To our knowledge, this study is the first to report tendon hypertrophy following resistance training. Further, the data show that the human PT CSA varies along the length of the tendon.

Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-prolinealanine (NPA) motifs, the central constriction, and an aromatic͞ arginine (ar͞R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar͞R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A͞ R195V), did not affect water permeability. The double mutant AQP1-H180A͞R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A͞H180A) enlarged the maximal diameter of the ar͞R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar͞R constriction in AQP1-R195V and AQP1-H180A͞R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar͞R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar͞R constriction. mutational analysis ͉ proton filter ͉ solute selectivity O rthodox aquaporins (AQPs) constitute one branch of water-conducting channels within the superfamily of major intrinsic proteins (1). In recent years, the protein structure of the prototypical mammalian AQP1 has been refined to 2.2-Å resolution (2-5). Two highly conserved structural features within the channel were proposed as filters that exclude the passage of solutes larger than water and of charged molecules, including protons. A central constriction is formed by the capping amino acids Asn-Pro-Ala (NPA constriction) at each positive end of two short ␣-helices, such that the two NPA motifs are pinched in the center of the pore. Proline and alanine are exchangeable to some extent, whereas asparagine is invariable (1). Extensive molecular dynamics͞quantum mechanical simulations suggest that the free energy barrier located at the NPA constriction predominates in the exclusion of protons (reviewed in ref. 6). Depending on the computational approach, a second significant energy barrier, termed aromatic͞arginine (ar͞R) constriction, exists. It is located below the channel mouth and is even narrower than the central NPA constriction (6, 7). It is formed by four amino acids (Phe-56, His-180, Cys-189, and Arg-195 in rat AQP1). The ensemble of Phe, His, and Arg is highly conserved in orthodox AQPs. Their side chains directly flank the pore, whereas the less-conserved C...

Using functional complementation and a yeast mutant deficient in ammonium (NH þ 4 ) transport (Dmep1-3), three wheat (Triticum aestivum) TIP2 aquaporin homologues were isolated that restored the ability of the mutant to grow when 2 mM NH

We have shown recently, in a yeast expression system, that some aquaporins are permeable to ammonia. In the present study, we expressed the mammalian aquaporins AQP8, AQP9, AQP3, AQP1 and a plant aquaporin TIP2;1 in Xenopus oocytes to study the transport of ammonia (NH3) and ammonium (NH4+) under open-circuit and voltage-clamped conditions. TIP2;1 was tested as the wild-type and in a mutated version (tip2;1) in which the water permeability is intact. When AQP8-, AQP9-, AQP3- and TIP2;1-expressing oocytes were placed in a well-stirred bathing medium of low buffer capacity, NH3 permeability was evident from the acidification of the bathing medium; the effects observed with AQP1 and tip2;1 did not exceed that of native oocytes. AQP8, AQP9, AQP3, and TIP2;1 were permeable to larger amides, while AQP1 was not. Under voltage-clamp conditions, given sufficient NH3, AQP8, AQP9, AQP3, and TIP2;1 supported inwards currents carried by NH4+. This conductivity increased as a sigmoid function of external [NH3]: for AQP8 at a bath pH (pH(e)) of 6.5, the conductance was abolished, at pH(e) 7.4 it was half maximal and at pH(e) 7.8 it saturated. NH4+ influx was associated with oocyte swelling. In comparison, native oocytes as well as AQP1 and tip2;1-expressing oocytes showed small currents that were associated with small and even negative volume changes. We conclude that AQP8, AQP9, AQP3, and TIP2;1, apart from being water channels, also support significant fluxes of NH3. These aquaporins could support NH4+ transport and have physiological implications for liver and kidney function.

