The peak force-resting membrane potential relationships of mouse fast- and slow-twitch muscle.

We endeavored to understand the factors determining the peak force-resting membrane potential (EM) relationships of isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles from mice (25°C), especially in relation to fatigue. Interrelationships between intracellular K+ activity ([Formula: see text]), extracellular K+ concentration ([K+]o), resting EM, action potentials, and force were studied. The large resting EM variation was mainly due to the variability of [Formula: see text]. Action potential overshoot-resting EM relationships determined at 4 and 8-10 mM [K+]o after short (<5 min) and prolonged (>50 min) depolarization periods revealed a constant overshoot from -90 to -70 mV providing a safety margin. Overshoot decline with depolarization beyond -70 mV was less after short than prolonged depolarization. Inexcitable fibers occurred only with prolonged depolarization. The overshoot decline during action potential trains (2 s) exceeded that during short depolarizations. Concomitant lower extracellular [Na+] and raised [K+]o depressed the overshoot in an additive manner and peak force in a synergistic manner. Raised [K+]o-induced force loss was exacerbated with transverse wire versus parallel plate stimulation in soleus, implicating action potential propagation failure in the surface membrane. Increasing stimulus pulse parameters restored tetanic force at 9-10 mM [K+]o in soleus but not EDL, indicative of action potential failure within trains. The peak tetanic force-resting EM relationships (determined with resting EM from deeper rather than surface fibers) were dynamic and showed pronounced force depression over -69 to -60 mV in both muscle types, implicating that such depolarization contributes to fatigue. The K+-Na+ interaction shifted this relationship toward less depolarized potentials, suggesting that the combined ionic effect is physiologically important during fatigue.

Amiloride modifies the progression of lithium-induced renal interstitial fibrosis.

AIM: Long-term administration of lithium has been associated with the development of a chronic interstitial fibrosis in addition to nephrogenic diabetes insipidus (NDI). Earlier studies have demonstrated that amiloride, by blocking the epithelial sodium channel ENaC and thus preventing lithium uptake into the principal cells of the collecting ducts, can partially reverse lithium-induced NDI. However, there are no long-term studies examining whether or not amiloride also modifies the progressive chronic interstitial fibrosis and tubular atrophy often evident with long-term lithium exposure. METHODS: Using an established animal model of lithium-induced chronic interstitial fibrosis, rats were treated with amiloride and lithium for 5 months following 1 month of exposure to lithium alone and compared with control animals and those given only lithium. RESULTS AND CONCLUSIONS: In this study, the 5 months of amiloride therapy partially mitigated the lithium-induced NDI and limited the further progression of lithium-induced kidney fibrosis. This improvement was associated with decreased expression of the pro-fibrotic connective tissue growth factor (CTGF), along with reduced myofibroblast infiltration and decreased collagen deposition around the distended cortical collecting ducts. This may, in part, be mediated by modifying lithium-induced alterations in ß-catenin activity through its effects on GSK-3ß.

Extracellular Ca2+-induced force restoration in K+-depressed skeletal muscle of the mouse involves an elevation of [K+]i: implications for fatigue.

We examined whether a Ca(2+)-K(+) interaction was a potential mechanism operating during fatigue with repeated tetani in isolated mouse muscles. Raising the extracellular Ca(2+) concentration ([Ca(2+)]o) from 1.3 to 10 mM in K(+)-depressed slow-twitch soleus and/or fast-twitch extensor digitorum longus muscles caused the following: 1) increase of intracellular K(+) activity by 20-60 mM (raised intracellular K(+) content, unchanged intracellular fluid volume), so that the K(+)-equilibrium potential increased by ∼10 mV and resting membrane potential repolarized by 5-10 mV; 2) large restoration of action potential amplitude (16-54 mV); 3) considerable recovery of excitable fibers (∼50% total); and 4) restoration of peak force with the peak tetanic force-extracellular K(+) concentration ([K(+)]o) relationship shifting rightward toward higher [K(+)]o. Double-sigmoid curve-fitting to fatigue profiles (125 Hz for 500 ms, every second for 100 s) showed that prior exposure to raised [K(+)]o (7 mM) increased, whereas lowered [K(+)]o (2 mM) decreased, the rate and extent of force loss during the late phase of fatigue (second sigmoid) in soleus, hence implying a K(+) dependence for late fatigue. Prior exposure to 10 mM [Ca(2+)]o slowed late fatigue in both muscle types, but was without effect on the extent of fatigue. These combined findings support our notion that a Ca(2+)-K(+) interaction is plausible during severe fatigue in both muscle types. We speculate that a diminished transsarcolemmal K(+) gradient and lowered [Ca(2+)]o contribute to late fatigue through reduced action potential amplitude and excitability. The raised [Ca(2+)]o-induced slowing of fatigue is likely to be mediated by a higher intracellular K(+) activity, which prolongs the time before stimulation-induced K(+) efflux depolarizes the sarcolemma sufficiently to interfere with action potentials.

