female rats were studied using standard plethysmography under control conditions and during acute hypoxia (inspired oxygen fraction = 0.12) and hypercapnia (inspired CO(2) fraction = 0.07). The rats were also studied during treadmill exercise. Both male and female BN rats had a significantly lower ventilatory response during 7% CO(2) compared with SS rats of the same gender. SS-6(BN) female rats had a significantly reduced ventilatory response, similar to BN rats due primarily to a reduced tidal volume. Male SS-6(BN) rats had a significantly reduced tidal volume response to hypercapnia but a slightly increased frequency response during hypercapnia. Gene(s) on the Y chromosome may play a role in this increased frequency response in the male rats because the SS-Y(BN) hypercapnic ventilatory response involves a significantly increased frequency response. Several chromosomal substitutions slightly altered the ventilatory responses to hypoxia and exercise. However, genes on chromosomes 6 and Y of those studied are of primary importance in aspects of ventilatory control currently studied.     

Congenic strains reveal the effect of the renin gene on skeletal muscle angiogenesis induced by electrical stimulation.

Previous studies have indicated the importance of angiotensin II (ANG II) in skeletal muscle angiogenesis. The present study explored the effect of regulation of the renin gene on angiogenesis induced by electrical stimulation with the use of physiological, pharmacological, and genetic manipulations of the renin-angiotensin system (RAS). Transfer of the entire chromosome 13, containing the physiologically regulated renin gene, from the normotensive inbred Brown Norway (BN) rat into the background of an inbred substrain of the Dahl salt-sensitive (SS/Mcwi) rat restored renin levels and the angiogenic response after electrical stimulation. This restored response was significantly attenuated when SS-13(BN)/Mcwi consomic rats were treated with lisinopril or high-salt diet. The role of ANG II on this effect was confirmed by the complete restoration of skeletal muscle angiogenesis in SS/Mcwi rats infused with subpressor doses of ANG II. Congenic strains derived from the SS-13(BN)/Mcwi consomic were used to further verify the role of the renin gene in this response. Microvessel density was markedly increased after stimulation in congenic strains that contained the renin gene from the BN rat (congenic lines A and D). This angiogenic response was suppressed in control strains that carried regions of the BN genome just above (congenic line C) or just below (congenic line B) the renin gene. The present study emphasizes the importance of maintaining normal renin regulation as well as ANG II levels during the angiogenesis process with a combination of physiological, genetic, and pharmacological manipulation of the RAS.     

Modest PGC-1alpha overexpression in muscle in vivo is sufficient to increase insulin sensitivity and palmitate oxidation in subsarcolemmal, not intermyofibrillar, mitochondria

PGC-1alpha overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1alpha overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1alpha overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1alpha overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1alpha was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5'-AMP-activated protein kinase, alpha2 subunit, and HSL. Transfection of PGC-1alpha into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1alpha protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5'-AMP-activated protein kinase, alpha2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1alpha overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1alpha overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.     

Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle.