K+ channels permit more than one ion within their conducting pathway at any given moment and show a saturating single-file behavior. The conduction of Rb+ shows an unusual behavior, a so-called “Rb+ anomaly,” and it has been used to probe the mechanism of the ion conduction through K+-selective channels. Under the bi-ionic condition of K+ and Rb+, we carried out patch-clamp single-channel current measurements in MaxiK+ channels from mouse submandibular acinar cells. Keeping only K+ on one side of the membrane while varying fractional Rb+ concentration on the opposite, we had a series of current-voltage relationships. It showed a characteristic inflection at which the ion conductance was divided into two components, one ascribed to pure K+ conduction and the other to K+ and Rb+ bi-ionic conduction. By analyzing the latter, we depicted that (1) the bi-ionic conductance showed a characteristic reduction curve as the Rb+ fractional concentration increased; (2) Rb+ can bind the channel more tightly when it accesses from the outside than from the inside. Thus we conclude that such asymmetry of the Rb+ binding determines the pattern of bi-ionic conductance reduction in K-selective channels.
This study examined the effects of a different number of eccentric contractions (ECs) on histological characteristics, surface electromyogram (EMG) parameters (integral EMG, iEMG; muscle fiber conduction velocity, MFCV; and action potential waveform), and isometric peak torque using the rat EC model. Male Wistar rats (n = 40) were anesthetized, and ECs were initiated in the tibialis anterior muscle via electrical stimulation while the muscle was being stretched by electromotor. The rats were grouped according to the number of ECs (EC1, EC5, EC10, EC20, EC30, EC40, and EC100). Three days after the ECs, surface EMG signals and isometric peak torque were measured during evoked twitch contractions via electrical stimulation of the peroneal nerve. The muscle damage was evaluated from hematoxylin-eosin (HE) stained cross sections as a relative number of damaged fibers to intact fibers. Intense histological muscle damage (approximately 50% to 70% of the fiber), loss of isometric peak torque, disturbance of action potential waveform, and depression of iEMG (approximately -60% to -70%) were observed at EC20, EC30, EC40, and EC100. On the other hand, the MFCV did not change in any EC group. Although muscle damage and pathological surface EMG signals were not found at EC10, isometric peak torque was reduced significantly. In conclusion, the extent of histological muscle damage is not proportionally related to the number of ECs. Muscle damage was reflected by iEMG and action potential waveforms, but not by MFCV, which remained unaffected even though approximately 50% to 70% of the fiber demonstrated injury.
The oligomeric structure and the residual propeptide are distinct characteristics of cathepsin C from other members in the papain superfamily. In this study, we examined the physiological role of the cathepsin C propeptide. The stable overexpression of cathepsin C propeptide significantly decreased the activities of intestinal alkaline phosphatase (IAP) and sucrase in human Caco-2 intestinal epithelial cells, whereas it did not change the proliferation and cathepsin C activity. The overexpression of cathepsin C propeptide significantly decreased the amounts of IAP protein in differentiated Caco-2 cells, compared with the transfection of mock vector, whereas the amounts of IAP transcripts were not changed. Pulse-chase analysis confirmed that the reduction in IAP activity was due to an increase in IAP degradation, but not a decrease in IAP expression. For the mechanism of the enhanced IAP degradation, we identified proteins interacting with cathepsin C propeptide in Caco-2 cells by immunoprecipitation and mass spectrometry. Cathepsin C propeptide interacted with proteins with a molecular mass of approximately 70 kDa, including IAP and heat shock cognate protein 70. Our present results suggest that the propeptide of cathepsin C may stimulate the sorting to the lysosome, at least in part, contributing to the degradation of IAP in Caco-2 cells.
We investigated the pivotal roles of glucose and its transporter in the regulation of mechanical activity of isolated rat thoracic ducts and then examined whether mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) are involved in those responses. In the absence of extracellular glucose, the thoracic ducts showed pump activity during 120 min. Extracellular glucose caused a dose-dependent increase in the frequency of pump activity and a constriction in the thoracic ducts. Pump activity of the thoracic ducts in 0 mm glucose was completely inhibited in the presence of chlorogenic acid (an inhibitor of glucose-6-phosphatase). Cytochalasin B, an inhibitor of facilitative glucose transporter (GLUT), or phlorizin, an inhibitor of sodium-dependent glucose cotransporter (SGLT), significantly reduced the frequency of pump activity and dilated the thoracic ducts. A decrease in the frequency of pump activity induced by 5-hydroxydecanoate (5-HD, a selective blocker of mitoK(ATP)) was completely reversed by ruthenium red (an inhibitor of Ca(2+) uniporter in mitochondria). Diazoxide (a selective opener of mitoK(ATP)) significantly increased the frequency of pump activity. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, a protonophore of mitochondrial proton pump action) significantly reduced the frequency of pump activity and dilated the thoracic ducts. Collectively, these findings suggest that glucose derived from intracellular glycogen and/or through GLUT/SGLT in lymphatic smooth muscles contributes to the regulation of the pump activity of isolated rat thoracic ducts, and that mitoK(ATP) in the cells may partially serve as a modulator of the mechanical functions associated with mitochondrial Ca(2+) uptake.
