Testosterone: the culprit for producing splenocyte immune depression after trauma hemorrhage

Studies indicate that, whereas immune functions in males aredepressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se isresponsible for producing the immune depression. To study this, femaleC3H/HeN mice (n = 7 animals/group)were pretreated with 5-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock(blood pressure 35 ± 5 mmHg for 90 min) followed by fluidresuscitation or sham operation. Nontreated males underwent eithertrauma hemorrhage or sham operation. Twenty-four hours thereafter,splenocyte immune functions as well as plasma DHT, estradiol, andcorticosterone levels were measured. DHT-pretreated females hadsignificantly (P < 0.05) increasedDHT levels, comparable to those seen in males. Conversely, estradiollevels in such females were similar to control males. Splenocyteproliferation as well as interleukin-2 and interleukin-3 release werenot depressed in vehicle-treated females, whereas it was in DHT-treatedfemales after trauma hemorrhage, comparable to hemorrhaged males. Thushigh testosterone and/or low estradiol levels appear to beresponsible for producing splenocyte immune depression in males aftertrauma hemorrhage. Agents that block testosterone receptors or increaseestradiol levels may therefore be helpful in improving depressed immunefunctions in male trauma patients.

     

Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging

Previous studies have demonstrated that hemorrhagic shockproduces immunodepression in young male mice, whereas theimmunoresponsivness in young proestrus female mice is enhanced undersuch conditions. This sexually dimorphic immune response to hemorrhageappears to be related to high estrogen and testosterone levels infemales and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2-3 mo) and aged (18-19 mo) male and female CBA/J NIA mice were subjected tolaparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon- (IFN-) release] were depressed in youngmales and enhanced in young females after trauma-hemorrhage. Incontrast, in the aged male and female groups these parameters ofsplenocyte function were reversed after trauma-hemorrhage (i.e.,increased proliferation and IL-2 release in aged males compared withsuppressed proliferation and IFN- release in aged females).Furthermore, the release of the immunosuppressive cytokine IL-10inversely correlated with the age- and gender-related changes insplenocyte responses after trauma-hemorrhage. Thus the sexuallydimorphic immune response in young males and females totrauma-hemorrhage appears to reverse as sex hormone levels decline with age.

     

Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL^+/+ and transgenic (PRL^–/–) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL^–/– mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL^+/+ mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL^+/+ mice. Splenocyte proliferation in male PRL^–/– mice was significantly lower than in PRL^+/+ mice after T-H. Marginal differences between PRL^+/+ and PRL^–/– mice were observed in the release of IL-2 and IFN- by splenocytes, while the release of IL-10 was significantly higher in PRL^–/– than in PRL^+/+ mice. A significant observation of our study is the release of a 25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL^+/+ and PRL^–/– mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL^–/– transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice. Nb2 cells; cytokines; immune functions     

Age-dependent response of the electrocardiogram to K(+) channel blockers in mice

Developmental changes in electrocardiogram (ECG) andresponse to selective K⁺ channelblockers were assessed in conscious, unsedated neonatal (days 1, 7, 14) and adult male mice(>60 days of age). Mean sinus R-R interval decreased from 120 ± 3 ms in day 1 to 110 ± 3 ms inday 7, 97 ± 3 ms inday 14, and 81 ± 1 ms in adultmice (P < 0.001 by ANOVA; all 3 groups different from day 1). Inparallel, the mean P-R interval progressively decreased duringdevelopment. Similarly, the mean Q-T interval decreased from 62 ± 2 ms in day 1 to 50 ± 2 ms inday 7, 47 ± 8 ms inday 14 neonatal mice, and 46 ± 2 ms in adult mice (P < 0.001 byANOVA; all 3 groups are significantly different fromday 1).Q-T_c was calculated asQ- interval.Q-T_c significantly shortened from179 ± 4 ms in day 1 to 149 ± 5 ms in day 7 mice(P < 0.001). In addition, the J junction-S-T segment elevation observed in day1 neonatal mice resolved by day14. Dofetilide (0.5 mg/kg), the selective blocker ofthe rapid component of the delayed rectifier(I_Kr) abolished S-T segment elevation and prolonged Q-T andQ-T_c intervals in day 1 neonates but not in adult mice.In contrast, 4-aminopyridine (4-AP, 2.5 mg/kg) had no effect onday 1 neonates but in adults prolongedQ-T and Q-T_c intervals andspecifically decreased the amplitude of a transiently repolarizingwave, which appears as an r' wave at the end of the apparent QRSin adult mice. In conclusion, ECG intervals and configuration changeduring normal postnatal development in the mouse.K⁺ channel blockers affect themouse ECG differently depending on age. These data are consistent withthe previous findings that the dofetilide-sensitiveI_Kr is dominantin day 1 mice, whereas 4-AP-sensitivecurrents, the transiently repolarizingK⁺ current, and the rapidlyactivating, slowly inactivating K⁺current are the dominant K⁺currents in adult mice. This study provides background information useful for assessing abnormal development in transgenic mice.

