NEDD4-2 and salt-sensitive hypertension.

PURPOSE OF REVIEW: NEDD4-2 is an ubiquitin-protein ligase that was originally identified as an interactor of the epithelial Na+ channel (ENaC); this interaction is defective in Liddle's syndrome, causing elevated ENaC activity and salt-sensitive hypertension. In this review we aim to highlight progress achieved in recent years demonstrating that NEDD4-2 is involved in the control of Na+ transporters that are different from ENaC, but which also play a role in salt-sensitive hypertension. RECENT FINDINGS: It has been shown that NEDD4-2 interacts with ubiquitylates and negatively regulates the thiazide-sensitive NCC (Na+,Cl- -cotransporter), both in vitro and in vivo in inducible, nephron-specific Nedd4-2 knockout mice. Moreover, evidence has been provided that NEDD4-2 is also involved in the regulation of human NHE3 (Na+,H+-exchanger 3) and NKCC2 (Na+,K+,2Cl- -cotransporter 2). SUMMARY: The emerging role of NEDD4-2 in the regulation of different Na+ transporters along the nephron and the identification of human polymorphisms in the NEDD4-2 gene (Nedd4L) related to salt-sensitive hypertension makes this ubiquitin-protein ligase an interesting target for the development of antihypertensive drugs.

The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice.

Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl- in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl-/HCO3- exchanger (NDCBE/SLC4A8) and the Na+-independent Cl-/HCO3- exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.

Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype.

The expression of the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by mineralocorticoids and, in turn, upregulates the renal epithelial Na(+) channel (ENaC). Total inactivation of Sgk1 has been associated with transient urinary Na(+) wasting with a low-Na(+) diet, while the aldosterone-mediated ENaC channel activation was unchanged in the collecting duct. Since Sgk1 is ubiquitously expressed, we aimed to study the role of renal Sgk1 and generated an inducible kidney-specific knockout (KO) mouse. We took advantage of the previously described TetOn/CreLoxP system, in which rtTA is under the control of the Pax8 promotor, allowing inducible inactivation of the floxed Sgk1 allele in the renal tubules (Sgk1fl/fl/Pax8/LC1 mice). We found that under a standard Na(+) diet, renal water and Na(+)/K(+) excretion had a tendency to be higher in doxycycline-treated Sgk1 KO mice compared with control mice. The impaired ability of Sgk1 KO mice to retain Na(+) increased significantly with a low-salt diet despite higher plasma aldosterone levels. On a low-Na(+) diet, the Sgk1 KO mice were also hyperkaliuric and lost body weight. This phenotype was accompanied by a decrease in systolic and diastolic blood pressure. At the protein level, we observed a reduction in phosphorylation of the ubiquitin protein-ligase Nedd4-2 and a decrease in the expression of the Na(+)-Cl(-)-cotransporter (NCC) and to a lesser extent of ENaC.

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter

The human and shark Na–K–Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark–human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86Rb influx with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect Km(Cl), demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark–human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp.

We have used capacitance measurements with a 1-microsecond voltage clamp technique to probe electrogenic ion-transporter interactions in giant excised membrane patches. The hydrophobic ion dipicrylamine was used to test model predictions for a simple charge-moving reaction. The voltage and frequency dependencies of the apparent dipicrylamine-induced capacitance, monitored by 1-mV sinusoidal perturbations, correspond to single charges moving across 76% of the membrane field at a rate of 9500 s-1 at 0 mV. For the cardiac Na,K pump, the combined presence of cytoplasmic ATP and sodium induces an increase of apparent membrane capacitance which requires the presence of extracellular sodium. The dependencies of capacitance changes on frequency, voltage, ATP, and sodium verify that phosphorylation enables a slow, 300- to 900-s-1, pump transition (the E1-E2 conformational change), which in turn enables fast, electrogenic, extracellular sodium binding reactions. For the GAT1 (gamma-aminobutyric acid,Na,Cl) cotransporter, expressed in Xenopus oocyte membrane, we find that chloride binding from the cytoplasmic side, and probably sodium binding from the extracellular side, results in a decrease of membrane capacitance monitored with 1- to 50-kHz perturbation frequencies. Evidently, ion binding by the GAT1 transporter suppresses an intrinsic fast charge movement which may originate from a mobility of charged residues of the transporter binding sites. The results demonstrate that fast capacitance measurements can provide new insight into electrogenic processes closely associated with ion binding by membrane transporters.