The mechanisms of the protective effect conferred by heat shock preconditioning (HS) are currently unknown. The purpose of this study was to determine the effect of HS on muscle injury after downhill running and to address the mechanism of the effect. Female Wistar rats were assigned to three groups: HS, downhill running (E), and downhill running after heat shock preconditioning (HS + E). The HS and HS + E rats were placed in a heat chamber for 60 min (ambient temperature 42 +/- 1.0 degrees C) 48 h before downhill running. Reactive oxygen species (ROS) scavenging activity was determined by electron spin resonance (ESR), and heat shock protein 72 (HSP72) mRNA expression was measured in rat quadriceps femoris. Leukocyte infiltration and degenerated muscle fibers were determined histopathologically. ROS scavenging activity significantly increased at 3 days after HS (151 +/- 18%) and HSP72 mRNA expression increased immediately after HS (1750 +/- 1914%). No decrease in ROS scavenging activity was observed in the HS + E rats at 2 days after exercise compared with the E rats (102 +/- 9% vs. 79 +/- 5%). Degenerated muscle fibers in HS + E rats were significantly less than in E rats at 2, 3, and 7 days after exercise (0.8 +/- 1.0 vs. 2.8 +/- 1.6, 0.8 +/- 1.0 vs. 1.8 +/- 1.6, 0 vs. 0.3 +/- 0.6, respectively). These data demonstrated that HS can reduce muscle injury after downhill running, and this effect may be mediated by increased ROS scavenging activity. Furthermore, HS may protect the antioxidant defense system in skeletal muscle by enhancing the adaptive HSP72 mRNA response.
We tested the hypothesis that the biphasic ventilatory response to poikilocapnic hypoxia shows circadian variation and contribution of histamine H1 receptors in mice. Initial increases in ventilation were augmented during dark periods. H1 receptors had no major relationship with circadian variation, but affected the declined phase.
The Purkinje fibers are located in the ventricular walls of the heart, just beneath the endocardium and conduct excitation from the right and left bundle branches to the ventricular myocardium. Recently, anatomists succeeded in photographing the Purkinje fibers of a sheep, which clearly showed the mesh structure of the Purkinje fibers. In this study, we present a technique for modeling the mesh structure of Purkinje fibers semiautomatically using an extended L-system. The L-system is a formal grammar that defines the growth of a fractal structure by generating rules (or rewriting rules) and an initial structure. It was originally formulated to describe the growth of plant cells, and has subsequently been applied for various purposes in computer graphics such as modeling plants, buildings, streets, and ornaments. For our purpose, we extended the growth process of the L-system as follows: 1) each growing branch keeps away from existing branches as much as possible to create a uniform distribution, and 2) when branches collide, we connect the colliding branches to construct a closed mesh structure. We designed a generating rule based on observations of the photograph of Purkinje fibers and manually specified three terminal positions on a three-dimensional (3D) heart model: those of the right bundle branch, the anterior fascicle, and the left posterior fascicle of the left branch. Then, we grew fibers starting from each of the three positions based on the specified generating rule. We achieved to generate 3D models of Purkinje fibers of which physical appearances closely resembled the real photograph. The generation takes a few seconds. Variations of the Purkinje fibers could be constructed easily by modifying the generating rules and parameters.
Nicotine, like several other abused drugs, is known to act on the reward system in the brain. Smoking-associated cues produce smoking urges and cravings accompanied by autonomic dysfunction to these cues in smokers. The present study was aimed at investigating whether cues related to smoking elicit the autonomic response in smokers. The subjective and physiological reactivity of 7 smokers and 12 nonsmokers in a supine position to smoking-related visual cues was assessed under indirect dim light using a self-assessment manikin and a specially designed pupillometer. The experimental procedure consisted of the elicitation and measurement of pupil size (PS) while the subjects viewed a smoking image and images from three valence-defined categories (i.e., pleasant, unpleasant, and neutral), based on normative affective ratings selected from the International Affective Picture System. Both groups produced significantly larger PS increases in response to pleasant or unpleasant images compared to neutral images. Smokers, viewing smoking-related visual cues but no other affective images, produced significantly larger PS's compared to nonsmokers. Moreover, smokers rated the smoking image with more pleasure and arousal than nonsmokers. These findings suggest that cues related to smoking induce not only a subjective emotional alteration, but also sympathetic activation, measured by the time-series PS data in smokers.