Effects of chronic lithium administration on renal acid excretion in humans and rats.

Lithium therapy's most common side effects affecting the kidney are nephrogenic diabetes insipidus (NDI) and chronic kidney disease. Lithium may also induce a distal renal tubular acidosis. This study investigated the effect of chronic lithium exposure on renal acid-base homeostasis, with emphasis on ammonia and citrate excretion. We compared 11 individuals on long-term lithium therapy with six healthy individuals. Under basal conditions, lithium-treated individuals excreted significantly more urinary ammonia than did control subjects. Following an acute acid load, urinary ammonia excretion increased approximately twofold above basal rates in both lithium-treated and control humans. There were no significant differences between lithium-treated and control subjects in urinary pH or urinary citrate excretion. To elucidate possible mechanisms, rats were randomized to diets containing lithium or regular diet for 6 months. Similar to humans, basal ammonia excretion was significantly higher in lithium-treated rats; in addition, urinary citrate excretion was also significantly greater. There were no differences in urinary pH. Expression of the critical ammonia transporter, Rhesus C Glycoprotein (Rhcg), was substantially greater in lithium-treated rats than in control rats. We conclude that chronic lithium exposure increases renal ammonia excretion through mechanisms independent of urinary pH and likely to involve increased collecting duct ammonia secretion via the ammonia transporter, Rhcg.

Chronic interstitial fibrosis in the rat kidney induced by long-term (6-mo) exposure to lithium.

There is a lack of suitable animal models that replicate the slowly progressive chronic interstitial fibrosis that is characteristic of many human chronic nephropathies. We describe a chronic long-term (6-mo) model of lithium-induced renal fibrosis, with minimal active inflammation, which mimics chronic kidney interstitial fibrosis seen in the human kidney. Rats received lithium via their chow (60 mmol lithium/kg food) daily for 6 mo. No animals died during the exposure. Nephrogenic diabetes insipidus was established by 3 wk and persisted for the 6 mo. Following metabolic studies, the animals were killed at 1, 3, and 6 mo and the kidneys were processed for histological and immunohistochemical studies. Progressive interstitial fibrosis, characterized by increasing numbers of myofibroblasts, enhanced transforming growth factor-ß(1) expression and interstitial collagen deposition, and a minimal inflammatory cellular response was evident. Elucidation of the underlying mechanisms of injury in this model will provide a greater understanding of chronic interstitial fibrosis and allow the development of intervention strategies to prevent injury.

Exacerbated potassium-induced paralysis of mouse soleus muscle at 37°C vis-à-vis 25°C: implications for fatigue. K+ -induced paralysis at 37°C.

The main aim was to investigate the effects of raised [K+](o) on contraction of isolated non-fatigued skeletal muscle at 37°C and 25°C to assess the physiological significance of K+ in fatigue. Mouse soleus muscles equilibrated at 25°C had good mechanical stability when temperature was elevated to 37°C. The main findings at 37°C vis-à-vis 25°C were as follows. When [K+](o) was raised from 4 to 7 mM, there was greater twitch potentiation, but no significant difference in peak tetanic force. At 10 mM [K+](o) there was (1) a faster time course for the decline of peak tetanic force, (2) a greater steady-state depression of twitches and tetani, (3) an increase of peak force over 50-200 Hz (whereas it decreased at 25°C), (4) significant tetanus restoration when stimulus pulse duration increased from 0.1 to 0.25 ms and (5) greater depolarisation of layer-2 fibres, with no repolarisation of surface fibres. These combined data strengthen the proposal that a large run-down of the K+ gradient contributes to severe fatigue at physiological temperatures via depolarisation and impaired sarcolemmal excitability. Moreover, terbutaline, a ß(2)-adrenergic agonist, induced a slightly greater and more rapid, but transient, restoration of peak tetanic force at 10 mM [K+](o) at 37°C vis-à-vis 25°C. A right shift of the twitch force-stimulation strength relationship at 10 mM [K+](o) was partially reversed with terbutaline to confer the protective effect. Thus, catecholamines are likely to stimulate the Na+ -K+ pump more powerfully at 37°C to restore excitability and attenuate, but not prevent, the detrimental effects of K+.