No abstract

Muscle mass accretion is accomplished by heavy-load resistance training. The effect of light-load resistance exercise has been far more sparsely investigated with regard to potential effect on muscle size and contractile strength. We applied a resistance exercise protocol in which the same individual trained one leg at 70% of one-repetition maximum (1RM) (heavy load, HL) while training the other leg at 15.5% 1RM (light load, LL). Eleven sedentary men (age 25 +/- 1 yr) trained for 12 wk at three times/week. Before and after the intervention muscle hypertrophy was determined by magnetic resonance imaging, muscle biopsies were obtained bilaterally from vastus lateralis for determination of myosin heavy chain (MHC) composition, and maximal muscle strength was assessed by 1RM testing and in an isokinetic dynamometer at 60 degrees /s. Quadriceps muscle cross-sectional area increased (P < 0.05) 8 +/- 1% and 3 +/- 1% in HL and LL legs, respectively, with a greater gain in HL than LL (P < 0.05). Likewise, 1RM strength increased (P < 0.001) in both legs (HL: 36 +/- 5%, LL: 19 +/- 2%), albeit more so with HL (P < 0.01). Isokinetic 60 degrees /s muscle strength improved by 13 +/- 5% (P < 0.05) in HL but remained unchanged in LL (4 +/- 5%, not significant). Finally, MHC IIX protein expression was decreased with HL but not LL, despite identical total workload in HL and LL. Our main finding was that LL resistance training was sufficient to induce a small but significant muscle hypertrophy in healthy young men. However, LL resistance training was inferior to HL training in evoking adaptive changes in muscle size and contractile strength and was insufficient to induce changes in MHC composition.

In skeletal muscle and tendon the extracellular matrix confers important tensile properties and is crucially important for tissue regeneration after injury. Musculoskeletal tissue adaptation is influenced by mechanical loading, which modulates the availability of growth factors, including growth hormone (GH) and insulin-like growth factor-I (IGF-I), which may be of key importance. To test the hypothesis that GH promotes matrix collagen synthesis in musculotendinous tissue, we investigated the effects of 14 day administration of 33-50 μg kg −1 day −1 recombinant human GH (rhGH) in healthy young individuals. rhGH administration caused an increase in serum GH, serum IGF-I, and IGF-I mRNA expression in tendon and muscle. Tendon collagen I mRNA expression and tendon collagen protein synthesis increased by 3.9-fold and 1.3-fold, respectively (P < 0.01 and P = 0.02), and muscle collagen I mRNA expression and muscle collagen protein synthesis increased by 2.3-fold and 5.8-fold, respectively (P < 0.01 and P = 0.06). Myofibrillar protein synthesis was unaffected by elevation of GH and IGF-I. Moderate exercise did not enhance the effects of GH manipulation. Thus, increased GH availability stimulates matrix collagen synthesis in skeletal muscle and tendon, but without any effect upon myofibrillar protein synthesis. The results suggest that GH is more important in strengthening the matrix tissue than for muscle cell hypertrophy in adult human musculotendinous tissue.

The adaptive response of connective tissue to loading requires increased synthesis and turnover of matrix proteins, with special emphasis on collagen. Collagen formation and degradation in the tendon increases with both acute and chronic loading, and data suggest that a gender difference exists, in that females respond less than males with regard to an increase in collagen formation after exercise. It is suggested that estrogen may contribute toward a diminished collagen synthesis response in females. Conversely, the stimulation of collagen synthesis by other growth factors can be shown in both animal and human models where insulin‐like growth factor 1 (IGF‐I) and transforming growth factor‐β‐1 (TGF‐β‐1) expression increases to accompany or precede an increase in procollagen expression and collagen synthesis. In humans, it can be demonstrated that an increase in the interstitial concentration of TGF‐β, PGE2, IGF‐I plus its binding proteins and interleukin‐6 takes place after exercise. The increase in IGF‐I expression in tendon includes the isoform that has so far been thought only to exist in skeletal muscle (mechano growth factor). The increase in IGF‐I and procollagen expression showed a similar response whether the tendon was stimulated by concentric, isometric or eccentric muscle contraction, suggesting that strain rather that stress/torque determines the collagen‐synthesis stimulating response seen with exercise. The adaptation time to chronic loading is longer in tendon tissue compared with contractile elements of skeletal muscle or the heart, and only with very prolonged loading are significant changes in gross dimensions of the tendon observed, suggesting that habitual loading is associated with a robust change in the size and mechanical properties of human tendons. An intimate interplay between mechanical signalling and biochemical changes in the matrix is needed in tendon, such that chemical changes can be converted into adaptations in the morphology, structure and material properties.