     

Effect of IBMX and alkaline phosphatase inhibitors on Cl- secretion in G551D cystic fibrosis mutant mice

Some cysticfibrosis transmembrane conductance regulator (CFTR) mutations, such asG551D, result in a correctly localized Cl channel at the cellapical membrane, albeit with markedly reduced function. Patch-clampstudies have indicated that both phosphatase inhibitors and3-isobutyl-1-methylxanthine (IBMX) can induceCl secretion through theG551D mutant protein. We have now assessed whether these agents caninduce Cl secretion incftr^G551D mutantmice. No induction of Clsecretion was seen with the alkaline phosphatase inhibitorsbromotetramisole or levamisole in either the respiratory or intestinaltracts of wild-type orcftr^G551D mice.In contrast, in G551D intestinal tissues, IBMX was able to produce asmall CFTR-related secretory response [means ± SE: jejunum,1.8 ± 0.9 µA/cm²,n = 7; cecum, 3.7 ± 0.8 µA/cm²,n = 7; rectum (in vivo),1.9 ± 0.9 mV, n = 5]. Thiswas approximately one order of magnitude less than the wild-typeresponse to this agent and, in the cecum, was significantly greaterthan that seen in null mice(cftr^UNC). Inthe trachea, IBMX produced a transientCl secretory response (37.3 ± 14.7 µA/cm²,n = 6) of a magnitude similar to thatseen in wild-type mice (33.7 ± 4.7 µA/cm²,n = 9). This response was also presentin null mice and therefore is likely to be independent of CFTR. Noeffect of IBMX on Clsecretion was seen in the nasal epithelium ofcftr^G551D mice.We conclude that IBMX is able to induce detectable levels ofCFTR-related Cl secretionin the intestinal tract but not the respiratory tract through the G551Dmutant protein.

     

Immunomodulatory effects of dehydroepiandrosterone in proestrus female mice after trauma-hemorrhage.

Studies indicate that administration of the adrenal steroid dehydroepiandrosterone (DHEA) after trauma-hemorrhage in male mice improved cellular immune functions and reduced mortality rates from subsequent sepsis. There is evidence, however, that DHEA is converted to estrogens in males and that estrogens are immunoprotective after trauma-hemorrhage (TH). In contrast, DHEA in females can be converted to testosterone that has deleterious effects on immune functions. The aim of our study, therefore, was to determine whether administration of DHEA in proestrus females after TH would deteriorate immune responses. Proestrus female C3H/HeN mice (age 7-8 wk) were subjected to laparotomy (i.e., soft tissue trauma induced) and hemorrhagic shock (35 +/- 5 mmHg for 90 min) or sham operation. The mice then received DHEA (100 micro/25 g body wt) or vehicle subcutaneously followed by fluid resuscitation (4x the shed blood volume). Plasma IL-6, splenocyte proliferation, splenocyte IL-2, IL-3, IFN-gamma, IL-10 release, and splenic Mphi IL-1beta, IL-6, IL-10, and IL-12 release were determined 24 h after TH. Plasma IL-6 levels were significantly increased in vehicle-treated females, and DHEA administration markedly attenuated this response. In vehicle-treated females, splenocyte proliferation, IL-2, IL-3, and IFN-gamma release, and splenic Mphi IL-1 beta, IL-6, and IL-12 release were maintained or slightly enhanced after TH. In DHEA-treated females, however, these immune functional parameters were either unaltered compared with vehicle-treated animals or even further enhanced, but surprisingly were not depressed. Moreover, DHEA reduced splenocyte and splenic M phi anti-inflammatory cytokine (i.e., IL-10) production after TH compared with vehicle-treated females. Because DHEA further enhances the immune responsiveness in proestrus females after TH, this hormone might be a useful adjunct even in females for further enhancing immune responses and decreasing the mortality rate after trauma and severe blood loss.     

