Recovery of radial PO(2) profiles from phosphorescence quenching measurements in microvessels

Work by previous investigators has indicated that a substantial amount of oxygen diffuses from the precapillary circulation. These losses imply that there should be radial gradients of oxygen tension (PO(2)) in arterioles, leading to a non-uniform distribution of oxygen within these microvessels. We have employed the phosphorescence quenching method to measure oxygen, allowing us to evaluate the heterogeneity of PO(2) inside short segments of microvessels. The phosphorescence decay curve contains information about the distribution of oxygen within the excited volume and the distribution can be represented as a histogram, by decomposing the decay curve into several components with weights proportional to the volume fraction of plasma with different PO(2), under the condition of a high signal-to-noise ratio. Furthermore, the histogram can be converted into a radial profile of PO(2), based on the assumptions of a circular vascular lumen, axisymmetric distribution of oxygen and monotonic PO(2) profile. Albumin-bound Pd-porphyrin phosphor was infused into the circulation of hamsters and excited by flash illumination at 10 Hz, with a square region of excitation light just covering the entire lumen, (i.e. width of region equaled luminal diameter) of microvessels in the hamster mesentery. A set of 50 curves (5 s of data) was averaged to obtain a decay curve with low noise. Curves were analyzed with the above histogram procedure, and this analysis allowed us to distinguish between PO(2) values originating from intra and extravascular subvolumes. The intravascular PO(2) in these microvessels was very heterogeneous, which could be explained by the existence of significant radial PO(2) gradients. The radial PO(2) gradients were estimated to be approximately 1 mmHg/microm.     

PO2 measurements in the microcirculation using phosphorescence quenching microscopy at high magnification

In phosphorescence quenching microscopy (PQM), the multiple excitation of a reference volume produces the integration of oxygen consumption artifacts caused by individual flashes. We analyzed the performance of two types of PQM instruments to explain reported data on Po2 in the microcirculation. The combination of a large excitation area (LEA) and high flash rate produces a large oxygen photoconsumption artifact manifested differently in stationary and flowing fluids. A LEA instrument strongly depresses Po2 in a motionless tissue, but less in flowing blood, creating an apparent transmural Po2 drop in arterioles. The proposed model explains the mechanisms responsible for producing apparent transmural and longitudinal Po2 gradients in arterioles, a Po2 rise in venules, a hypothetical high respiration rate in the arteriolar wall and mesenteric tissue, a low Po2 in lymphatic microvessels, and both low and uniform tissue Po2. This alternative explanation for reported paradoxical results of Po2 distribution in the microcirculation obviates the need to revise the dominant role of capillaries in oxygen transport to tissue. Finding a way to eliminate the photoconsumption artifact is crucial for accurate microscopic oxygen measurements in microvascular networks and tissue. The PQM technique that employs a small excitation area (SEA) together with a low flash rate was specially designed to avoid accumulated oxygen photoconsumption in flowing blood and lymph. The related scanning SEA instrument provides artifact-free Po2 measurements in stationary tissue and motionless fluids. Thus the SEA technique significantly improves the accuracy of microscopic Po2 measurements in the microcirculation using the PQM.     

Microvascular flow and tissue PO(2) in skeletal muscle of chronic reduced renal mass hypertensive rats

This study determined whether arteriolar blood flow, capillary red blood cell (RBC) velocity, capillary hematocrit (Hct(cap)), and tissue PO(2) are altered in cremaster muscles of rats with chronic reduced renal mass hypertension (RRM-HT) relative to normotensive rats on high- or low-salt (NT-HS vs. NT-LS) diet. The blood flow in first- through third-order arterioles was not different between NT and HT rats, either at rest or during maximal relaxation of the vessels with 10(-4) M adenosine. Capillary RBC velocity was similar between the groups at rest but was elevated in RRM-HT and NT-HS rats during adenosine superfusion. Hct(cap) was reduced at rest in RRM-HT and NT-HS rats compared with NT-LS and was reduced in RRM-HT rats during adenosine-induced dilation. Tissue PO(2) was reduced in RRM-HT and NT-HS rats compared with NT-LS rats during control conditions and was lower in RRM-HT than in NT-LS rats during adenosine-induced dilation. These results indicate that both RRM-HT and chronic exposure of normotensive rats to a high-salt diet lead to reduced tissue oxygenation, despite the maintenance of normal arteriolar blood flow.     

Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers

Values of skeletal muscle intracellularPO₂ during conditions ranging fromrest to maximal metabolic rates have been difficult to quantify. Amethod for measurement of intracellular PO₂ in isolated single skeletalmuscle fibers by using O₂-dependent quenching of aphosphorescent-O₂probe is described. Intact single skeletal muscle fibersfrom Xenopus laevis were dissectedfrom the lumbrical muscle and mounted in a glass chamber containingRinger solution at 20°C. The chamber was placed on the stage of aninverted microscope configured for epi-illumination. A solutioncontaining palladium-meso-tetra(4-carboxyphenyl) porphine bound to bovine serum albumin was injectedinto single fibers by micropipette pressure injection.Phosphorescence-decay curves (average of 10 rapid flashes) wererecorded every 7 s from single cells(n = 24) in which respiration had beeneliminated with NaCN, while the PO₂of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculatedat the varied extracellular PO₂ byobtaining a best-fit estimate by using a monoexponential function. Thephosphorescence lifetime varied from 40 to 70 µs at an extracellularPO₂ of 159 Torr to 650-700 µsat 0 Torr. The phosphorescent lifetimes for the variedPO₂ were used to calculate, by usingthe Stern-Volmer relationship, the phosphorescence-quenching constant(100 Torr¹ · s¹),and the phosphorescence lifetime in azero-O₂ environment (690 µs) forthe phosphor within the intracellular environment. This techniquedemonstrates a novel method for determining intracellular PO₂ in isolated single skeletalmuscle fibers.     

Cellular PO2 as a determinant of maximal mitochondrial O(2) consumption in trained human skeletal muscle.

Previously, by measuring myoglobin-associated PO(2) (P(Mb)O(2)) during maximal exercise, we have demonstrated that 1) intracellular PO(2) is 10-fold less than calculated mean capillary PO(2) and 2) intracellular PO(2) and maximum O(2) uptake (VO(2 max)) fall proportionately in hypoxia. To further elucidate this relationship, five trained subjects performed maximum knee-extensor exercise under conditions of normoxia (21% O(2)), hypoxia (12% O(2)), and hyperoxia (100% O(2)) in balanced order. Quadriceps O(2) uptake (VO(2)) was calculated from arterial and venous blood O(2) concentrations and thermodilution blood flow measurements. Magnetic resonance spectroscopy was used to determine myoglobin desaturation, and an O(2) half-saturation pressure of 3.2 Torr was used to calculate P(Mb)O(2) from saturation. Skeletal muscle VO(2 max) at 12, 21, and 100% O(2) was 0.86 +/- 0.1, 1.08 +/- 0.2, and 1.28 +/- 0.2 ml. min(-1). ml(-1), respectively. The 100% O(2) values approached twice that previously reported in human skeletal muscle. P(Mb)O(2) values were 2.3 +/- 0.5, 3.0 +/- 0.7, and 4.1 +/- 0.7 Torr while the subjects breathed 12, 21, and 100% O(2), respectively. From 12 to 21% O(2), VO(2) and P(Mb)O(2) were again proportionately related. However, 100% O(2) increased VO(2 max) relatively less than P(Mb)O(2), suggesting an approach to maximal mitochondrial capacity with 100% O(2). These data 1) again demonstrate very low cytoplasmic PO(2) at VO(2 max), 2) are consistent with supply limitation of VO(2 max) of trained skeletal muscle, even in hyperoxia, and 3) reveal a disproportionate increase in intracellular PO(2) in hyperoxia, which may be interpreted as evidence that, in trained skeletal muscle, very high mitochondrial metabolic limits to muscle VO(2) are being approached.     

Dissociation between skeletal muscle microvascular PO2 and hypoxia-induced microvascular inflammation.