Femur bone densities and tibialis anterior muscle properties of type II collagen-induced arthritic mice were determined. Furthermore, voluntary running activities of arthritic mice were compared with those of controls. Arthritis was induced by an intradermal injection of type II collagen in the adjuvant. Body and muscle weights were lower in arthritic mice than in controls. Cortical and trabecular bone densities and muscle fiber cross-sectional areas were decreased by arthritis. After classifying the arthritic severity into slight, intermediate, and severe levels based on the degree of knuckle swelling, cortical and trabecular bone densities, fiber cross-sectional areas, and fiber succinate dehydrogenase activities were lowest when arthritis was most severe. Furthermore, arthritic mice, especially those with intermediate and severe levels, showed lower voluntary running activities. These findings indicate that lower bone density and muscle atrophy of type II collagen-induced arthritic mice are related to arthritic severity and decreased motor activity.
This study was performed to examine whether electroacupuncture potentiates the neostigmine-induced antiallodynia in neuropathic pain rats. Although intrathecal neostigmine (0.05, 0.1, and 0.3 microg) dose-dependently relieved cold allodynia, 0.3 microg neostigmine caused side effects. The coapplication of 0.1 microg neostigmine and electroacupuncture, however, produced potent antiallodynia, which was parallel to the effect of 0.3 microg neostigmine, without side effects. These results indicate that electroacupuncture can enhance the antiallodynic action of intrathecal neostigmine.
A head-down tilt posture, the Trendelenburg position, which could facilitate venous return from the splanchnic organs and lower extremities, is recommended for the treatment of anaphylactic shock. However, few data of animal studies support its effectiveness. We examined the effects of a head-down tilt maneuver on anaphylactic hypotension in BALB/c mice and Sprague-Dawley rats. We measured systemic arterial pressure (Sap) and portal venous pressure (Pvp) in spontaneously breathing anesthetized animals sensitized with ovalbumin. At either supine (control) or a 30-degree head-down tilt position, anaphylactic hypotension was induced by an intravenous injection of antigen. In the control rats, an increase in Sap by 66 mmHg and a decrease in Pvp by 11.5 cmH2O were observed at 2.5 and 6 min, respectively, after antigen. In contrast, in control mice injected with antigen, Sap decreased similarly, but Pvp increased by only 4 cmH2O. A head-down tilt maneuver in mice substantially attenuated the antigen-induced decrease in Sap throughout the 60 min measurements, though it aggravated slightly, but significantly, only at the late phase of after 25 min in rats. We conclude that a head-down tilt maneuver attenuates anaphylactic hypotension in anesthetized mice and rats. These beneficial effects were smaller in rats than in mice probably because of substantial portal hypertension, which might prevent the head-down tilt-induced increase in venous return from the splanchnic vascular bed.
Cutaneous receptors stimulated by ice-water immersion of one hand will increase sympathetic nerve activity to the palm skin in the nonimmersed contralateral hand and reduce blood flow, reflecting on a decrease in skin surface temperature under a constant ambient environment. To test the hypothesis that gender might affect the contralateral vasoconstrictor response, we analyzed the spatiotemporal pattern of palm skin surface temperature during ice-water immersion for 10 min using thermography in eight males and eight females. As soon as the left hand was immersed in ice-water, palm skin temperature in the nonimmersed right hand quickly decreased in all subjects, particularly in the periphery of the digits and palm. The reduction in skin temperature was short-lasting in 63% of males and 38% of females, but it lasted throughout immersion in the remaining subjects. The average decrease in palm skin temperature was not significantly different between males and females, though it tended to be greater in males. The mean arterial blood pressure significantly increased and heart rate decreased during immersion in males, whereas no substantial cardiovascular changes were observed in females. Cold sensation was well coincident with the appearance of a reduction in the palm skin temperature. In consideration of all these results, we suggest that cutaneous cold stimuli increased skin sympathetic nerve activity in the nonimmersed hand and reduced skin blood flow. We also contend that gender difference in the contralateral vasoconstrictor response was denied because the time course and magnitude of the decrease in palm skin temperature were not different between males and females.