Male sex steroids are responsible for depressing macrophage immune function after trauma-hemorrhage

Recent studiessuggest beneficial effects of castration before soft tissue trauma andhemorrhagic shock on splenocyte immune functions. Nonetheless, itremains unknown whether this effect of testosterone depletion islimited to splenocytes or is a generalized effect on immune function.The present study was therefore carried out to determine whetherandrogen depletion before trauma-hemorrhage also has salutary effectson splenic and peritoneal macrophage as well as on Kupffer cellfunction, as indicated by interleukin (IL)-1 and IL-6 release. MaleC3H/HeN mice were castrated or sham-castrated 2 wk before theexperiment and were killed at 24 h after trauma-hemorrhage andresuscitation. Significant depression of macrophage IL-1 and IL-6release was only observed in sham-castrated mice, as opposed to normallevels of cytokine release from castrated animals after trauma-hemorrhage. In addition, only sham-castrated animals showed significantly increased levels of IL-6 release from Kupffer cells, which is believed to contribute to the systemic inflammatory response to trauma-hemorrhage. These observations suggest that the beneficial effects of androgen depletion before trauma-hemorrhage are not limitedto splenocyte immune functions but are more global in nature. Theseresults in surgically castrated animals suggest that androgen-blockingagents should be studied for their potential to reverse theimmunodepression associated with trauma-hemorrhage.

     

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon

The action of the isoflavonegenistein on the cystic fibrosis transmembrane conductance regulator(CFTR) has been studied in many cell systems but not in intact murinetissues. We have investigated the action of genistein on murine tissuesfrom normal and cystic fibrosis (CF) mice. Genistein increased theshort-circuit current (I_sc) in tracheal(16.4 ± 2.8 µA/cm²) and colonic (40.0 ± 4.4 µA/cm²) epithelia of wild-type mice. This increase wasinhibited by furosemide, diphenylamine-2-carboxylate, andglibenclamide, but not by DIDS. In contrast, genistein produced nosignificant change in the I_sc of the trachealepithelium (0.9 ± 1.1 µA/cm²) and decreased theI_sc of colons from CF null (13.1 ± 2.3 µA/cm²) and F508 mice (10.3 ± 1.3 µA/cm²). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored thegenistein response in the tracheas to wild-type levels. Tracheas fromF508 mice were also studied: 46% of trachea showed no response togenistein, whereas 54% gave an increase in I_scsimilar to that in wild type. We conclude that genistein activatesCFTR-mediated Cl secretion in the murine trachea anddistal colon.

     

Progressive decrease of intramyocellular accumulation of H+ and Pi in human skeletal muscle during repeated isotonic exercise

