Recovery Half-time Research Articles

Kinetics of desensitization and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells.

Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human alpha4beta2-nicotinic acetylcholine receptors (nAChR) heterologously expressed in the SH-EP1 human epithelial cell line. Whole-cell patch clamp recordings were performed to evaluate alpha4beta2-nAChR currents. Application of 0.1 micromol/L nicotine or 1 mmol/L acetylcholine (ACh) for 1 s or longer induced two phases, with time constants of approximately 70 and approximately 700 ms, for the onset of alpha4beta2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of alpha4beta2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs(1) from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human alpha4beta2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings(2) using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. These findings indicate the existence of alpha4beta2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.

Becoming stable and strong: The interplay between vinculin exchange dynamics and adhesion strength during adhesion site maturation

The coordinated formation and release of focal adhesions is necessary for cell attachment and migration. According to current models, these processes are caused by temporal variations in protein composition. Protein incorporation into focal adhesions is believed to be controlled by phosphorylation. Here, we tested the exchange dynamics of GFP-vinculin as marker protein of focal adhesions using the method of Fluorescence Recovery After Photobleaching. The relevance of the phosphorylation state of the protein, the age of focal adhesions and the acting force were investigated. For stable focal adhesions of stationary keratinocytes, we determined an exchangeable vinculin fraction of 52% and a recovery halftime of 57 s. Nascent focal adhesions of moving cells contained a fraction of exchanging vinculin of 70% with a recovery halftime of 36 s. Upon maturation, mean saturation values and recovery halftimes decreased to levels of 49% and 42 s, respectively. Additionally, the fraction of stably incorporated vinculin increased with cell forces and decreased with vinculin phosphorylation within these sites. Experiments on a nonphosphorylatable vinculin mutant construct at phosphorylation site tyr1065 confirmed the direct interplay between phosphorylation and exchange dynamics of adhesion proteins during adhesion site maturation.

Dynamic Organization of Aminoacyl-tRNA Synthetase Complexes in the Cytoplasm of Human Cells

The localization in space and in time of proteins within the cytoplasm of eukaryotic cells is a central question of the cellular compartmentalization of metabolic pathways. The assembly of proteins within stable or transient complexes plays an essential role in this process. Here, we examined the subcellular localization of the multi-aminoacyl-tRNA synthetase complex in human cells. The sequestration of its components within the cytoplasm rests on the presence of the eukaryotic-specific polypeptide extensions that characterize the human enzymes, as compared with their prokaryotic counterparts. The cellular mobility of several synthetases, assessed by measuring fluorescence recovery after photobleaching, suggested that they are not freely diffusible within the cytoplasm. Several of these enzymes, isolated by tandem affinity purification, were copurified with ribosomal proteins and actin. The capacity of aminoacyl-tRNA synthetases to interact with polyribosomes and with the actin cytoskeleton impacts their subcellular localization and mobility. Our observations have conceptual implications for understanding how translation machinery is organized in vivo.

Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an "open" conformation, or "lock" the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.

Sex differences in calf muscle hemoglobin oxygen saturation in patients with intermittent claudication