This study was performed to investigate the heterogeneity of physiological and pharmacological properties in segments of the facial veins with special reference to selective brain cooling. Canine facial veins were isolated and the isometric tension of each segment was measured using the organ bath technique. Vessels in the segments of the facial veins that run opposite to the buccal cavity automatically produced myogenic tone and tended to show spontaneous contractions, but vessels in other segments did not. When no contractile agent was used for precontraction, noradrenaline and adrenaline produced dose-dependent relaxations in the former venous segments, but contractions in the latter ones. A Schild plot analysis for metoprolol against denopamine and for ICI118,551 against salbutamol showed that the venous segments running opposite the buccal cavity contained both beta(1)- and beta(2)-adrenoceptors, but the other venous segments contained only beta(2)-adrenoceptors. Electrical field stimulation-induced tetrodotoxin-sensitive relaxations in the former venous segments were diminished by pretreatment with metoprolol, but not with ICI118,551, indicating that the electrical stimulation-induced relaxation may be related to the activation of beta(1)-adrenoceptors in the venous smooth muscles. In conclusion, the heterogeneity of the functional properties, especially in the distribution of beta-adrenoceptors, in different segments of canine facial veins was observed in the present study, and autoregulatory mechanisms, humoral mechanisms, and neural mechanisms were suggested to affect cranial venous drainage.
This review discusses the present knowledge on the oxygen uptake kinetics at the onset of exercise in skeletal muscle and the contribution of a previously developed computer model of oxidative phosphorylation in intact skeletal muscle to the understanding of the factors determining this kinetics on the biochemical level. It has been demonstrated recently that an increase in the total creatine pool [PCr + Cr] and in glycolytic ATP supply lengthen the half-transition time of the VO2 on-kinetics, while an increase in mitochondria content, in parallel activation of ATP supply and ATP usage, in muscle oxygen concentration, in proton leak, in resting energy demand, in resting cytosolic pH, and in initial alkalization diminish this parameter. It has also been shown that the half-transition time is near-linearly proportional to the absolute difference between the phosphocreatine concentration during work and at rest (deltaPCr). The present review discusses whether the V/O2 on-kinetics on the muscle level is strictly or only approximately exponential. Finally, it is postulated that a short transition time of the VO2 on-kinetics in itself does not need be profitable for the skeletal muscle functioning during exercise, but usually a short transition time is correlated with factors that improve exercise capacity. The transition time is a phenomenological parameter resulting from the biochemical properties of the system and not a physical factor that can cause anything in the system.
The purpose of the present study was to examine the effect of unilateral lower limb suspension (ULLS) deconditioning on oxygen uptake kinetics. Eight healthy males underwent ULLS for 20 days and performed a series of 6-min square-wave transitions from rest to 60-W single-leg cycling exercises just before and after ULLS. To characterize the kinetics of the oxygen uptake response, a single exponential model was applied to the data until the end of the fast component omitted the first 15 s of the on-transit using a nonlinear least-squares fitting procedure. The following results were found: (i) the time constant of oxygen uptake was unchanged before and after ULLS; (ii) although there was no significant difference in the baseline and the asymptotic amplitude of the fast component, the asymptote, i.e., the absolute asymptotic amplitude of the fast component (the sum of the baseline and the asymptotic amplitude), and the end exercise oxygen uptake were decreased after ULLS; (iii) the contribution of the slow component to the total response of oxygen uptake was unchanged at pre- and post-ULLS. In conclusion, the asymptote in the fast component and the end exercise oxygen uptake were decreased after 20-d ULLS, though the response speed and the amplitude of the slow component of oxygen uptake were not changed. It is suggested that deconditioning as a result of limb disuse affects oxygen uptake response.
A chronic administration of isoproterenol hydrochloride (60 mg/kg body weight; 30 days) alters the collagen metabolism in denervated gastrocnemius muscle of mice. Hydroxyproline assay for collagen showed an increase in collagen content by 47%, 44%, and 61% in innervated gastrocnemius + drug, denervated control, and denervated + drug, respectively, in gastrocnemius muscles after 30 days of drug administration. Collagen proliferation is beta-agonist (isoproterenol) specific confirmed with the simultaneous administration of beta-antagonist propranolol (100 mg/kg body weight; 30 days). Van Gieson staining showed heavy collagen proliferation in the epimysium region of the muscle section and adventitia of blood vessels and some specialized regions. However, denervated gastrocnemius muscle represented a heavy collagen proliferation in the endomysium region, which also is probably responsible for extensive collagen proliferation in denervated muscle after drug administration. The SDS-PAGE of pepsin-soluble collagen revealed five bands from origin to the point of migration, gamma, beta1, beta2, alpha1, and alpha2. The SDS-PAGE of CNBr-treated pepsin-insoluble collagen pointed toward the more prominent remodeling of collagen metabolism in the beta-agonist-induced denervated gastrocnemius muscle after drug administration. From the present study, we can conclude that beta-agonist, isoproterenol hydrochloride, augments collagen proliferation in innervated as well as in denervated gastrocnemius muscle.
