Journal of Physiology

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Determination of cable parameters in skeletal muscle fibres during repetitive firing of action potentials

15 October 2014

Recent studies in rat muscle fibres show that repetitive firing of action potentials causes changes in fibre resting membrane conductance (Gm) that reflect regulation of ClC-1 Cl– and KATP K+ ion channels. Methodologically, these findings were obtained by inserting two microelectrodes at close proximity in the same fibres enabling measurements of fibre input resistance (Rin) in between action potential trains. Since the fibre length constant () could not be determined, however, the calculation of Gm relied on the assumptions that the specific cytosolic resistivity (Ri) and muscle fibre volume remained constant during the repeated action potential firing. Here we present a three-microelectrode technique that enables determinations of multiple cable parameters in action potential-firing fibres including Rin and as well as waveform and conduction velocities of fully propagating action potentials. It is shown that in both rat and mouse extensor digitorum longus (EDL) fibres, action potential firing leads to substantial changes in both muscle fibre volume and Ri. The analysis also showed, however, that regardless of these changes, rat and mouse EDL fibres both exhibited initial decreases in Gm that were eventually followed by a ~3-fold, fully reversible increase in Gm after the firing of 1450–1800 action potentials. Using this three-electrode method we further show that the latter rise in Gm was closely associated with excitation failures and loss of action potential signal above –20 mV.

Fluorescence recovery after photobleaching reveals regulation and distribution of connexin36 gap junction coupling within mouse islets of Langerhans

15 October 2014
Key points

  • Gap junctions provide electrical coupling that is critical to the function of pancreatic islets. Disruptions to connexin36 (Cx36) have been suggested to occur in diabetes.

  • No accurate and non-invasive technique has yet been established to quantify changes in Cx36 gap junction coupling in the intact islet.

  • This study developed fluorescence recovery after photobleaching (FRAP) as a non-invasive technique for quantifying Cx36 gap junction coupling in living islets.

  • The study identified treatments that modulate gap junction coupling, confirmed that the cellular distribution of coupling throughout the islet is highly heterogeneous and confirmed that α cells and β cells do not form functional Cx36 gap junctions.

  • This technique will enable future studies to examine the regulation of Cx36 gap junction coupling and its disruption in diabetes, and to uncover potential novel therapeutic targets associated with gap junction coupling.

  • Abstract

    The pancreatic islets are central to the maintenance of glucose homeostasis through insulin secretion. Glucose-stimulated insulin secretion is tightly linked to electrical activity in β cells within the islet. Gap junctions, composed of connexin36 (Cx36), form intercellular channels between β cells, synchronizing electrical activity and insulin secretion. Loss of gap junction coupling leads to altered insulin secretion dynamics and disrupted glucose homeostasis. Gap junction coupling is known to be disrupted in mouse models of pre-diabetes. Although approaches to measure gap junction coupling have been devised, they either lack cell specificity, suitable quantification of coupling or spatial resolution, or are invasive. The purpose of this study was to develop fluorescence recovery after photobleaching (FRAP) as a technique to accurately and robustly measure gap junction coupling in the islet. The cationic dye Rhodamine 123 was used with FRAP to quantify dye diffusion between islet β cells as a measure of Cx36 gap junction coupling. Measurements in islets with reduced Cx36 verified the accuracy of this technique in distinguishing between distinct levels of gap junction coupling. Analysis of individual cells revealed that the distribution of coupling across the islet is highly heterogeneous. Analysis of several modulators of gap junction coupling revealed glucose- and cAMP-dependent modulation of gap junction coupling in islets. Finally, FRAP was used to determine cell population specific coupling, where no functional gap junction coupling was observed between α cells and β cells in the islet. The results of this study show FRAP to be a robust technique which provides the cellular resolution to quantify the distribution and regulation of Cx36 gap junction coupling in specific cell populations within the islet. Future studies utilizing this technique may elucidate the role of gap junction coupling in the progression of diabetes and identify mechanisms of gap junction regulation for potential therapies.