We tested the hypotheses that women have greater impairment in calf muscle hemoglobin oxygen saturation (StO(2)) in response to exercise than men, and that the sex-related difference in calf muscle StO(2) would partially explain the shorter claudication distances of women. The study comprised 27 men and 24 women with peripheral arterial disease limited by intermittent claudication. Patients were characterized on calf muscle StO(2) before, during, and after a graded treadmill test, as well as on demographic and cardiovascular risk factors, ankle-brachial index (ABI), ischemic window, initial claudication distance (ICD), and absolute claudication distance (ACD). Women had a 45% lower ACD than men (296 +/- 268 m vs 539 +/- 288 m; P = .001) during the treadmill test. Calf muscle StO(2) declined more rapidly during exercise in women than in men; the time to reach minimum StO(2) occurred 54% sooner in women (226 +/- 241 vs 491 +/- 426 seconds; P = .010). The recovery time for calf muscle StO(2) to reach the resting value after treadmill exercise was prolonged in women (383 +/- 365 vs 201 +/- 206 seconds; P = .036). Predictors of ACD included the time from start of exercise to minimum calf muscle StO(2), the average rate of decline in StO(2) from rest to minimum StO(2) value, the recovery half-time of StO(2), and ABI (R(2) = 0.70; P < .001). The ACD of women remained lower after adjusting for ABI (mean difference, 209 m; P = .003), but was no longer significantly lower (mean difference, 72 m; P = .132) after further adjustment for the StO(2) variables for the three calf muscles. In patients limited by intermittent claudication, women have lower ACD and greater impairment in calf muscle StO(2) during and after exercise than men, the exercise-mediated changes in calf muscle StO(2) are predictive of ACD, and women have similar ACD as men after adjusting for calf StO(2) and ABI measures.

Phosphorus-31 magnetic resonance spectroscopy of skeletal muscle in maternally inherited diabetes and deafness A3243G mitochondrial mutation carriers

To investigate high-energy phosphate metabolism in striated skeletal muscle of patients with Maternally Inherited Diabetes and Deafness (MIDD) syndrome. In 11 patients with the MIDD mutation (six with diabetes mellitus [DM] and five non-DM) and eight healthy subjects, phosphocreatine (PCr) and inorganic phosphate (Pi) in the vastus medialis muscle was measured immediately after exercise using (31)P-magnetic resonance spectroscopy (MRS). The half-time of recovery (t1/2) of monoexponentially fitted (PCr+Pi)/PCr was calculated from spectra obtained every 4 seconds after cessation of exercise. A multiple linear regression model was used for statistical analysis. Patients with the MIDD mutation showed a significantly prolonged t1/2 (PCr+Pi)/PCr after exercise as compared to controls (13.6+/-3.0 vs. 8.7+/-1.3 sec, P = 0.01). No association between the presence of DM and t1/2 (PCr + Pi)/PCr was found (P = 0.382). MIDD patients showed impaired mitochondrial oxidative phosphorylation in skeletal muscle shortly after exercise, irrespective of the presence of DM.

JNK-mediated Phosphorylation of Paxillin in Adhesion Assembly and Tension-induced Cell Death by the Adenovirus Death Factor E4orf4

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.

Increased Recovery Rates of Phosphocreatine and Inorganic Phosphate after Isometric Contraction in Oxidative Muscle Fibers and Elevated Hepatic Insulin Resistance in Homozygous Carriers of the A-allele ofFTOrs9939609

Recent studies identified the rs9939609 A-allele of the FTO (fat mass and obesity associated) gene as being associated with obesity and type 2 diabetes. We studied the role of the A-allele in the regulation of peripheral organ functions involved in the pathogenesis of obesity and type 2 diabetes. Forty-six young men underwent a hyperinsulinemic euglycemic clamp with excision of skeletal muscle biopsies, an iv glucose tolerance test, 31phosphorous magnetic resonance spectroscopy, and 24-h whole body metabolism was measured in a respiratory chamber. The FTO rs9939609 A-allele was associated with elevated fasting blood glucose and plasma insulin, hepatic insulin resistance, and shorter recovery half-times of phosphocreatine and inorganic phosphate after exercise in a primarily type I muscle. These relationships--except for fasting insulin--remained significant after correction for body fat percentage. The risk allele was not associated with fat distribution, peripheral insulin sensitivity, insulin secretion, 24-h energy expenditure, or glucose and fat oxidation. The FTO genotype did not influence the mRNA expression of FTO or a set of key nuclear or mitochondrially encoded genes in skeletal muscle during rest. Increased energy efficiency--and potentially increased mitochondrial coupling--as suggested by faster recovery rates of phosphocreatine and inorganic phosphate in oxidative muscle fibers may contribute to the increased risk of obesity and type 2 diabetes in homozygous carriers of the FTO A-risk allele. Hepatic insulin resistance may represent the key metabolic defect responsible for mild elevations of fasting blood glucose associated with the FTO phenotype.