BACKGROUNDHypoxia-inducible factor (HIF)-1alpha regulates the transcription of lines of genes, including vascular endothelial growth factor (VEGF), a major gene responsible for angiogenesis. Several recent studies have demonstrated that a nonhypoxic pathway via nitric oxide (NO) is involved in the activation of HIF-1alpha. However, there is no direct evidence demonstrating the release of angiogenic factors by cardiomyocytes through the nonhypoxic induction pathway of HIF-1alpha in the heart. Therefore we assessed the effects of an NO donor, S-Nitroso-N-acetylpenicillamine (SNAP) on the induction of VEGF via HIF-1alpha under normoxia, using primary cultured rat cardiomyocytes (PRCMs).METHODS AND RESULTSPRCMs treated with acetylcholine (ACh) or SNAP exhibited a significant production of NO. SNAP activated the induction of HIF-1alpha protein expression in PRCMs during normoxia. Phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation was induced by SNAP and was completely blocked by wortmannin, a PI3K inhibitor, and NG-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor. The SNAP treatment also increased VEGF protein expression in PRCMs. Furthermore, conditioned medium derived from SNAP-treated cardiomyocytes phosphorylated the VEGF type-2 receptor (Flk-1) of human umbilical vein endothelial cells (a fourfold increase compared to the control group, p < 0.001, n = 5) and accelerated angiogenesis.CONCLUSIONOur results suggest that cardiomyocytes produce VEGF through a nonhypoxic HIF-1alpha induction pathway activated by NO, resulting in angiogenesis.
There is a well-characterized membrane chloride current (ICl,cAMP) in the heart that can be activated by beta-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17beta-estradiol (E2) modulates ICl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 microM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 +/- 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 microM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked ICl,cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICl 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICl 182,780 (100 microM) significantly reduced the increase in ICl,cAMP evoked by 10 microM E2 to 1.46 +/- 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-omega-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl,cAMP by 30 microM E2, to 1.93 +/- 0.06 (p < 0.02), and for 10 microM E2, to 1.32 +/- 0.05 (p < 0.002). E2 also increased ICl,cAMP evoked by bath application of 0.5 microM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl,cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl,cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.
We hypothesized that a freely paced 10,000 m running race would induce a smaller physiological strain (heart rate and oxygen uptake) compared with one performed at the same average speed but with an imposed constant pace. Furthermore, we analyzed the scaling properties with a wavelet transform algorithm computed log2 (wavelet transform energy) vs. log2 (scale) to get slope alpha, which is the scaling exponent, a measure of the irregularity of a time series. HR was sampled beat by beat and V2O, breath by breath. The enforced constant pace run elicited a significantly higher mean VO2 value (53 +/- 4 vs. 48 +/- 5 ml kg(-1) min(-1), P < 0.001), HR (169 +/- 13 vs. 165 +/- 14 bpm, P < 0.01), and blood lactate concentration (6.6 +/- 0.9 vs. 7.5 +/- 1 mM, P < 0.001) than the freely paced run. HR and VO2 signals showed a scaling behavior, which means that the signals have a similar irregularity (a self-similarity) whatever the scale of analysis may be, in both constant and free-paced 10,000 m runs. The scaling exponent was not significantly different according to the type of run (free vs. constant, P > 0.05) and the signal (HR vs. VO2, P > 0.05). The higher metabolic cost of constant vs. free paced run did not affect the self-similarity of HR and VO2, in either run. The HR signal only kept its scaling behavior only with a distance run, no matter the type of run (free or constant). The results suggest that the larger degree of pace variation in freely paced races may be an intentionally chosen strategy designed to minimize the physiological strain during severe exercise and to prevent a premature termination of effort, even if the variability of the heart rate and VO2, are comparable in an enforced constant vs. a freely paced run and if HR keeps the same variability until the arrival.