Systemic hypoxia (SHx) produces microvascular inflammation in mesenteric, cremasteric, and pial microcirculations. In anesthetized rats, SHx lowers arterial blood pressure (MABP), which may alter microvascular blood flow and microvascular Po(2) (Pm(O(2))) and influence SHx-induced leukocyte-endothelial adherence (LEA). These experiments attempted to determine the individual contributions of the decreases in Pm(O(2)), venular blood flow and shear rate, and MABP to the hypoxia-induced increase in LEA. Cremaster microcirculation of anesthetized rats was visualized by intravital microscopy. Pm(O(2)) was measured by a phosphorescence-quenching method. SHx [inspired Po(2) of 70 Torr for 10 min, MABP of 65 +/- 3 mmHg, arterial Po(2) (Pa(O(2))) of 33 +/- 1 Torr] and cremaster ischemia (MABP of 111 +/- 7 mmHg, Pa(O(2)) of 86 +/- 3 Torr) produced similar Pm(O(2)): 7 +/- 2 and 6 +/- 2 Torr, respectively. However, LEA increased only in SHx (1.9 +/- 0.9 vs. 11.2 +/- 1.1 leukocytes/100 microm, control vs. SHx, P < 0.05). Phentolamine-induced hypotension (MABP of 55 +/- 4 mmHg) in normoxia lowered Pm(O(2)) to 26 +/- 6 Torr but did not increase LEA. Cremaster equilibration with 95% N(2)-5% CO(2) during air breathing (Pa(O(2)) of 80 +/- 1 Torr) lowered Pm(O(2)) to 6 +/- 1 Torr but did not increase LEA. On the other hand, when cremaster Pm(O(2)) was maintained at 60-70 Torr during SHx (Pa(O(2)) of 35 +/- 1 Torr), LEA increased from 2.1 +/- 1.1 to 11.1 +/- 1.5 leukocytes/100 microm (P < 0.05). The results show a dissociation between Pm(O(2)) and LEA and support the idea that SHx results in the release of a mediator responsible for the inflammatory response.     

Fall in intracellular PO(2) at the onset of contractions in Xenopus single skeletal muscle fibers.

It remains uncertain whether the delayed onset of mitochondrial respiration on initiation of muscle contractions is related to O(2) availability. The purpose of this research was to measure the kinetics of the fall in intracellular PO(2) at the onset of a contractile work period in rested and previously worked single skeletal muscle fibers. Intact single skeletal muscle fibers (n = 11) from Xenopus laevis were dissected from the lumbrical muscle, injected with an O(2)-sensitive probe, mounted in a glass chamber, and perfused with Ringer solution (PO(2) = 32 +/- 4 Torr and pH = 7.0) at 20 degrees C. Intracellular PO(2) was measured in each fiber during a protocol consisting sequentially of 1-min rest; 3 min of tetanic contractions (1 contraction/2 s); 5-min rest; and, finally, a second 3-min contractile period identical to the first. Maximal force development and the fall in force (to 83 +/- 2 vs. 86 +/- 3% of maximal force development) in contractile periods 1 and 2, respectively, were not significantly different. The time delay (time before intracellular PO(2) began to decrease after the onset of contractions) was significantly greater (P < 0.01) in the first contractile period (13 +/- 3 s) compared with the second (5 +/- 2 s), as was the time to reach 50% of the contractile steady-state intracellular PO(2) (28 +/- 5 vs. 18 +/- 4 s, respectively). In Xenopus single skeletal muscle fibers, 1) the lengthy response time for the fall in intracellular PO(2) at the onset of contractions suggests that intracellular factors other than O(2) availability determine the on-kinetics of oxidative phosphorylation and 2) a prior contractile period results in more rapid on-kinetics.     

Effect of varied extracellular PO2 on muscle performance in Xenopus single skeletal muscle fibers.