Regulation of blood pressure and renal function by NCC and ENaC: lessons from genetically engineered mice.

The activity of the thiazide-sensitive Na(+)/Cl(-) cotransporter (NCC) and of the amiloride-sensitive epithelial Na(+) channel (ENaC) is pivotal for blood pressure regulation. NCC is responsible for Na(+) reabsorption in the distal convoluted tubule (DCT) of the nephron, while ENaC reabsorbs the filtered Na(+) in the late DCT and in the cortical collecting ducts (CCD) providing the final renal adjustment to Na(+) balance. Here, we aim to highlight the recent advances made using transgenic mouse models towards the understanding of the regulation of NCC and ENaC function relevant to the control of sodium balance and blood pressure. We thus like to pave the way for common mechanisms regulating these two sodium-transporting proteins and their potential implication in structural remodeling of the nephron segments and Na(+) and Cl(-) reabsorption.

The composition of mineral waters sourced from Europe and North America in respect to bone health: composition of mineral water optimal for bone.

The consumption of mineral waters is increasing in industrialised countries. High intakes of Ca and other alkalising cations as well as a low acid intake are beneficial to bone. We examined which components of mineral waters are conditioning their Ca content and their alkalinising power, in order to define the optimal profile. European mineral waters were randomly selected on the Internet: 100 waters with less than 200 mg Ca/l (9.98 mEq/l) and fifty with more than 200 mg/l, all with complete data for SO4, P, Cl, Na, K, Mg and Ca, and most also for HCO3. For comparison, forty North American mineral waters were randomly chosen. The potential renal acid load (PRAL) was calculated for each mineral water. North American waters did not reveal significant results because of their low mineralisation. We performed correlations between all eight components in order to explore the properties of the mineral waters. In the European waters, twenty-six out of twenty-eight correlations showed a P value of <or= 0.01. In waters with PRAL >0 (acidifying waters), PRAL was positively correlated with SO4, Ca, K and Mg (P < 0.001). In those with PRAL < 0 (alkalinising waters), PRAL was negatively correlated with HCO3, Na, Mg, Ca, K, Cl and SO4 (P < 0.001). SO4 and HCO3 were not found together in high quantities in the same water for geochemical reasons. A high Ca content is associated with either a high SO4 or a high HCO3 content. SO4 theoretically increases Ca excretion, while HCO3 and low PRAL values are associated with positive effects on bone. Therefore, the best waters for bone health are rich in both HCO3 and Ca, and by consequence low in SO4.

Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

Geochemistry and stable isotope composition of fresh alkaline porphyry copper tailings: Implications on sources and mobility of elements during transport and early stages of deposition

Prevention of acid mine drainage (AMD) in sulfide-containing tailings requires the identification of the geochemical processes and element pathways in the early stages of tailing deposition. However, analyses of recently deposited tailings in active tailings impoundments are scarce because mineralogical changes occur near the detection limits of many assays. This study shows that a detailed geochemical study which includes stable isotopes of water (delta H-2, delta O-18), dissolved sulfates (delta S-34, delta O-18) and hydrochernical parameter (pH, Eh, DOC, major and trace elements) from tailings samples taken at different depths in rainy and dry seasons allows the understanding of weathering (oxidation, dissolution, sorption, and desorption), water and element pathways, and mixing processes in active tailings impoundments. Fresh alkaline tailings (pH 9.2-10.2) from the Cu-Mo porphyry deposit in El Teniente, Chile had low carbonate (0.8-1.1 Wt-% CaCO3 equivalent) and sulfide concentrations (0.8-1.3 wt.%, mainly as pyrite). In the alkaline tailings water, Mo and Cu (up to 3.9 mg/L Mo and 0.016 mg/L Cu) were mobile as MoO42- and Cu (OH)(2)(0). During the flotation, tailings water reached equilibrium with gypsum (up to 738 mg/L Ca and 1765 mg/ L SO4). The delta S-34 VS. delta O-18 covariations of dissolved sulfate (2.3 to 4.5% delta S-34 and 4.1 to 6.0 % delta O-18) revealed the sulfate sources: the dissolution of primary sulfates (12.0 to 13.2%. delta S-34, 7.4 to 10.9%.delta O-18) and oxidation of primary sulfides (-6.7 to 1.7%. delta S-34). Sedimented tailings in the tailings impoundment can be divided into three layers with different water sources, element pathways, and geochemical processes. The deeper sediments (> 1 m depth) were infiltrated by catchment water, which partly replaced the original tailings water, especially during the winter season. This may have resulted in the change from alkaline to near-neutral pH and towards lower concentrations of most dissolved elements. The neutral pH and high DOC (up to 99.4 mg/L C) of the catchment water mobilized Cu (up to 0.25 mg/L) due to formation of organic Cu complexes; and Zn (up to 130 mg/L) due to dissolution of Zn oxides and desorption). At I m depth, tailings pore water obtained during the winter season was chemically and isotopically similar to fresh tailings water (pH 9.8-10.6, 26.7-35.5 mg/L Cl, 2.3-6.0 mg/L Mo). During the summer, a vadose zone evolved locally and temporarily up to 1.2 m depth. resulting in a higher concentration of dissolved solids in the pore water due to evaporation. During periodical new deposition of fresh tailings, the geochemistry of the surface layer was geochemically similar to fresh tailings. In periods without deposition, sulfide oxidation was suggested by decreasing pH (7.7-9.5), enrichment of MoO42- and SO42-, and changes in the isotopic composition of dissolved sulfates. Further enrichment for Na, K, Cl, SO4, Mg, Cu, and Mo (up to 23.8 mg/L Mo) resulted from capillary transport towards the surface followed by evaporation and the precipitation of highly soluble efflorescent salts (e.g., mirabilite, syngenite) at the tailing surface during summer. (C) 2008 Elsevier B.V. All rights reserved.