The purpose of this study was to evaluatethe hypotheses that accumulation of hydrogen ions and/or inorganicphosphate (Pi) in skeletal muscle increases with repeated bouts ofisotonic exercise. ³¹P-Magnetic resonance spectroscopy wasused to examine the gastrocnemius muscle of seven highly aerobicallytrained females during four bouts of isotonic plantar flexion. Theexercise bouts (EX1-4) of 3 min and 18 swere separated by 3 min and 54 s of complete rest. Muscle ATP did notchange during the four bouts. Phosphocreatine (PCr) degradation duringEX1 (13.3 ± 2.4 mmol/kg wet weight) was higher(P < 0.01) compared with EX3-4(9.7 ± 1.6 and 9.6 ± 1.8 mmol/kg wet weight, respectively).The intramyocellular pH at the end of EX1 (6.87 ± 0.05) was significantly lower (P < 0.001) than thoseof EX2 (6.97 ± 0.02), EX3 (7.02 ± 0.01), and EX4 (7.02 ± 0.02). Total Pi anddiprotonated Pi were significantly higher (P < 0.001)at the end of EX1 (17.3 ± 2.7 and 7.8 ± 1.6 mmol/kg wet weight, respectively) compared with the values at the end of EX3 and EX4. The monoprotonated Pi at the endof EX1 (9.5 ± 1.2 mmol/kg wet weight) was alsosignificantly higher (P < 0.001) than that afterEX4 (7.5 ± 1.1 mmol/kg wet weight). Subjects' ratingof perceived exertion increased (P < 0.001) towardexhaustion as the number of exercises progressed (7.1 ± 0.4, EX1; 8.0 ± 0.3, EX2; 8.5 ± 0.3, EX3; and 9.0 ± 0.4, EX4; scale from 0 to10). The present results indicate that human muscle fatigue during repeated intense isotonic exercise is not due to progressive depletion of high energy phosphates nor to intracellular accumulation of hydrogenions, total, mono-, or diprotonated Pi.

     

Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice

Blank, Sally E., T. Bucky Jones, Eric G. Lee, C. JayneBrahler, Randle M. Gallucci, Marne L. Fox, and Gary G. Meadows. Modulation of NK cell cytolytic activity by macrophages in chronically exercise-stressed mice. J. Appl.Physiol. 83(3): 845-850, 1997.This study wasdesigned to investigate the effects of moderate-intensity endurancetraining on basal natural killer (NK) cell cytolytic activity in murinesplenocytes that were enriched for1)NK1.1⁺ cells or2) macrophages andNK1.1⁺ cells. Mice were assignedto sedentary (Sed), treadmill control (TM), or treadmill-trained (Trn)groups. Splenocyte number, the percentages ofNK1.1⁺, large granular lymphocytes(NK1.1⁺, LGL-1⁺),and other subpopulations did not change in Trn mice. Approximately 70%of cells enriched for NK1.1⁺expressed this surface antigen. Lytic units (LU) expressed per LGL-1⁺ cell were significantlylower in Trn [83.9 ± 3.2 (SE)] compared with Sed (109.5 ± 7.5) and TM (101.3 ± 6.4) groups. When macrophages remainedin the in vitro assay, LU perLGL-1⁺ cell did not differ acrossgroups. The results indicate that highly enrichedNK1.1⁺ cells from Trn mice hadlower NK cell activity compared with Sed mice. No differences in NKcell activity were observed when cells were enriched forNK1.1⁺ cells and macrophages.These findings support the hypothesis that macrophage modulation of NKcells may be one mechanism contributing to augmented basal NK cellactivity in endurance-trained individuals.

     

Decreased [3H]ouabain binding sites in skeletal muscle of rats with chronic heart failure

Pickar, Joel G., John P. Mattson, Steve Lloyd, and TimothyI. Musch. Decreased[³H]ouabainbinding sites in skeletal muscle of rats with chronic heart failure.J. Appl. Physiol. 83(1): 323-329, 1997.Abnormalities intrinsic to skeletal muscle are thought tocontribute to decrements in exercise capacity found in individualswith chronic heart failure (CHF).Na⁺-K⁺-adenosinetriphosphatase(the Na⁺ pump) is essential formaintaining muscle excitability and contractility. Therefore, weinvestigated the possibility that the number and affinity ofNa⁺ pumps in locomotor muscles ofrats with CHF are decreased. Myocardial infarction (MI) was induced in8 rats, and a sham operation was performed in 12 rats. The degree ofCHF was assessed ~180 days after surgery. Soleus and plantarismuscles were harvested, and Na⁺pumps were quantified by using a[³H]ouabain bindingassay. At the time of muscle harvest, MI and sham-operated rats weresimilar in age (458 ± 54 vs. 447 ± 34 days old, respectively).Compared with their sham-operated counterparts, MI rats had asignificant amount of heart failure, right ventricular-to-body weightratio was greater (48%), and the presence of pulmonary congestion wassuggested by an elevated lung-to-body weight ratio (29%). Leftventricular end-diastolic pressure was significantly increased in theMI rats (11 ± 1 mmHg) compared with the sham-operated controls (1 ± 1 mmHg). In addition, mean arterial blood pressure was lower inthe MI rats compared with their control counterparts. [³H]ouabain bindingsites were reduced 18% in soleus muscle (136 ± 12 vs. 175 ± 13 pmol/g wet wt, MI vs. sham, respectively) and 22% in plantaris muscle(119 ± 12 vs. 147 ± 8 pmol/g wet wt, MI vs. sham,respectively). The affinity of these[³H]ouabain bindingsites was similar for the two groups. The relationship between thereduction in Na⁺ pump number andthe reduced exercise capacity in individuals with CHF remains to bedetermined.