Forchlorfenuron Alters Mammalian Septin Assembly, Organization, and Dynamics

Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

Niemann-Pick C1-like 1 (NPC1L1) is a recently identified protein that mediates intestinal cholesterol absorption and regulates biliary cholesterol excretion. The itineraries and kinetics of NPC1L1 trafficking remain uncertain. In this study, we have visualized movement of NPC1L1-enhanced green fluorescent protein (NPC1L1-EGFP) and cholesterol analogs in hepatoma cells. At steady state, about 42% of NPC1L1 resided in the transferrin (Tf)-positive, sterol-enriched endocytic recycling compartment (ERC), whereas time-lapse microscopy demonstrated NPC1L1 traffic between the plasma membrane and the ERC. Fluorescence recovery after photobleaching revealed rapid recovery (half-time approximately 2.5 min) of about 35% of NPC1L1 in the ERC, probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells, NPC1L1 resided almost exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between the cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1-mediated cellular sterol uptake.

Pilot Study of Peripheral Muscle Function in Primary Biliary Cirrhosis: Potential Implications for Fatigue Pathogenesis

Primary biliary cirrhosis (PBC) is characterized in 95% of patients by autoantibody responses directed against the mitochondrial antigen pyruvate dehydrogenase complex (PDC). Although anti-PDC inhibits PDC function in vitro, mitochondrial function in vivo in PBC has not been examined. (31)P magnetic resonance spectroscopy was performed in PBC patients (n = 15) and fatigued (chronic fatigue syndrome/myalgic encephalomyelitis, n = 8), cholestatic (primary sclerosing cholangitis [PSC], n = 4), and normal (n = 8) controls to define mitochondrial function and pH regulation in peripheral muscle during exercise at 25% and 35% of maximum voluntary contraction. Normal, chronic fatigue syndrome/myalgic encephalomyelitis, and PSC subjects all showed close correlation between kinetics of adenosine diphosphate (ADP) and phosphocreatine (PCr) recovery after low-impact exercise, reflecting the normal tight regulation of PCr "response" by mitochondria to ADP "drive." This relationship was lost in PBC patients, indicating mitochondrial dysfunction (normal r(2) = 0.78, P < .005; PBC r(2) = 0.007, P = ns). Ratio between PCr and ADP recovery half-times (constant in controls, indicating normal mitochondrial responsivity) was significantly elevated in PBC patients (but not PSC) and was associated with anti-PDC levels. At higher levels of exercise PBC (but not PSC) patients showed excess muscle acidosis, with pH correlating with elevation of PCr/ADP recovery ratio, indicating a link to mitochondrial dysfunction. PBC patients alone also showed significant prolongation of muscle pH recovery time after exercise (unrelated to mitochondrial function), which correlated with clinical fatigue. PBC patients exhibit a variable degree of muscle mitochondrial dysfunction that manifests as excess acidosis after exercise. The extent to which patients can recover rapidly from acidosis appears to determine whether they are clinically fatigued.

Lower Intrinsic ADP-Stimulated Mitochondrial Respiration Underlies In Vivo Mitochondrial Dysfunction in Muscle of Male Type 2 Diabetic Patients

