Carnitine metabolism has been previously shown to change with exercise in normal subjects, and in patients with ischemic muscle diseases. To characterize carnitine metabolism further during exercise, six normal male subjects performed constantload exercise on a bicycle ergometer on two separate occasions. Low-intensity exercise was performed for 60 min at a work load equal to 50% of the lactate threshold, and high-intensity exercise was performed for 30 min at a work load between the lactate threshold and maximal work capacity for the individual. Low-intensity exercise was not associated with a change in muscle (vastus lateralis) carnitine metabolism. In contrast, from rest to 10 min of high-intensity exercise, muscle shortchain acylcarnitine content increased 5.5-fold while free carnitine content decreased 66%, and muscle total carnitine content decreased by 19% (all P < 0.01). These changes in skeletal muscle carnitine metabolism were present at the completion of 30 min of high-intensity exercise, and persisted through a 60-min recovery period. With 30 min of high-intensity exercise, plasma short-chain and long-chain acylcarnitine concentrations increased by 46% and 23%, respectively. Neither exercise state was associated with a change in the urine excretion rates of free carnitine or acylcarnitines. Thus, alterations in skeletal muscle carnitine metabolism, characterized by an increase in acylcarnitines and a decrease in free and total carnitine, are dependent on the work load and, therefore, the metabolic state associated with the exercise, and are poorly reflected in the plasma and urine carnitine pools.
Increased production of endothelin-1 (ET-1) has been detected in lungs of fawn-hooded rats (FHR) with idiopathic pulmonary hypertension. Accelerated pulmonary artery (PA) smooth muscle cell (SMC) proliferation contributes to vascular remodeling in these rats. We hypothesized that PA SMC would be an important site of enhanced ET-1 expression in FHR lung, that these SMC would have increased growth compared with cells from a normotensive strain, and that this locally produced ET-1 would contribute to the increased growth of these cells. We found that isolated FHR PASMC overexpressed preproET-1 mRNA and produced more ET-1 peptide compared with cells from normotensive Sprague-Dawley control rats (SDR). PA SMC from FHR had increased growth compared with control cells under conditions of serum withdrawal (0.1%), submaximal serum stimulation (0.3%; a condition previously found to be required for detection of growth in response to the comitogen, ET-1), and maximal serum stimulation (10%). Enhanced growth of FHR PA SMC in the presence of 0.3% serum, but not under the other test conditions, was inhibited by the ETA receptor antagonist, BQ-123. In summary, PA SMC from rats with idiopathic pulmonary hypertension overproduce ET-1. This overproduction contributes to the enhanced growth of FHR PA SMC in the presence of 0.3% serum. These cells also possess other unique growth characteristics that are independent of ET-1. Together, these ET-1-dependent and -independent growth properties likely contribute to the hyperplasia of FHR PA SMC found in vivo.
Growth properties retained and acquired by immature pulmonary artery (PA) smooth muscle cells (SMC) in vivo after chronic exposure to hypoxia and the mechanisms that regulate hypoxia-induced change in proliferative phenotype are not known. We tested the hypothesis that PA SMC from neonatal calves exposed to hypoxia after birth would both retain fetal-like and acquire new growth characteristics and that these changes would be at least partially dependent on protein kinase C (PKC), a key proproliferative signal transduction pathway. Like fetal cells, PA SMC from hypoxic calves grew faster in the presence and absence of serum and were more responsive to insulin-like growth factor I and platelet-derived growth factor-BB than control neonatal and adult cells. PA SMC from hypoxic calves also acquired other growth properties (i.e., including increased hypoxic growth after PKC activation) that were new compared with those observed for fetal cells. The proliferative response to hypoxia was first detectable in the neonatal period and was further increased in cells from hypoxic calves. SMC from fetuses and hypoxic calves were more susceptible to the growth-inhibiting effects of PKC antagonists (dihydrosphingosine and calphostin C) than control neonatal and adult cells. To test if the Ca(2+)-dependent isozymes of PKC were uniquely important in the developmental and acquired growth changes observed, the antagonistic effect of the specific, but isozyme nonselective, PKC inhibitor Ro-81-8220 was then compared with GF-109203X, a structural analog with relative specificity for the Ca(2+)-dependent isozymes of PKC (alpha and beta in PA SMC). The faster growing PA SMC from bovine fetuses and hypoxia-exposed calves again demonstrated greater growth inhibition in response to both inhibitors. GF-109203X was equipotent to Ro-31-8220, and its antiproliferative effects were shown to not be due to an increase in apoptosis. Phorbol ester-induced PKC downregulation, another inhibitor strategy that selectively depletes bovine PA SMC of PKC-alpha, but not -beta, mimicked the antiproliferative effects of GF-109203X. Whole cellular PKC catalytic activity paralleled the pattern of peptide-induced growth and susceptibility to PKC inhibition. These results suggest that PA SMC from hypoxia-exposed neonatal calves retain enhanced fetal-like proliferative capacity and acquire new growth properties that are at least partially dependent on the Ca(2+)-regulated isozymes of PKC and in particular PKC-alpha.
Carnitine has been used to enhance human exercise performance. To test the hypothesis that carnitine can directly modify skeletal muscle function, fatigue of isolated rat skeletal muscle strips was studied in vitro. Carnitine (10 mM) did not modify the initial force of soleus contraction. The time over which force declined by 50% during repetitive electrical stimulation of the soleus muscle (fiber type I) was prolonged 25% in the presence of 10 mM carnitine. In contrast, carnitine had no effect on the fatigue of extensor digitorum longus muscle strips (fiber type II). The beneficial effect of carnitine on soleus muscle strips was not observed if the routine 30-min preincubation in the presence of carnitine was decreased to 5 min; it was associated with a five- to sixfold increase in muscle total carnitine content and a 50-150% increase in muscle long-chain acylcarnitine content. Carnitine did not consistently modify lactate accumulation or glycogen depletion during the fatigue protocol. Incubation with propionyl-L-carnitine resulted in a decreased initial force of contraction and a delay in reaching maximal contractile force. Thus, carnitine can directly improve the fatigue characteristics of muscles enriched in type I fibers.
We sought to determine which isozymes of protein kinase C (PKC) contribute to the increased proliferation of immature bovine pulmonary artery (PA) adventitial fibroblasts. Seven were identified in lysates of neonatal PA fibroblasts by Western blot: three Ca2+ dependent (α, βI, and βII) and four Ca2+ independent (δ, ε, ζ, and μ). Four isozymes (γ, η, θ, and ι) were not detected in fibroblasts isolated at any developmental stage. Of the seven detected isozymes, only PKC-α and -βII protein levels were higher in fetal and neonatal cells compared with adult fibroblasts. Their role in the enhanced growth of immature fibroblasts was then evaluated. The isozyme nonselective PKC inhibitor Ro-31-8220 was first compared with GF-109203X, a structural analog of Ro-31-8220 with relative specificity for the Ca2+-dependent isozymes of PKC. GF-109203X selectively inhibited the growth of immature cells and was nearly as potent as Ro-31-8220. Go-6976, a more specific inhibitor of the Ca2+-dependent isozymes, mimicked the antiproliferative effect of GF-109203X. PKC downregulation with 1 μM phorbol 12-myristate 13-acetate had the same selective antiproliferative effect on immature fibroblasts as GF-109203X and Go-6976. The protein levels of PKC-α and -βII, but not of PKC-βI, were completely degraded in response to phorbol 12-myristate 13-acetate pretreatment. These results suggest that PKC-α and -βII are important in the augmented growth of immature bovine PA adventitial fibroblasts.
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