     

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.

     

Microdialysis ethanol removal reflects probe recovery rather than local blood flow in skeletal muscle

The present study compared the microdialysis ethanoloutflow-inflow technique for estimating blood flow (BF) in skeletalmuscle of humans with measurements by Doppler ultrasound of femoralartery inflow to the limb(BF_FA). The microdialysis probeswere inserted in the vastus lateralis muscle and perfused with a Ringeracetate solution containing ethanol,[2-³H]adenosine (Ado),andD-[¹⁴C(U)]glucose.BF_FA at rest increased from0.16 ± 0.02 to 1.80 ± 0.26 and 4.86 ± 0.53 l/minwith femoral artery infusion of Ado (Ado_FA,i) at 125 and 1,000 µg · min¹ · l¹thigh volume (low dose and high dose, respectively;P < 0.05) and to 3.79 ± 0.37 and6.13 ± 0.65 l/min during one-legged, dynamic, thigh muscle exercisewithout and with high Ado_FA,i,respectively (P < 0.05). The ethanoloutflow-to-inflow ratio (38.3 ± 2.3%) and the probe recoveries(PR) for [2-³H]Ado(35.4 ± 1.6%) and forD-[¹⁴C(U)]glucose(15.9 ± 1.1%) did not change withAdo_FA,i at rest (P = not significant). During exercisewithout and with Ado_FA,i, theethanol outflow-to-inflow ratio decreased(P < 0.05) to a similar level of17.5 ± 3.4 and 20.6 ± 3.2%, respectively(P = not significant), respectively,while the PR increased (P < 0.05) toa similar level (P = not significant)of 55.8 ± 2.8 and 61.2 ± 2.5% for[2-³H]Ado and to 42.8 ± 3.9 and 45.2 ± 5.1% forD-[¹⁴C(U)]glucose.Whereas the ethanol outflow-to-inflow ratio and PR correlated inverselyand positively, respectively, to the changes in BF during muscularcontractions, neither of the ratio nor PR correlated tothe Ado_FA,i-induced BF increase.Thus the ethanol outflow-to-inflow ratio does not represent skeletalmuscle BF but rather contraction-induced changes in molecular transport in the interstitium or over the microdialysis membrane.

     

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.

     

Cytokine response to eccentric exercise in young and elderly humans

To examine the plasma interleukin(IL)-6 response in elderly (E) and young (Y) humans, 10 E and 10 Ysubjects completed 60 min of eccentric lower limb exercise at the samerelative oxygen uptake. Plasma IL-6 was measured before, immediatelyafter, and 5 days into recovery from exercise, as were the biochemicalmarkers of muscle damage, creatine kinase (CK), and myoglobin. In both groups, IL-6 increased (P < 0.05) immediately afterexercise and peaked 4 h after exercise at 4.35 ± 1.7 vs.5.05 ± 3.17 pg/ml for E and Y subjects, respectively. However,the increase in IL-6 in both groups was modest relative to theincreases in CK peaking at 539 ± 413 vs. 10,301 ± 5,863 U/lfor E and Y subjects, respectively. In addition, the increase in IL-6was less pronounced (P < 0.05) in E subjects comparedwith Y subjects. These results suggest that IL-6 increasesprogressively after eccentric exercise, suggesting that this increaseis related to muscle damage. However, the modest increase in IL-6,despite large increases in CK, suggests that the IL-6 response tomuscle damage does not make an important contribution to the largeincrease in IL-6 observed during concentric exercise of long duration.Our data also suggest that aging may be associated with impaired repairmechanisms for exercise-induced muscle damage.