OBJECTIVE—A lower in vivo mitochondrial function has been reported in both type 2 diabetic patients and first-degree relatives of type 2 diabetic patients. The nature of this reduction is unknown. Here, we tested the hypothesis that a lower intrinsic mitochondrial respiratory capacity may underlie lower in vivo mitochondrial function observed in diabetic patients.RESEARCH DESIGN AND METHODS—Ten overweight diabetic patients, 12 first-degree relatives, and 16 control subjects, all men, matched for age and BMI, participated in this study. Insulin sensitivity was measured with a hyperinsulinemic-euglycemic clamp. Ex vivo intrinsic mitochondrial respiratory capacity was determined in permeabilized skinned muscle fibers using high-resolution respirometry and normalized for mitochondrial content. In vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time after exercise using 31P-magnetic resonance spectroscopy.RESULTS—Insulin-stimulated glucose disposal was lower in diabetic patients compared with control subjects (11.2 ± 2.8 vs. 28.9 ± 3.7 μmol · kg−1 fat-free mass · min−1, respectively; P = 0.003), with intermediate values for first-degree relatives (22.1 ± 3.4 μmol · kg−1 fat-free mass · min−1). In vivo mitochondrial function was 25% lower in diabetic patients (P = 0.034) and 23% lower in first-degree relatives, but the latter did not reach statistical significance (P = 0.08). Interestingly, ADP-stimulated basal respiration was 35% lower in diabetic patients (P = 0.031), and fluoro-carbonyl cyanide phenylhydrazone–driven maximal mitochondrial respiratory capacity was 31% lower in diabetic patients (P = 0.05) compared with control subjects with intermediate values for first-degree relatives.CONCLUSIONS—A reduced basal ADP-stimulated and maximal mitochondrial respiratory capacity underlies the reduction in in vivo mitochondrial function, independent of mitochondrial content. A reduced capacity at both the level of the electron transport chain and phosphorylation system underlies this impaired mitochondrial capacity.

Vanishing Act

Potassium channels play an important role in the regulation of vascular smooth muscle tone and, thus, contribute to the regulation of blood pressure, blood flow, and microvascular exchange.1 These channels importantly participate in the determination of vascular smooth muscle cell (VSMC) membrane potential,1,2 which, in turn, controls Ca2+ influx through voltage-gated Ca2+ channels1,2 and has been implicated in the control of Ca2+ release and Ca2+ sensitivity of VSMCs.1 VSMCs express a diverse array of K+ channels that contribute to the regulation of VSMC function,1 including ≥1 type of vascular ATP-sensitive K+ (KATP) channels.2,3 KATP channels consist of a tetramer of α-pore–forming subunits from the KIR6.X family of inwardly rectifying K+ channels, along with complimentary sulfonylurea receptor (SUR) subunits that are members of the ATP-binding cassette family of proteins.2 The SUR subunits are essential for normal trafficking of KATP channels, modulate channel function, and are the binding sites for sulfonylurea antagonists of these channels, such as glibenclamide.2 Vascular smooth muscle KATP channels appear to be composed of KIR6.1 and SUR2B subunits,2 although some VSMCs may also express KATP channels composed of KIR6.2/SUR2B.3 As originally described, KATP channels open during hypoxia or ischemic conditions when cellular ATP levels fall, decreasing cell excitability and protecting energy-limited cells.2 However, both in vitro and in vivo studies suggest that VSMC KATP channels may be open under resting conditions and contribute to the regulation of VSMC membrane potential and vascular tone.1,2 Importantly, the activity of KATP channels can be modulated by a number of physiologically relevant vasoactive substances and conditions. As their name implies, KATP channels may be activated by decreases in intracellular …

Predicting Effects of Exercise Training in Patients With Heart Failure Secondary to Ischemic or Idiopathic Dilated Cardiomyopathy