The purpose of this study was to examine the development of fatigue in isolated, single skeletal muscle fibers when O2 availability was reduced but not to levels considered rate limiting to mitochondrial respiration. Tetanic force was measured in single living muscle fibers (n = 6) from Xenopus laevis while being stimulated at increasing contraction rates (0.25, 0.33, 0.5, and 1 Hz) in a sequential manner, with each stimulation frequency lasting 2 min. Muscle fatigue (determined as 75% of initial maximum force) was measured during three separate work bouts (with 45 min of rest between) as the perfusate PO2 was switched between values of 30 +/- 1.9, 76 +/- 3.0, or 159 Torr in a blocked-order design. No significant differences were found in the initial peak tensions between the high-, intermediate-, and low-PO2 treatments (323 +/- 22, 298 +/- 27, and 331 +/- 24 kPa, respectively). The time to fatigue was reached significantly sooner (P < 0.05) during the 30-Torr treatment (233 +/- 39 s) compared with the 76- (385 +/- 62 s) or 159-Torr (416 +/- 65 s) treatments. The calculated critical extracellular PO2 necessary to develop an anoxic core within these fibers was 13 +/- 1 Torr, indicating that the extracellular PO2 of 30 Torr should not have been rate limiting to mitochondrial respiration. The magnitude of an unstirred layer (243 +/- 64 micron) or an intracellular O2 diffusion coefficient (0.45 +/- 0.04 x 10(-5) cm2/s) necessary to develop an anoxic core under the conditions of the study was unlikely. The earlier initiation of fatigue during the lowest extracellular PO2 condition, at physiologically high intracellular PO2 levels, suggests that muscle performance may be O2 dependent even when mitochondrial respiration is not necessarily compromised.     

Involuntary leg movements affect interstitial nutrient gradients and blood flow in rat skeletal muscle.

To evaluate the effect of passive muscle shortening and lengthening (PSL) on the transcapillary exchange of glucose, lactate, and insulin in the insulin-stimulated state, microdialysis was performed in rat quadriceps muscle. Electrical pulsatile stimulation (0.1 ms, 0.3-0.6 V, 1 Hz) was performed on the sciatic nerve in one leg to induce passive tension on the quadriceps during a hyperinsulinemic-euglycemic clamp (10 mU x kg(-1) x min(-1)). In the non-insulin-stimulated (basal) state, the muscle arterial-interstitial (A-I) concentration difference of glucose was 1.6 +/- 0.3 mM (P < 0.01). During insulin infusion, it remained unaltered in resting muscle (1.3 +/- 0.3 mM) but diminished during PSL. In the basal state there was no A-I concentration difference of lactate, whereas in the insulin infusion state it increased significantly and was significantly greater in moving (2.8 +/- 0.5 mM, P < 0.01) than in resting muscle (0.7 +/- 0.4 mM). The A-I concentration difference of insulin was equal in resting and moving muscle: 86 +/- 7 and 100 +/- 8 microU/ml, respectively. Muscle blood flow estimated by use of radiolabeled microspheres increased during PSL from 17 +/- 4 to 34 +/- 6 ml x 100 g(-1) x min(-1) (P < 0.05). These results confirm that diffusion over the capillary wall is partly rate limiting for the exchange of insulin and glucose and lactate in resting muscle. PSL, in addition to insulin stimulation, increases blood flow and capillary permeability and, as a result, diminishes the A-I concentration gradient of glucose but not that of insulin or lactate.     

Effect of training on H(2)O(2) release by mitochondria from rat skeletal muscle

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.     

Effects of age and resistance exercise on skeletal muscle interstitial prostaglandin F(2alpha)

Prostaglandin (PG) F2alpha has been shown to contribute to the anabolic events in skeletal muscle. We measured the skeletal muscle interstitial concentration of PGF2alpha at rest and following a standard bout of resistance exercise in eight young (27+/-2 year) and eight old (75+/-4 year) men. Interstitial PGF2alpha concentration was determined from microdialysate samples obtained from two microdialysis probes placed in the vastus lateralis. Microdialysates were collected 1h pre- and 5-6, 8-9, and 24-25 h postexercise. The exercise bout consisted of 4 exercises (3 sets of 8 replications at 80% 1 RM per exercise) emphasizing the quadriceps. Interstitial PGF2alpha levels were not different (P>0.05) between young and old at rest (1.50+/-0.35 vs. 1.52+/-0.30 ng ml-1) or at any time point following the resistance exercise bout. For the young and old combined there was a change (P<0.05) in PGF2alpha levels at 5-6 h (93%) and 8-9 h (95%), which had returned to preexercise levels by 24-25 h. These results show that PGF2alpha is increased in skeletal muscle following a standard bout of resistance exercise and aging does not alter interstitial levels of this PG at rest or after exercise. These data, coupled with previous findings, suggest that the anabolic factor PGF2alpha should be considered when discussing the complex processes that regulate muscle mass in young and old individuals.     

Bicarbonate measurements in rat liver, brain, heart, and skeletal muscle.