Lithium-induced nephrogenic diabetes insipidus: renal effects of amiloride.

BACKGROUND AND OBJECTIVES: Polyuria, polydipsia, and nephrogenic diabetes insipidus have been associated with use of psychotropic medications, especially lithium. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The impact of psychotropic medications on urinary concentrating ability and urinary aquaporin 2 (AQP2) excretion was investigated after overnight fluid deprivation, and over 6 h after 40 microg of desmopressin (dDAVP), in patients on lithium (n = 45), compared with those on alternate psychotropic medications (n = 42). RESULTS: Those not on lithium demonstrated normal urinary concentrating ability (958 +/- 51 mOsm/kg) and increased urinary excretion of AQP2 (98 +/- 21 fmol/micromol creatinine) and cAMP (410 +/- 15 pmol/micromol creatinine). Participants taking lithium were divided into tertiles according to urinary concentrating ability: normal, >750 mOsm/kg; partial nephrogenic diabetes insipidus (NDI), 750 to 300 mOsm/kg; full NDI, <300 mOsm/kg. Urinary AQP2 concentrations were 70.9 +/- 13.6 fmol/micromol creatinine (normal), 76.5 +/- 10.4 fmol/micromol creatinine (partial NDI), and 27.3 fmol/micromol creatinine (full NDI). Impaired urinary concentrating ability and reduced urinary AQP2, cAMP excretion correlated with duration of lithium therapy. Other psychotropic agents did not impair urinary concentrating ability. Eleven patients on lithium were enrolled in a randomized placebo-controlled crossover trial investigating the actions of amiloride (10 mg daily for 6 wk) on dDAVP-stimulated urinary concentrating ability and AQP2 excretion. Amiloride increased maximal urinary osmolality and AQP2 excretion. CONCLUSIONS: By inference, amiloride-induced reduction of lithium uptake in the principal cells of the collecting duct improves responsiveness to AVP-stimulated translocation of AQP2 to the apical membrane of the principal cells.

Amiloride restores renal medullary osmolytes in lithium-induced nephrogenic diabetes insipidus.

In lithium-induced nephrogenic diabetes insipidus (NDI), alterations in renal medullary osmolyte concentrations have been assumed but never investigated. Amiloride can modify lithium-induced NDI, but the impact of amiloride in lithium-induced NDI on renal medullary osmolytes, aquaporins, and urea transporters is unknown and is the basis of this study. Rats fed lithium (60 mmol/kg dry food) over 4 wk developed NDI. Urine osmolality fell to 287 +/- 19 mosmol/kgH(2)O (controls 1,211 +/- 90 mosmol/kgH(2)O). Organic osmolytes in the renal medulla showed significant decreases compared with controls [inositol 221 +/- 35 to 85 +/- 10 mmol/kg protein; sorbitol 35 +/- 9 to 3 +/- 1 mmol/kg protein; glycerophosphorylcholine (GPC) 352 +/- 80 to 91 +/- 20 mmol/kg protein; and glycine betaine 69 +/- 11 to 38 +/- 38 mmol/kg protein]. Medullary urea content fell from 2,868 +/- 624 to 480 +/- 117 mmol/kg protein. Concurrent administration of amiloride (0.2 mmol/l) in the drinking water restored urine osmolality (1,132 +/- 154 mosmol/kgH(2)O), and reduced urine volume. Medullary osmolyte content were restored to control values (inositol, 232 +/- 12; sorbitol 32 +/- 6; GPC, 244 +/- 26; glycine betaine, 84 +/- 5 mmol/kg protein). Medullary urea rose to 2,122 +/- 305 mmol/kg protein. Reduced AQP2, AQP3, and urea transporter (UT-A1) expression was significantly reversed following amiloride therapy. Data presented here provide further understanding of how amiloride may substantially restore the lithium-induced impaired renal concentrating mechanism.