The purpose of this study was to investigate which patient characteristics may predict training effects on maximal and submaximal exercise performance in patients with heart failure. Together with commonly used clinical and performance-related variables, oxygen uptake kinetics during exercise recovery were included as possible predictors. Fifty patients with heart failure (New York Heart Association class II or III) performed a 12-week training program (cycle interval and resistance training). Training effects were expressed as changes in peak oxygen uptake (Vo(2)), Vo(2) at ventilatory threshold (VT), and the time constant of Vo(2) recovery after submaximal exercise (tau-rec). After training, peak Vo(2), Vo(2) at VT, and tau-rec improved significantly, with a wide variety in training responses. Changes in peak Vo(2) were related to changes in VT (r = 0.79, p <0.001), but both changes were not related to changes in tau-rec. Using multivariate regression analyses, post-training changes in peak Vo(2) could be predicted by recovery halftime of peak Vo(2) (T1/2), peak Vo(2) (percentage of predicted), and peak respiratory exchange ratio (R(2) = 36%). Post-training changes in VT could be predicted by T1/2 and VT (predicted) (R(2) = 29%), whereas changes in tau-rec could be predicted only by tau-rec at baseline (R(2) = 34%). In conclusion, oxygen recovery kinetics after maximal and submaximal exercise substantially add to the prediction of training effects in patients with heart failure, presumably because of their relations with, respectively, central and peripheral impairments of exercise capacity. However, the explained variance in training effects is not sufficient to make a definite distinction between training responders and nonresponders.

The Pleckstrin Homology Domain of the Arf6-specific Exchange Factor EFA6 Localizes to the Plasma Membrane by Interacting with Phosphatidylinositol 4,5-Bisphosphate and F-actin

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

Calf muscle hemoglobin oxygen saturation characteristics and exercise performance in patients with intermittent claudication

This study was conducted to determine the association between the characteristics of calf muscle hemoglobin oxygen saturation (StO(2)) and exercise performance in patients with intermittent claudication. The study comprised 39 patients with peripheral arterial disease limited by intermittent claudication. Patients were characterized on calf muscle StO(2) before, during, and after a graded treadmill test, as well as on demographic and cardiovascular risk factors, ankle-brachial index (ABI), ischemic window, initial claudication distance (ICD), and absolute claudication distance (ACD). Calf muscle StO(2) decreased 72%, from 55% +/- 18% (mean +/- SD) saturation at rest to the minimum value of 17% +/- 19% saturation attained 459 +/- 380 seconds after the initiation of exercise. After exercise, recovery half-time of calf muscle StO(2) was attained at 129 +/- 98 seconds, whereas full recovery to the resting value was reached at 225 +/- 140 seconds. After adjusting for sex, race, and grouping according to the initial decline constant in calf muscle StO(2) during exercise, the exercise time to minimum calf muscle StO(2) was correlated with the ischemic window (r = -0.493, P = .002), ICD (r = 0.339, P = .043), and ACD (r = 0.680, P < .001). After treadmill exercise, the recovery half-time of calf muscle StO(2) was correlated with the ischemic window (r = 0.531, P < .001), ICD (r = -0.598, P < .001), and ACD (r = -0.491, P = .003). In patients limited by intermittent claudication, shorter ICD and ACD values are associated with reaching a minimum value in calf muscle StO(2) sooner during treadmill exercise and with having a delayed recovery in calf muscle StO(2) after exercise.

The Insulin-Sensitizing Effect of Rosiglitazone in Type 2 Diabetes Mellitus Patients Does Not Require Improved in Vivo Muscle Mitochondrial Function

Our objective was to investigate whether improved in vivo mitochondrial function in skeletal muscle and intramyocellular lipids (IMCLs) contribute to the insulin-sensitizing effect of rosiglitazone. Eight overweight type 2 diabetic patients (body mass index = 29.3 +/- 1.1 kg/m(2)) were treated with rosiglitazone for 8 wk. Before and after treatment, insulin sensitivity was determined by a hyperinsulinemic euglycemic clamp. Muscular mitochondrial function (half-time of phosphocreatine recovery after exercise) and IMCL content were measured by magnetic resonance spectroscopy. Insulin sensitivity improved after rosiglitazone (glucose infusion rate: 19.9 +/- 2.8 to 24.8 +/- 2.1 micromol/kg.min; P < 0.05). In vivo mitochondrial function (phosphocreatine recovery half-time: 23.8 +/- 3.5 to 20.0 +/- 1.7 sec; P = 0.23) and IMCL content (0.93 +/- 0.18% to 1.37 +/- 0.40%; P = 0.34) did not change. Interestingly, the changes in PCr half-time correlated/tended to correlate with changes in fasting insulin (R(2) = 0.50; P = 0.05) and glucose (R(2) = 0.43; P = 0.08) levels. Changes in PCr half-time did not correlate with changes in glucose infusion rate (R(2) = 0.08; P = 0.49). The rosiglitazone-enhanced insulin sensitivity does not require improved muscular mitochondrial function.