A method is described for measuring total CO₂ and HCO₃^? in tissues rapidly frozen in liquid nitrogen. The method is a modification of the procedure of D. D. Van Slyke and J. M. Neill (1924, J. Biol. Chem.61, 523–573) for use with freeze-clamped tissue where anoxic changes have not occurred. The HCO₃^? content exclusive of tissue CO₂ in fed rats was found to be: liver, 19.4 ± 1.0; brain, 20.2 ± 0.9; thigh, 16.2 ± 0.8; and heart, 15.4 ± 1.4 μmol/g.     

New fiber formation in the interstitial spaces of rat skeletal muscle during postnatal growth.

The purpose of this study was to determine whether fiber hyperplasia occurs in the rat plantaris muscle during postnatal weeks 3-20. Total muscle fiber number, obtained via the nitric acid digestion method, increased by 28% during the early postnatal rapid growth phase (3-10 weeks), whereas the number of branched fibers was consistently low. Whole-muscle mitotic activity and amino acid uptake levels showed an inverse relationship to the increase in total fiber number. The expression of MyoD mRNA (RT-PCR) levels decreased from 3 to 20 weeks of age, as did the detection of anti-BrdU- and MyoD-positive cells in histological sections. Immunohistochemical staining patterns for MyoD, myogenin, or developmental myosin heavy chain on sections stained for laminin (identification of the basal lamina) and electron micrographs clearly indicate that de novo fiber formation occurred in the interstitial spaces. Myogenic cells in the interstitial spaces were negative for the reliable specific satellite cell marker M-cadherin. In contrast, CD34 (an established marker for hematopoietic stem cells)-positive cells were located only in the interstitial spaces, and their frequency and location were similar to those of MyoD- and/or myogenin-positive cells. These findings are consistent with fiber hyperplasia occurring in the interstitial spaces of the rat plantaris muscle during the rapid postnatal growth phase. Furthermore, these data suggest that the new fibers may be formed from myogenic cells in the interstitial spaces of skeletal muscle and may express CD34 that is distinct from satellite cells.     

Intracellular PO2 kinetics at different contraction frequencies in Xenopus single skeletal muscle fibers.

Increasing contraction frequency in single skeletal muscle fibers has been shown to increase the magnitude of the fall in intracellular Po(2) (Pi(O(2))), reflecting a greater metabolic rate. To test whether Pi(O(2)) kinetics are altered by contraction frequency through this increase in metabolic stress, Pi(O(2)) was measured in Xenopus single fibers (n = 11) during and after contraction bouts at three different frequencies. Pi(O(2)) was measured via phosphorescence quenching at 0.16-, 0.25-, and 0.5-Hz tetanic stimulation. The kinetics of the change in Pi(O(2)) from resting baseline to end-contraction values and end contraction to rest were described as a mean response time (MRT) representing the time to 63% of the change in Pi(O(2)). As predicted, the fall in Pi(O(2)) from baseline following contractions was progressively greater at 0.5 and 0.25 Hz than at 0.16 Hz (32.8 +/- 2.1 and 29.3 +/- 2.0 Torr vs. 23.6 +/- 2.2 Torr, respectively) since metabolic demand was greater. The MRT for the decrease in Pi(O(2)) was progressively faster at the higher frequencies (0.5 Hz: 45.3 +/- 4.5 s; 0.25 Hz: 63.3 +/- 4.1 s; 0.16 Hz: 78.0 +/- 4.1 s), suggesting faster accumulation of stimulators of oxidative phosphorylation. The MRT for Pi(O(2)) off-kinetics (0.5 Hz: 84.0 +/- 11.7 s; 0.25 Hz: 79.1 +/- 8.4 s; 0.16 Hz: 81.1 +/- 8.3 s) was not different between trials. These data demonstrate in single fibers that the rate of the fall in Pi(O(2)) is dependent on contraction frequency, whereas the rate of recovery following contractions is independent of either the magnitude of the fall in Pi(O(2)) from baseline or the contraction frequency. This suggests that stimulation frequency plays an integral role in setting the initial metabolic response to work in isolated muscle fibers, possibly due to temporal recovery between contractions, but it does not determine recovery kinetics.     