cAMP-dependent chloride secretion mediates tubule enlargement and cyst formation by cultured mammalian collecting duct cells.

Polycystic kidney diseases result from disruption of the genetically defined program that controls the size and geometry of renal tubules. Cysts which frequently arise from the collecting duct (CD) result from cell proliferation and fluid secretion. From mCCD(cl1) cells, a differentiated mouse CD cell line, we isolated a clonal subpopulation (mCCD-N21) that retains morphogenetic capacity. When grown in three-dimensional gels, mCCD-N21 cells formed highly organized tubular structures consisting of a palisade of polarized epithelial cells surrounding a cylindrical lumen. Subsequent addition of cAMP-elevating agents (forskolin or cholera toxin) or of membrane-permeable cAMP analogs (CPT-cAMP) resulted in rapid and progressive dilatation of existing tubules, leading to the formation of cystlike structures. When grown on filters, mCCD-N21 cells exhibited a high transepithelial resistance as well as aldosterone- and/or vasopressin-induced amiloride-sensitive and -insensitive current. The latter was in part inhibited by Na(+)-K(+)-2Cl(-) cotransporter (bumetanide) and chloride channel (NPPB) inhibitors. Real-time PCR analysis confirmed the expression of NKCC1, the ubiquitous Na(+)-K(+)-2Cl(-) cotransporter and cystic fibrosis transmembrane regulator (CFTR) in mCCD-N21 cells. Tubule enlargement and cyst formation were prevented by inhibitors of Na(+)-K(+)-2Cl(-) cotransporters (bumetanide or ethacrynic acid) or CFTR (NPPB or CFTR inhibitor-172). These results further support the notion that cAMP signaling plays a key role in renal cyst formation, at least in part by promoting chloride-driven fluid secretion. This new in vitro model of tubule-to-cyst conversion affords a unique opportunity for investigating the molecular mechanisms that govern the architecture of epithelial tubes, as well as for dissecting the pathophysiological processes underlying cystic kidney diseases.

Proposal for an index to classify irrigation water quality: a case study in northeastern Brazil

One way of classifying water quality is by means of indices, in which a series of parameters analyzed are joined a single value, facilitating the interpretation of extensive lists of variables or indicators, underlying the classification of water quality. The objective of this study was to develop a statistically based index to classify water according to the Irrigation Water Quality Index (IWQI), to evaluate the ionic composition of water for use in irrigation and classify it by its source. For this purpose, the database generated during the Technology Generation and Adaptation (GAT) program was used, in which, as of 1988, water samples were collected monthly from water sources in the states of Paraíba, Rio Grande do Norte and Ceará. To evaluate water quality, the electrical conductivity (EC) of irrigation water was taken as a reference, with values corresponding to 0.7 dS m-1. The chemical variables used in this study were: pH, EC, Ca, Mg, Na, K, Cl, HCO3, CO3, and SO4. The data of all characteristics evaluated were standardized and data normality was confirmed by Lilliefors test. Then the irrigation water quality index was determined by an equation that relates the standardized value of the variable with the number of characteristics evaluated. Thus, the IWQI was classified based on indices, considering normal distribution. Finally, these indices were subjected to regression analysis. The method proposed for the IWQI allowed a satisfactory classification of the irrigation water quality, being able to estimate it as a function of EC for the three water sources. Variation in the ionic composition was observed among the three sources and within a single source. Although the water quality differed, it was good in most cases, with the classification IWQI II.