Effects of dry needling at tender points for neck pain (Japanese: katakori): near-infrared spectroscopy for monitoring muscular oxygenation of the trapezius

BackgroundNeck pain (katakori) is a common symptom in adult Japanese people. However, the pathophysiological aspect of this condition has not been well documented to date. The purpose of this study was to investigate the effects of tender point dry needling to the trapezius muscles and the resultant changes in muscular hemodynamics. Methods“Neck pain” patients were defined as those complaining of dull pain or discomfort mainly along the trapezius muscles without serious spinal or shoulder disorders. We used near-infrared spectroscopy to monitor the changes of oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb) of the trapezius muscles and a Visual Analogue Scale (VAS) to assess subjective neck pain intensity. Experiment I: Nine subjects with “neck pain” and four control subjects were recruited. Total hemoglobin (Hb) and SdO2 [= oxyHb/(oxyHb + deoxyHb)] were measured before and immediately after needling for 15 min. We compared these parameters and VAS before and immediately after needling. Experiment II: Thirteen subjects with “neck pain” were instructed to perform isometric contraction of their trapezius muscles for 1 min; the half-recovery time of SdO2 (defined as TR) was measured. After that, all subjects underwent needling. On the next day, we repeated the measurements of TR after the same voluntary contraction of the trapezius muscle in the same patients. We compared TR and VAS before and on the day after needling. ResultsExperiment I: All subjects with “neck pain” reported significant pain relief (P = 0.0147) measured by VAS immediately after needling, but total Hb and SdO2 exhibited no significant change after needling. Experiment II: TR was shortened on the day after needling in 10 of 13 patients (P = 0.0043), and neck pain was decreased in 12 patients (P = 0.0158). ConclusionsAfter dry needling, total Hb and SdO2 did not change in real time, but TR was shortened on the next day. These results showed that the shortening of TR would provide a measure by which to assess the effectiveness of treatment for neck pain.

Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.

Pre‐ and postsynaptic excitation and inhibition at octopus optic lobe photoreceptor terminals; implications for the function of the ‘presynaptic bags’

Synaptic transmission was examined in the plexiform zone of Octopus vulgaris optic lobes using field-potential recording from optic lobe slices. Stimulation of the optic nerve produced pre- and postsynaptic field potentials. Transmission was abolished in calcium-free seawater, L- glutamate or the AMPA/Kainate receptor blocker CNQX (EC(50), 40 microm), leaving an intact presynaptic field potential. ACh markedly reduced or blocked and d-tubocurarine augmented both pre- and postsynaptic field potentials, while alpha-bungarotoxin and atropine were without effect. Paired-pulse stimulation showed short-term depression of pre- and postsynaptic components with a half-time of recovery of approximately 500 ms. The depression was partially relieved in the presence of d-tubocurarine (half-time of recovery, 350 ms). No long-term changes in synaptic strength were induced by repetitive stimulation. A polyclonal antibody raised against a squid glutamate receptor produced positive staining in the third radial layer of the plexiform zone. No positive staining was observed in the other layers. Taking into account previous morphological data and our results, we propose that the excitatory terminations of the photoreceptors are in the innermost layer of the plexiform zone where the transmitter is likely to be glutamate and postsynaptic receptors are AMPA/kainate-like. Thus, the function of the terminal bags is to provide a location for a presynaptic cholinergic inhibitory shunt. The results imply that this arrangement provides a temporal filter for visual processing and enhances the perception of moving vs. stationary objects.