Phosphorescence quenching microrespirometry of skeletal muscle in situ

We have developed an optical method for the evaluation of the oxygen consumption (Vo(2)) in microscopic volumes of spinotrapezius muscle. Using phosphorescence quenching microscopy (PQM) for the measurement of interstitial Po(2), together with rapid pneumatic compression of the organ, we recorded the oxygen disappearance curve (ODC) in the muscle of the anesthetized rats. A 0.6-mm diameter area in the tissue, preloaded with the phosphorescent oxygen probe, was excited once a second by a 532-nm Q-switched laser with pulse duration of 15 ns. Each of the evoked phosphorescence decays was analyzed to obtain a sequence of Po(2) values that constituted the ODC. Following flow arrest and tissue compression, the interstitial Po(2) decreased rapidly and the initial slope of the ODC was used to calculate the Vo(2). Special analysis of instrumental factors affecting the ODC was performed, and the resulting model was used for evaluation of Vo(2). The calculation was based on the observation of only a small amount of residual blood in the tissue after compression. The contribution of oxygen photoconsumption by PQM and oxygen inflow from external sources was evaluated in specially designed tests. The average oxygen consumption of the rat spinotrapezius muscle was Vo(2) = 123.4 ± 13.4 (SE) nl O(2)/cm(3) · s (N = 38, within 6 muscles) at a baseline interstitial Po(2) of 50.8 ± 2.9 mmHg. This technique has opened the opportunity for monitoring respiration rates in microscopic volumes of functioning skeletal muscle.     

Cortisol binding in rat skeletal muscle.

Studies of the reversible binding of [3H]cortisol by rat gastrocnemius muscle cytoplasm in vitro reveal specific binding in the 27,000 times g supernatant fraction at 0 degrees. The [3H]cortisol-binding molecule had an apparant Kd value of 1.7 times 10-7 M and the number of binding sites was 0.99 pmol per mg of cytosol protein. Only a single class of [3H]cortisol-binding sites could be detected, whose protein nature was suggested by its susceptibility to nagarse. The [3H]cortisol-protein complex sedimented at similar to 4 S in a 5 to 20% sucrose gradient either in the presence or absence of 0.3 M KCl. Binding increased more than 2-fold in adrenalectomized rats and was markedly reduced in the muscle of rats pretreated with cortisol. In contrast to the binding of [3H]dexamethasone and [3H]triamcinolone acetonide to receptor proteins in muscle, no correlation was found between the ability of various steroids to complete wtth [3H]cortisol binding and their glucocorticoid potency: [3H]cortisol binding was inhibited by a 1000-fold higher concentration of unlabeled cortisol and progesterone but not by dexamethasone or triamcinolone acetonide. It is therefore suggested that the [3H]cortisol-binding reaction is not directly involved in the biological effects of all potent glucocorticoids in skeletal muscle. The [3H]cortisol-binding protein in muscle cytosol could not be unequivocally distinguished from rat plasma corticosteroid-binding globulin, because both had similar steroid specificity and temperature stability, were not markedly affected by--SH reagents, and displayed similar sedimentation properties.     

Catecholamines inhibit Ca(2+)-dependent proteolysis in rat skeletal muscle through beta(2)-adrenoceptors and cAMP

Overall proteolysis and the activity of skeletal muscle proteolytic systems were investigated in rats 1, 2, or 4 days after adrenodemedullation. Adrenodemedullation reduced plasma epinephrine by 95% and norepinephrine by 35% but did not affect muscle norepinephrine content. In soleus and extensor digitorum longus (EDL) muscles, rates of overall proteolysis increased by 15-20% by 2 days after surgery but returned to normal levels after 4 days. The rise in rates of protein degradation was accompanied by an increased activity of Ca(2+)-dependent proteolysis in both muscles, with no significant change in the activity of lysosomal and ATP-dependent proteolytic systems. In vitro rates of Ca(2+)-dependent proteolysis in soleus and EDL from normal rats decreased by ~35% in the presence of either 10(-5) M clenbuterol, a beta(2)-adrenergic agonist, or epinephrine or norepinephrine. In the presence of dibutyryl cAMP, proteolysis was reduced by 62% in soleus and 34% in EDL. The data suggest that catecholamines secreted by the adrenal medulla exert an inhibitory control of Ca(2+)-dependent proteolysis in rat skeletal muscle, mediated by beta(2)-adrenoceptors, with the participation of a cAMP-dependent pathway.