     

Contractile function and low-intensity exercise effects of old dystrophic (mdx) mice

Oldmdx mice display a severe myopathyalmost identical to Duchenne's muscular dystrophy. This study examinedthe contractile properties of old mdxmuscles and investigated any effects of low-intensity exercise.Isometric contractile properties of the extensor digitorum longus (EDL)and soleus muscles were tested in adult (8-10 mo) and old (24 mo,split into sedentary and exercised groups)mdx mice. The EDL and soleus from oldmdx mice exhibited decreased absolutetwitch and tetanic forces, and the soleus exhibited a >50% decreasein relative forces (13.4 ± 0.4 vs. 6.0 ± 0.9 N/cm²) compared with adult mice.Old mdx muscles also showed longer contraction times and a higher percentage of type I fibers. Normal andmdx mice completed 10 wk of swimming,but mdx mice spent significantly lesstime swimming than normal animals (7.8 ± 0.4 vs. 15.8 ± 1.1 min, respectively). However, despite their severe dystrophy,mdx muscles responded positively tothe low-intensity exercise. Relative tetanic tensions were increased(~25% and ~45% for the EDL and soleus, respectively) after theswimming, although absolute forces were unaffected. Thus these resultsindicate that, even with a dystrophin-deficient myopathy,mdx muscles can still respond to low-intensity exercise. This study shows that the contractile functionof muscles of old mdx mice displaysmany similarities to that of human dystrophic patients and providesfurther evidence that the use of non-weight-bearing, low-intensityexercises, such as swimming, has no detrimental effect on dystrophicmuscle and could be a useful therapeutic aid for sufferers of musculardystrophy.

     

Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

K+ supplementation increases muscle [Na+-K+-ATPase] and improves extrarenal K+ homeostasis in rats

Bundgaard, Henning, Thomas A. Schmidt, Jim S. Larsen, andKeld Kjeldsen. K⁺supplementation increases muscle[Na⁺-K⁺-ATPase]and improves extrarenal K⁺homeostasis in rats. J. Appl. Physiol.82(4): 1136-1144, 1997.Effects ofK⁺ supplementation (~200 mmolKCl/100 g chow) on plasma K⁺,K⁺ content, andNa⁺-K⁺-adeonsinetriphosphatase(ATPase) concentration([Na⁺-K⁺-ATPase])in skeletal muscles as well as on extrarenalK⁺ clearance were evaluated inrats. After 2 days of K⁺supplementation, hyperkalemia prevailed(K⁺-supplemented vs.weight-matched control animals) [5.1 ± 0.2 (SE) vs. 3.2 ± 0.1 mmol/l, P < 0.05, n = 5-6], and after 4 daysa significant increase in K⁺content was observed in gastrocnemius muscle (104 ± 2 vs. 97 ± 1 µmol/g wet wt, P < 0.05, n = 5-6). After 7 days ofK⁺ supplementation, a significantincrease in[³H]ouabain bindingsite concentration (344 ± 5 vs. 239 ± 8 pmol/g wet wt,P < 0.05, n = 4) was observed in gastrocnemiusmuscle. After 2 wk, increases in plasmaK⁺,K⁺ content, and[³H]ouabain bindingsite concentration in gastrocnemius muscle amounted to 40, 8, and 68%(P < 0.05) above values observed inweight-matched control animals, respectively. The latter change wasconfirmed by K⁺-dependentp-nitrophenyl phosphatase activitymeasurements. Fasting for 1 day reduced plasmaK⁺ andK⁺ content in gastrocnemius musclein rats that had been K⁺supplemented for 2 wk by 3.1 ± 0.3 mmol/l(P < 0.05, n = 5) and 15 ± 2 µmol/g wet wt(P < 0.05, n = 5), respectively. After induction of anesthesia, arterial plasma K⁺was measured during intravenous KCl infusion (0.75 mmolKCl · 100 g bodywt¹ · h¹).The K⁺-supplemented fasted groupdemonstrated a 42% (P < 0.05) lower plasma K⁺ rise, associated with asignificantly higher increase inK⁺ content in gastrocnemius muscleof 7 µmol/g wet wt (P < 0.05, n = 5) compared with their controlanimals. In conclusion, K⁺supplementation increases plasmaK⁺,K⁺ content, and[Na⁺-K⁺-ATPase]in skeletal muscles and improves extrarenalK⁺ clearance capacity.