    Fine spatiotemporal activity in contracting myometrium revealed by motion-corrected calcium imaging

    15 October 2014

    Successful childbirth depends on the occurrence of precisely coordinated uterine contractions during labour. Calcium indicator fluorescence imaging is one of the main techniques for investigating the mechanisms governing this physiological process and its pathologies. The effective spatiotemporal resolution of calcium signals is, however, limited by the motion of contracting tissue: structures of interest in the order of microns can move over a hundred times their width during a contraction. The simultaneous changes in local intensity and tissue configuration make motion tracking a non-trivial problem in image analysis and confound many of the standard techniques. This paper presents a method that tracks local motion throughout the tissue and allows for the almost complete removal of motion artefacts. This provides a stabilized calcium signal down to a pixel resolution, which, for the data examined, is in the order of a few microns. As a byproduct of image stabilization, a complete kinematic description of the contraction–relaxation cycle is also obtained. This contains novel information about the mechanical response of the tissue, such as the identification of a characteristic length scale, in the order of 40–50 μm, below which tissue motion is homogeneous. Applied to our data, we illustrate that the method allows for analyses of calcium dynamics in contracting myometrium in unprecedented spatiotemporal detail. Additionally, we use the kinematics of tissue motion to compare calcium signals at the subcellular level and local contractile motion. The computer code used is provided in a freely modifiable form and has potential applicability to in vivo calcium imaging of neural tissue, as well as other smooth muscle tissue.

    Cooperative subunit interactions mediate fast C-type inactivation of hERG1 K+ channels

    15 October 2014

    At depolarized membrane potentials, the conductance of some voltage-gated K+ channels is reduced by C-type inactivation. This gating process is voltage independent in Kv1 and involves a conformational change in the selectivity filter that is mediated by cooperative subunit interactions. C-type inactivation in hERG1 K+ channels is voltage-dependent, much faster in onset and greatly attenuates currents at positive potentials. Here we investigate the potential role of subunit interactions in C-type inactivation of hERG1 channels. Point mutations in hERG1 known to eliminate (G628C/S631C), inhibit (S620T or S631A) or enhance (T618A or M645C) C-type inactivation were introduced into subunits that were combined with wild-type subunits to form concatenated tetrameric channels with defined subunit composition and stoichiometry. Channels were heterologously expressed in Xenopus oocytes and the two-microelectrode voltage clamp was used to measure the kinetics and steady-state properties of inactivation of whole cell currents. The effect of S631A or T618A mutations on inactivation was a graded function of the number of mutant subunits within a concatenated tetramer as predicted by a sequential model of cooperative subunit interactions, whereas M645C subunits increased the rate of inactivation of concatemers, as predicted for subunits that act independently of one another. For mutations located within the inactivation gate proper (S620T or G628C/S631C), the presence of a single subunit in a concatenated hERG1 tetramer disrupted gating to the same extent as that observed for mutant homotetramers. Together, our findings indicate that the final step of C-type inactivation of hERG1 channels involves a concerted, all-or-none cooperative interaction between all four subunits, and that probing the mechanisms of channel gating with concatenated heterotypic channels should be interpreted with care, as conclusions regarding the nature of subunit interactions may depend on the specific mutation used to probe the gating process.

    Action of the isolated canine diaphragm on the lower ribs at high lung volumes

    15 October 2014

    The normal diaphragm has an inspiratory action on the lower ribs, but subjects with chronic obstructive pulmonary disease commonly have an inward displacement of the lateral portions of the lower rib cage during inspiration. This paradoxical displacement, conventionally called ‘Hoover's sign’, has traditionally been attributed to the direct action of radially oriented diaphragmatic muscle fibres. In the present study, the inspiratory intercostal muscles in all interspaces in anaesthetized dogs were severed so that the diaphragm was the only muscle active during inspiration. The displacements of the lower ribs along the craniocaudal and laterolateral axes and the changes in pleural pressure (Ppl) and transdiaphragmatic pressure were measured during occluded breaths and mechanical ventilation at different lung volumes between functional residual capacity (FRC) and total lung capacity. From these data, the separate effects on rib displacement of Ppl and of the force exerted by the diaphragm on the ribs were determined. Isolated spontaneous diaphragm contraction at FRC displaced the lower ribs cranially and outward, but this motion was progressively reversed into a caudal and inward motion as lung volume increased. However, although the force exerted by the diaphragm on the ribs decreased with increasing volume, it continued to displace the ribs cranially and outward. These observations suggest that Hoover's sign is usually caused by the decrease in the zone of apposition and, thus, by the dominant effect of Ppl on the lower ribs, rather than an inward pull from the diaphragm.

    Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation

    15 October 2014

    The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ~33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge.

    Hypercapnia attenuates ventilator-induced lung injury via a disintegrin and metalloprotease-17

    15 October 2014
    Key points

  • Hypercapnia is common in mechanically ventilated patients with lung injury; while CO2 can ameliorate experimental lung injury, it can also cause harm.