Organic osmolytes in the developing kidney of the Australian brush-tailed possum, Trichosurus vulpecula.

Developmental changes in the plasma and urine composition of 120 immature possums, Trichosurus vulpecula, were investigated and correlated with changes in the osmolyte composition of the kidneys. In early life, when the animal is confined in the pouch, the urine is more or less isosmotic with the plasma (300+/-70 mOsm kg(-1)), but rises steeply in animals more than 90 days old, up to 667+/-53 mOsm kg(-1) in the oldest age group. In parallel with this change there are corresponding increases in medullary osmolytes. Measured as mmol kg(-1) wet weight, sorbitol was found to increase from 2.1+/-0.8 to 9.7+/-2.2, myo-inositol from 10.9+/-6.2 to 33.6+/-11.7, while the methylamines glycerophosphorylcholine and betaine rose from 6.2+/-0.5 to 15.3+/-3.1 and 3.9+/-2.2 to 9.4+/-2.5 mmol kg(-1) wet weight respectively. Medullary taurine showed no significant changes with age in young possums, while urea increased from 11+/-4.6 to 49+/-7.1 mmol kg(-1) wet weight. These values are similar to, but less than, those found in adult kidneys, but probably further refinement of the concentrating ability of the kidney occurs after the young animal becomes independent.

Changes in fluid secretion rate alter net transepithelial transport of MRP2 and P-glycoprotein substrates in Malpighian tubules of Drosophila melanogaster.

The effects of stimulants of fluid secretion on net transepithelial transport of the MRP2 substrate Texas Red and the p-glycoprotein substrate daunorubicin were examined in Malpighian tubules of Drosophila melanogaster. Fluid secretion rates were determined using the Ramsay assay and secreted fluid concentrations of Texas Red and daunorubicin were determined using a microfluorometric technique. Nanoliter droplets of secreted fluid were collected in optically flat glass capillaries and dye concentration was determined from fluorescence intensity measured by confocal laser scanning microscopy. Net transepithelial flux of each compound was then calculated as the product of its concentration in the secreted fluid and the fluid secretion rate. Net transepithelial flux of Texas Red increased when fluid secretion was stimulated by tyramine, cyclic AMP or hypoosmotic saline. Net flux decreased when fluid secretion rate of cAMP-stimulated tubules was reduced by elevating saline osmolality with sucrose. Net transepithelial flux of daunorubicin increased when fluid secretion was stimulated by cAMP. Significant increases in dye flux were seen only when the dyes were present at concentrations close to or greater than the concentration required for half maximal transport. Regression analyses showed that 57- 88% of the change in dye flux was attributable to the change in fluid secretion rate when tubules were stimulated with cAMP, cGMP, or tyramine. The results do not suggest that the effects of tyramine and cAMP are mediated through changes in transepithelial potential, nor do they indicate the direct effects of the stimulants on MRP2-like or p-glycoprotein-like transporters (e.g., via protein kinases). Instead, the results suggest that increases in fluid secretion rate minimize diffusive backflux of these dyes and, thus, facilitate higher rates of net transepithelial transport indirectly.

Ultraviolet photography of the in vivo human cornea unmasks the Hudson-Stähli line and physiologic vortex patterns.

PURPOSE: To perform ultraviolet (UV) macrophotography of the normal in vivo human cornea, establishing biometric data of the major component of UV absorption for comparison with the Hudson-Stähli (HS) line, the distribution of iron demonstrated by Perl stain, and cases of typical amiodarone keratopathy. METHODS: Nonrandomized comparative case series of UV photographs of 76 normal corneas (group 1) and 16 corneas with typical amiodarone keratopathy (group 2). Image-analysis software was used to grade the major component of UV absorption for slope and the coordinates of its points of intersection with the vertical corneal meridian and inflection. RESULTS: In group 1 the major component had a mean slope of 5.8 degrees, sloping down from nasal to temporal cornea. The mean coordinates of points of intersection with the vertical corneal meridian and inflection were (0, 0.30) and (0.02, 0.31), respectively. No significant differences between groups 1 and 2 were found for slope (P = 0.155), intersection with the vertical corneal meridian (P = 0.517), and point of inflection (P = 0.344). The major component of UV absorption was consistent with published characteristics of the HS line, and coincidence of UV absorption and Perl-stained iron was demonstrated in one corneal button. A vortex pattern of UV absorption was observed in all corneas. CONCLUSIONS: UV photography demonstrates subclinical corneal iron, confirming its deposition in an integrated HS line/vortex pattern. Coincident iron and amiodarone deposition occurs in amiodarone keratopathy.