Fluid evolution at the Variscan front in the vicinity of the Aachen thrust

Quartz-carbonate-chlorite veins were studied in borehole samples of the RWTH-1 well in Aachen. Veins formed in Devonian rocks in the footwall of the Aachen thrust during Variscan deformation and associated fluid flow. Primary fluid inclusions indicate subsolvus unmixing of a homogenous H(2)O-CO(2)-CH(4)-(N(2))-Na-(K)-Cl fluid into a H(2)O-Na-(K)-Cl solution and a vapour-rich CO(2)-(H(2)O, CH(4), N(2)) fluid. The aqueous end-member composition resembles that of metamorphic fluids of the Variscan front zone with salinities ranging from 4 to 7% NaCl equiv. and maximum homogenisation temperatures of close to 400A degrees C. Pressure estimates indicate a burial depth between 4,500 and 8,000 m at geothermal gradients between 50 and 75A degrees C/26 MPa, but pressure decrease to sublithostatic conditions is also indicated, probably as a consequence of fracture opening during episodic seismic activity. A second fluid system, mainly preserved in pseudo-secondary and secondary fluid inclusions, is characterised by fluid temperatures between 200 and 250A degrees C and salinities of < 5% NaCl equiv. Bulk stable isotope analyses of fluids released from vein quartz, calcite, and dolomite by decrepitation yielded delta D(H2O) values from -89 to -113 aEuro degrees, delta(13)C(CH4) from -26.9 to -28.9aEuro degrees (VPDB) and delta(13)C(CO2) from -12.8 to -23.3aEuro degrees (VPDB). The low delta D and delta(13)C range of the fluids is considered to be due to interaction with cracked hydrocarbons. The second fluid influx caused partial isotope exchange and disequilibrium. It is envisaged that an initial short lived flux of hot metamorphic fluids expelled from the epizonal metamorphic domains of the Stavelot-Venn massif. The metamorphic fluid was focused along major thrust faults of the Variscan front zone such as the Aachen thrust. A second fluid influx was introduced from formation waters in the footwall of the Aachen thrust as a consequence of progressive deformation. Mixing of the cooler and lower salinity formation water with the hot metamorphic fluid during episodic fluid trapping resulted in an evolving range of physicochemical fluid inclusion characteristics.

Teores foliares de nutrientes em genótipos de bananeira

A diagnose foliar é bastante útil na determinação do estado nutricional das plantas. As exigências nutricionais da bananeira podem diferir em função das suas características genéticas. O trabalho objetivou determinar os teores de macro e micronutrientes na terceira folha de genótipos de bananeira. Foram selecionados 24 genótipos de bananeira (triplóides e tetraplóides), em dois ciclos de produção (1999 e 2000), do Banco Ativo de Germoplasma de Banana da Embrapa Mandioca e Fruticultura Tropical, determinando-se os teores foliares de macronutrientes (N, P, K, Ca, Mg e S) e de micronutrientes (B, Cl, Cu, Fe, Mn e Zn). Os resultados indicaram que existe variação nos teores foliares entre plantas do mesmo grupo genômico e entre os ciclos de produção, com teores médios mais elevados no segundo ciclo para N, P, Ca, Mg, S, Cu e Mn e mais baixos para K, Cl, B, Fe e Zn. O N variou de 21,6 a 28,5 g kg-1, o K de 13,7 a 30,8 g kg-1 e foram os macronutrientes com teores mais elevados nas folhas. O teor de Cl variou de 10,4 a 24,7 g kg-1, o Mn de 43 a 574 mg kg-1 e o Fe de 56 a 212 mg kg-1, sendo os micronutrientes com teores mais elevados nas folhas.

β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline/β-agonists influence muscle performance during exercise/stress in humans.