  • Because hypercapnia can protect against ventilator-induced lung injury (VILI), understanding its impact on key signalling pathways may provide insight into the mechanisms of VILI.

  • We show that hypercapnia blocks stretch-mediated activation of p44/42 mitogen-activated protein kinase (MAPK) signalling in alveolar epithelial cells; this occurs through inhibition of sheddase (i.e. the metalloprotease, ADAM17), which releases ligands that bind to the epidermal growth factor receptor.

  • In vivo pharmacological blockade of ADAM17 reduces downstream MAPK activation and attenuates VILI in a two-hit mouse model.

  • Thus, hypercapnia uncovered a novel ADAM17-dependent mechanism of VILI, and this represents a potential therapeutic target.

  • Abstract

    Hypercapnic acidosis, common in mechanically ventilated patients, has been reported to exert both beneficial and harmful effects in models of lung injury. Understanding its effects at the molecular level may provide insight into mechanisms of injury and protection. The aim of this study was to establish the effects of hypercapnic acidosis on mitogen-activated protein kinase (MAPK) activation, and determine the relevant signalling pathways. p44/42 MAPK activation in a murine model of ventilator-induced lung injury (VILI) correlated with injury and was reduced in hypercapnia. When cultured rat alveolar epithelial cells were subjected to cyclic stretch, activation of p44/42 MAPK was dependent on epidermal growth factor receptor (EGFR) activity and on shedding of EGFR ligands; exposure to 12% CO2 without additional buffering blocked ligand shedding, as well as EGFR and p44/42 MAPK activation. The EGFR ligands are known substrates of the matrix metalloprotease ADAM17, suggesting stretch activates and hypercapnic acidosis blocks stretch-mediated activation of ADAM17. This was corroborated in the isolated perfused mouse lung, where elevated CO2 also inhibited stretch-activated shedding of the ADAM17 substrate TNFR1 from airway epithelial cells. Finally, in vivo confirmation was obtained in a two-hit murine model of VILI where pharmacological inhibition of ADAM17 reduced both injury and p44/42 MAPK activation. Thus, ADAM17 is an important proximal mediator of VILI; its inhibition is one mechanism of hypercapnic protection and may be a target for clinical therapy.

    Effects of hyperoxia and hypoxia on the physiological traits responsible for obstructive sleep apnoea

    15 October 2014

    Oxygen therapy is known to reduce loop gain (LG) in patients with obstructive sleep apnoea (OSA), yet its effects on the other traits responsible for OSA remain unknown. Therefore, we assessed how hyperoxia and hypoxia alter four physiological traits in OSA patients. Eleven OSA subjects underwent a night of polysomnography during which the physiological traits were measured using multiple 3-min ‘drops’ from therapeutic continuous positive airway pressure (CPAP) levels. LG was defined as the ratio of the ventilatory overshoot to the preceding reduction in ventilation. Pharyngeal collapsibility was quantified as the ventilation at CPAP of 0 cmH2O. Upper airway responsiveness was defined as the ratio of the increase in ventilation to the increase in ventilatory drive across the drop. Arousal threshold was estimated as the level of ventilatory drive associated with arousal. On separate nights, subjects were submitted to hyperoxia (n = 9; FiO2 ~0.5) or hypoxia (n = 10; FiO2 ~0.15) and the four traits were reassessed. Hyperoxia lowered LG from a median of 3.4 [interquartile range (IQR): 2.6–4.1] to 2.1 (IQR: 1.3–2.5) (P < 0.01), but did not alter the remaining traits. By contrast, hypoxia increased LG [median: 3.3 (IQR: 2.3–4.0) vs. 6.4 (IQR: 4.5–9.7); P < 0.005]. Hypoxia additionally increased the arousal threshold (mean ± s.d. 10.9 ± 2.1 l min–1 vs. 13.3 ± 4.3 l min–1; P < 0.05) and improved pharyngeal collapsibility (mean ± s.d. 3.4 ± 1.4 l min–1 vs. 4.9 ± 1.3 l min–1; P < 0.05), but did not alter upper airway responsiveness (P = 0.7). This study demonstrates that the beneficial effect of hyperoxia on the severity of OSA is primarily based on its ability to reduce LG. The effects of hypoxia described above may explain the disappearance of OSA and the emergence of central sleep apnoea in conditions such as high altitude.