Lithium-induced reduction in urinary concentrating ability and urinary aquaporin 2 (AQP2) excretion in healthy volunteers.

BACKGROUND: Lithium therapy is associated with the development of nephrogenic diabetes inspidus. Experimentally, lithium inhibits arginine vasopressin (AVP)-stimulated translocation of cytoplasmic aquaporin 2 (AQP2) to the apical membrane. Clinically, the actions of lithium on renal tubular function are less clearly established. This study examined the effects of four weeks of lithium therapy on desmopressin (dDAVP)-stimulated urinary concentrating ability in healthy volunteers. METHODS: Eleven healthy volunteers underwent baseline urinary concentrating ability studies which were repeated following 4 weeks therapy with lithium carbonate (250 mg twice a day). Urinary osmolality, urinary AQP2 and cyclic adenosine monophosphate (cAMP) levels were measured following overnight fluid deprivation and after the administration of 40 microg of dDAVP. Baseline values were compared with results after 4 weeks of lithium therapy. RESULTS: Four weeks of lithium therapy reduced dDAVP-stimulated urinary concentrating ability (996 +/- 27 to 945 +/- 26 mOsm/kg) (P < 0.05) and this was associated with significant reduction in urinary AQP2 excretion (99.2 +/- 10.0 to 77.8 +/- 7.4 fmol/micromol creatinine) (P < 0.05) and urinary cAMP excretion (3188 +/- 376 to 2212 +/- 378 units) (P < 0.01). CONCLUSION: Four weeks of lithium therapy in healthy volunteers produced a small but significant reduction in dDAVP-stimulated urinary concentrating ability, which appears to be mediated by the inhibition of AVP-stimulated translocation of cytoplasmic AQP2 to the collecting tubule apical membrane via inhibition of adenyl cyclase.

Aquaporin expression in normal human kidney and in renal disease.

Aquaporins (AQPs), membrane-inserted water channel proteins, play a highly important role in the reabsorption of water from the renal tubular fluid. Experimentally, both in rats and mice, failure to insert functional AQP molecules into renal tubular membranes leads to nephrogenic diabetes insipidus. In humans, most forms of renal disease lead to a reduction in the water handling capacity of the kidney. AQP distribution in various forms of human renal disease has not been documented. Immunohistochemical studies of biopsy samples from a wide range of renal diseases revealed a substantial and striking upregulation of AQP-1 in the glomeruli of most diseased kidneys. AQP-1 expression remained prominent in proximal tubules in all lesions. In contrast, there was judged qualitatively to be a reduction in the amounts of AQP-2 and AQP-3 expression, especially in lesions with substantial interstitial fibrosis and nephron loss, as compared with a healthy region of normal kidneys. The results were quantitatively confirmed by real-time reverse transcriptase-PCR. This is the first documentation of altered AQP expression in human renal disease. The significance of the increased AQP-1 expression requires further studies.

Urinary aquaporin-2 levels in healthy volunteers.

Renal water handling is regulated by the release of arginine vasopressin (AVP) and the subsequent insertion of aquaporin 2 (AQP2) in the apical membrane of collecting duct cells. This in turn increases the membrane permeability to water and the passive reabsorption of water down the concentration gradient present in the medulla. Aquaporin 2 can be detected in the urine under conditions of antidiuresis. We wish to validate an assay for urinary AQP2. Fourteen volunteers participated in studies of water loading and water deprivation followed by the administration of 1-deamino-8-D-arginine vasopressin (dDAVP). Urine osmolality was measured by vapour pressure osmometry. Urinary AQP2 was measured by using a chemiluminescent assay. Baseline correlations between serum AVP levels, urinary osmolality and urinary AQP2 levels were not significant. Following the administration of dDAVP, a positive correlation between urine osmolality and urinary AQP2 was evident (r = 0.762). For specific conditions where renal water retention is stimulated via AVP, urinary AQP2 measurements provide a reproducible measurement of the renal actions of AVP.