The present study examined the effect of high-intensity exercise training on muscle sarcolemmal lactate/H+ transport and the monocarboxylate transporters (MCT1 and MCT4) as well as lactate and H+ release during intense exercise in humans. One-legged knee-extensor exercise training was performed for 8 wk, and biopsies were obtained from untrained and trained vastus lateralis muscle. The rate of lactate/H+ transport determined in sarcolemmal giant vesicles was 12% higher ( P < 0.05) in the trained than in untrained muscle ( n = 7). The content of MCT1 and MCT4 protein was also higher (76 and 32%, respectively; n = 4) in trained muscle. Release of lactate and H+ from the quadriceps muscle at the end of intense exhaustive knee-extensor exercise was similar in the trained and untrained leg, although the estimated muscle intracellular-to-interstitial gradients of lactate and H+ were lower ( P < 0.05) in the trained than in the untrained muscle. The present data show that intense exercise training can increase lactate/H+transport capacity in human skeletal muscle as well as improve the ability of the muscle to release lactate and H+ during contractions.
Prostaglandin E2 (PGE2) is the major renal cyclooxygenase metabolite of arachidonic acid. Urinary excretion of PGE2 is increased by dietary salt restriction, as well in cirrhosis and congestive heart failure. To determine whether urinary PGE2 affects transport along the nephron, the actions of luminal PGE2 were studied in the isolated perfused rabbit cortical collecting duct (CCD). Luminal PGE2 transiently hyperpolarized transepithelial voltage (Vt) in a dose-dependent manner (half-maximal effect approximately 10(-8) M) in contrast to a sustained depolarization of Vt produced by basolateral PGE2. Luminal PGE2 (0.1 microM) also significantly stimulated osmotic water permeability in the CCD. In CCDs cultured on semipermeable supports, apical PGE2 stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production, suggesting the effects of luminal PGE2 are mediated by adenylyl cyclase-stimulating EP2 or EP4 receptors. Sulprostone, a PGE2 analogue selective for EP1 and EP3 receptors, affected Vt only when applied from the basolateral but not the luminal surface. Luminal application of the EP2 receptor agonist butaprost was also without effect. These results suggest that luminal PGE2 affects Vt via a butaprost-insensitive EP4 receptor. The Vt effect of luminal PGE2 was not blocked by pertussis toxin, also arguing against an EP3-mediated Gi-coupled effect. Finally, 1 microM luminal PGE2 only slightly increased CCD intracellular calcium concentration ([Ca2+]i), in contrast to the marked increase in [Ca2+]i produced by basolateral PGE2 (0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
We examined the mechanism by which the cytochrome P-450 metabolite of arachidonate, 5,6-epoxyeicosatrienoic acid (5,6-EET), modulates electrogenic transport in the rabbit cortical collecting duct (CCD). 5,6-EET depolarized transepithelial voltage (VT) in a concentration-dependent manner with a maximal effect at 1 microM. None of the other EET regioisomers (8,9-, 11,12-, or 14,15-EET; all at 1 microM) affected VT, This action was also stereoselective, with 5(S),6(R)-EET producing a 2.5-fold greater effect on VT than 5(R),6(S)-EET (1 microM each). Like basolateral prostaglandin E2 (PGE2), both luminal and basolateral 5,6-EET increased cytosolic Ca2+ concentration ([Ca2+]i) in the rabbit CCD. Pretreatment with cyclooxygenase inhibitors (10 microM ibuprofen or 5 microM indomethacin) completely blocked both the [Ca2+]i increase and the change in VT. Neither 5,6-epoxy-PGE1 nor 5-hydroxy-PGI1, cyclooxygenase metabolites of 5,6-EET, affected VT. However, when added to primary cultures of rabbit CCDs, 5,6-EET stimulated endogenous PGE2 synthesis. We propose that 5,6-EET stimulates endogenous prostaglandin synthesis, which inhibits electrogenic ion transport in the CCD.
The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.
Bovine epididymal spermatozoa were subjected to nitrogen cavitation (600 psi for 10 min) to remove plasma membrane. Examination of the cavitated cells by electron microscopy revealed that the plasma membrane was preferentially removed from the periacrosomal and flagellar regions. Nuclear, mitochondrial and acrosomal membranes remained intact and attached to the spermatozoa, but the cytoplasmic droplets were frequently disrupted and their internal membrane-bound vesicles were released. Lower pressures (less than 200 psi) were relatively ineffective in removing the periacrosomal plasma membrane, while an intermediate pressure (400 psi) removed this membrane from about 70% of the spermatozoa. No apparent selectivity for removal of the periacrosomal and flagellar plasma membrane was observed as a function of cavitation pressure. The cavitated cells were separated from the plasma membranes by differential followed by linear sucrose density gradient centrifugation. Two distinct membrane populations were resolved on sucrose gradients and were designated Band I and Band II. Band I contained only spherical vesicles which arose from the plasma membrane. Surface labeling of intact cells confirmed the plasma membrane as the origin of Band I. The membranes of higher density comprising Band II were heterogeneous consisting of both spherical and flattened vesicles. When purified cytoplasmic droplets were cavitated and centrifuged on the sucrose gradient only Band II was obtained. These studies indicate that nitrogen cavitation of bovine epididymal spermatozoa can result in significant contamination of plasma membrane fractions by cytoplasmic droplet membranes unless appropriate differential centrifugation is used to separate the membrane fractions.
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