     

Pulmonary emphysema decreases hamster skeletal muscle oxidative enzyme capacity

Skeletal muscle oxidative enzyme capacity is impaired inpatients suffering from emphysema and chronic obstructive pulmonary disease. This effect may result as a consequence of the physiological derangements because of the emphysema condition or, alternatively, as aconsequence of the reduced physical activity level in these patients.To explore this issue, citrate synthase (CS) activity was measured inselected hindlimb muscles and the diaphragm of Syrian Golden hamsters 6 mo after intratracheal instillation of either saline (Con,n = 7) or elastase [emphysema(Emp); 25 units/100 g body weight, n = 8]. Activity level was monitored, and no difference betweengroups was found. Excised lung volume increased with emphysema (Con,1.5 ± 0.3 g; Emp, 3.0 ± 0.3 g,P < 0.002). Emphysema significantly reduced CS activity in the gastrocnemius (Con, 45.1 ± 2.0; Emp, 39.2 ± 0.8 µmol · min¹ · gwet wt¹,P < 0.05) and vastus lateralis (Con,48.5 ± 1.5; Emp, 44.9 ± 0.8 µmol · min¹ · gwet wt¹,P < 0.05) but not in the plantaris(Con, 47.4 ± 3.9; Emp, 48.0 ± 2.1 µmol · min¹ · gwet wt¹,P < 0.05) muscle. In contrast, CSactivity increased in the costal (Con, 61.1 ± 1.8; Emp, 65.1 ± 1.5 µmol · min¹ · gwet wt¹,P < 0.05) and crural (Con, 58.5 ± 2.0; Emp, 65.7 ± 2.2 µmol · min¹ · gwet wt¹, P < 0.05) regions of the diaphragm. These data indicate that emphysema perse can induce decrements in the oxidative capacity of certainnonventilatory skeletal muscles that may contribute to exerciselimitations in the emphysematous patient.

     

Na+/H+ exchangers (NHE1-3) have similar turnover numbers but different percentages on the cell surface

NHE1, NHE2, andNHE3 are well-characterized cloned members of the mammalianNa⁺/H⁺exchanger (NHE) gene family. Given the specialized function and regulation of NHE1, NHE2, and NHE3, we compared basal turnover numbersof NHE1, NHE2, and NHE3 measured in the same cell system: PS120fibroblasts lacking endogenous NHEs. NHE1, NHE2, and NHE3 were epitopetagged with vesicular stomatitis virus glycoprotein (VSVG). Thefollowing characteristics were determined on the same passage of cellstransfected with NHE1V, NHE2V, or NHE3V:1) maximal reaction velocity(V_max) by²²Na⁺uptake and fluorometery, 2) totalamount of NHE protein by quantitative Western analysis with internalstandards of VSVG-tagged maltose-binding protein, and3) cell surface expression by cellsurface biotinylation. Cell surface expression (percentage of totalNHE) was 88.8 ± 3.5, 64.6 ± 3.3, 20.0 ± 2.6, and 14.0 ± 1.3 for NHE1V, 85- and 75-kDa NHE2V, and NHE3V, respectively. Despitethese divergent cell surface expression levels, turnover numbers forNHE1, NHE2, and NHE3 were similar (80.3 ± 9.6, 92.1 ± 8.6, and99.2 ± 9.1 s¹, whenV_max wasdetermined using ²²Na uptake at22°C and 742 ± 47, 459 ± 16, and 609 ± 39 s¹ whenV_max wasdetermined using fluorometry at 37°C). These data indicate that, inthe same cell system, intrinsic properties that determine turnovernumber are conserved among NHE1, NHE2, and NHE3.