Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Recently, it has been postulated that fatigue is a mechanism to protect the muscle fiber from deleterious ATP depletion and cell death. The ATP-sensitive potassium (KATP) channel is believed to play a major role in this mechanism. Under metabolic stress, the channels open, reducing membrane excitability, Ca2+ release and force production. This alleviates energy demand within the fiber, as activation of the channel reduces ATP consumption from cellular ATPases. Loss of KATP channel activity during fatigue results in excessive intracellular Ca2+ ([Ca2+]i) levels, likely entering the fiber through L-type Ca2+ channels. It has been demonstrated that when mouse muscle lacking functional KATP channels are stimulated to fatigue, ATP levels become significantly lower than wild type levels. Thus, it was hypothesized that a lack of KATP channel activity impairs energy metabolism, resulting in insufficient ATP production. The focus of work for this M.Sc. project was to test this hypothesis. Fatigue was elicited in Kir6.2-/- FDB muscles for three min followed by 15 min recovery. After 60 sec, a 2.6-fold greater glycogen breakdown was observed in Kir6.2-/- FDB compared to wild type FDB. However, this effect disappeared thereafter, as there were no longer any differences between wild type and Kir6.2-/- FDB in glycogen breakdown by 180 sec. Glucose oxidation after 60 sec was also greater in Kir6.2-/- FDB compared to wild type FDB. However, levels of oxidation failed to increase in Kir6.2-/- FDB from 60 to 180 sec. Calculated ATP production during the fatigue period was 2.7-times greater in Kir6.2-/- FDB, yet measured ATP levels during fatigue are much lower in Kir6.2-/- FDB compared to wild type FDB. Taken together, it appears that muscle energy metabolism is impaired in the absence KATP channel activity.

Animal and human studies have consistently demonstrated that recurrent hypoglycaemia (RH) blunts both hormonal and behavioral counter regulatory responses (CRR) to further episodes of hypoglycaemia. It is now well established that the brain is involved in regulating whole-body glucose homeostasis, including the CRR to hypoglycaemia. The aim of the current study was to investigate if adaptations occur, following RH, which are intrinsic to glucose-sensing neurons in the absence of synaptic/glial inputs or signals from the periphery. Utilising the GT1-7 hypothalamic mouse cell line as an in vitro model of homogenous glucose-excited neurons, the current study has demonstrated that recurrent low glucose exposure reprograms intracellular metabolism towards a “hypometabolic state”. This result occurs in conjunction with an attenuated ability of the cells to hyperpolarise in response to low glucose and a reduction in the sensitivity of the KATP channel to activation by MgADP. In an attempt to reverse the changes observed in KATP channel activity, the SUR1-selective KATP channel opener, NN414, was applied chronically to GT1-7 cells. However, chronic KATP channel activation severely reduced channel conductance and sensitivity to activation by MgADP and further NN414 application. These results suggest that chronic activation of the KATP channel leads to the induction of a negative feedback mechanism to reduce channel activity. This may be in an attempt to maintain neuronal membrane potential within a physiological range. These results also suggest activation of central KATP channels during RH may be driving the resulting defective CRR. However, adaptations in metabolism following RH may also be altering the function of central KATP channels.

Action potentials of striated muscle are created through movement of ions through membrane ion channels. ATP-sensitive potassium (KATP) channels are the only known channels that are gated by the intracellular energetic level ([ATP]/[ADP] ratio). KATP channels are both effectors and indicators of cellular metabolism as part of a negative feedback system. Decreased intracellular energetic level alters the gating of KATP channels, which is reflected in alterations of the action potential morphology. These changes protect the cell from exhaustion or injury by altering energy-consuming processes that are driven by membrane potential. Assessing the effects of KATP channel activation on resting membrane potential and action potential morphology, and the relationship to cellular stress is important to the understanding of normal cellular function. To better understand how muscle cells adapt to energetic stress, the monophasic action potential (MAP) electrode and floating microelectrode were used to record action potentials in intact hearts and skeletal muscles, respectively. Intact organs provide a more physiological environment for the study of energetics and membrane electrical phenomena. Utilizing these techniques, a stress on the intracellular energetic state resulted in greater and faster shortening of the duration of cardiac action potentials, and hyperpolarization of the membrane of skeletal muscle in a KATP channel dependent manner. Motion artifacts are a limitation to studying transmembrane action potentials, but the MAP and floating microelectrode techniques uniquely allow for reading of action potential morphology uncoupled from motion artifacts. The use of the floating microelectrode in skeletal muscles is a novel approach that provides previously unavailable data on skeletal muscle membrane potentials in situ.

No description available.

Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges and weakness/paralysis. It is a channelopathy that is caused by mutation in the SCN4A gene that encodes for the skeletal muscle Na+ channel isoform (Nav1.4) α-subunit. Limb muscles are severely affected while breathing musculature is rarely affected even though diaphragm expresses the Nav1.4 channel. The objective of this study was to investigate the mechanism(s) that render the HyperKPP diaphragm asymptomatic in order to find a novel long lasting therapeutic approach, to treat HyperKPP symptoms. A HyperKPP mouse model carrying the M1592V mutation was used because it has a similar phenotype to that of patients carrying the same mutation. HyperKPP diaphragm, the limb muscles soleus and EDL all had a higher tetrodotoxin (TTX) sensitive Na+ influx than wild type (WT), but only the soleus and EDL had a depolarized resting potential, lower force and greater K+-induced force loss when compared to WT. The lack of a membrane depolarization in HyperKPP diaphragm was because of greater electrogenic contribution of the Na+ K+ ATPase pump compared to WT while such increase was not observed in EDL and soleus. HyperKPP diaphragm also had greater action potential amplitude than EDL and soleus possibly because of higher Na+ K+ ATPase pump maintaining a low [Na+]i. An inhibition of PKA, but not of PKC, increased the sensitivity of the HyperKPP diaphragm to the K+-induced force depression. So, HyperKPP soleus was exposed to forskolin to increase cAMP levels in order to activate PKA to document whether greater activity of PKA will alleviate HyperKPP symptoms. At 4.7 mM K+, forskolin increased force production, but worsened the decrease in force at 8 and 11 mM K+. Forskolin also did not improve membrane excitability. Pinacidil a KATP channel opener, improved force production at all [K+]e by causing a hyperpolarization of resting EM which then allowed for greater action potential amplitude and more excitable fibers. It is concluded that the development of a better therapeutic approach to treat HyperKPP can include a mechanism which activates Na+ K+ ATPase pumps and KATP channels.

Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-­sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-­9216, on scalp follicle growth in organ culture was examined. NNC55-­9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.

<p>Catecholamine (CAT) secretion from adrenomedullary chromaffin cells (AMCs) is essential for survival of the fetus and for adaptation of the newborn to extrauterine life. CAT secretion protects the fetus from intrauterine hypoxia (low O₂) and is required for maintaining cardiac conduction and preparing the lungs for air breathing. Asphyxial stressors (e.g. hypoxia, hypercapnia (high PCO₂), and acidosis (low pH)) arising from labor contractions and postnatal apneas, are the main stimuli for the ‘non-neurogenic’ CAT release from perinatal AMCs. In the rat, the mechanisms of hypoxia chemosensitivity in AMCs involve inhibition of a variety of K⁺ channels, leading to membrane depolarization, voltage-gated Ca²⁺ entry, and CAT secretion. The magnitude of this depolarization is regulated by the simultaneous activation of ATP-sensitive K⁺ (K_ATP) channels, which tends to hyperpolarize the membrane potential during hypoxia. Interestingly, chemosensitivity of rat AMCs and CAT secretion in response to asphyxial stressors are markedly reduced postnatally following the development of functional innervation of these cells by the splanchnic nerve.</p> <p>The primary purpose of this thesis was to delineate molecular mechanisms involved in the suppression of hypoxia and hypercapnia chemosensitivity following splanchnic innervation in neonatal rat AMCs. Experiments were designed to test the general hypothesis that the ontogeny of O₂ and CO₂ sensitivity in AMCs is regulated by the activation of postsynaptic nicotinic ACh and opioid receptor signalling pathways following innervation. Previous studies in this laboratory showed that exposure of perinatal rat AMCs to nicotine <em>in utero </em>and <em>in vitro</em> resulted in the selective blunting of hypoxia (but <em>not</em> hypercapnia) chemosensitivity. The underlying mechanism was attributable to the increased membrane hyperpolarization caused by the functional upregulation of K_ATP channels. In Chapter 2, I report the results of investigations of molecular mechanisms involved in the nicotine-induced upregulation of K_ATP channels, using a rat fetal-derived, O₂- and CO₂-sensitive immortalized chromaffin cell line (MAH cells), as a model. Exposure of MAH cells to chronic nicotine (50 μM) for 7 days in culture caused an increase in the expression of the K_ATP channel subunit, Kir6.2. This effect was blocked by α-bungarotoxin, a blocker of homomeric α7 nicotinic acetylcholine receptors (α7 nAChRs). The upregulation of Kir6.2 in MAH cells was also dependent on the transcription factor, hypoxia inducible factor (HIF)-2α. First, whereas the upregulation of Kir6.2 was present in wild type and scrambled control MAH cells, it was absent in HIF-2α-deficient (shHIF-2α) MAH cells. Second, chronic nicotine caused a progressive, time-dependent increase in HIF-2α accumulation that occurred in parallel with the increase in Kir6.2 expression. Third, chromatin immunoprecipitation (ChIP) assays revealed the binding of HIF-2α to a hypoxia response element (HRE) in the promoter region of the Kir6.2 gene. These data suggest that chronic nicotine causes the accumulation of HIF-2α which results in the transcriptional upregulation of the Kir6.2 gene. These observations were validated in an <em>in vivo</em> model where rat pups were exposed to nicotine <em>in utero</em>. Western blot analysis of adrenal gland tissues from nicotine-exposed (relative to saline-exposed) pups revealed a significant increase in Kir6.2 subunit expression and HIF-2α accumulation, and both were restricted to the medullary (but not cortical) tissue.</p> <p>Chapter 3 tested the hypothesis that postnatal innervation causes the suppression of O₂- and CO₂-chemosensitivity in neonatal AMCs via opioid receptor signalling. It was found that chronic μ- and δ-opioid agonists (2 μM) <em>in vitro </em>led to the suppression of both O₂- and CO₂-chemosensitivity; this was correlated with the upregulation of K_ATP channel expression and the downregulation of carbonic anhydrase (CA) I and II respectively. The underlying molecular and signalling mechanisms were further investigated in Chapter 4. Using the MAH cell model, it was found that exposure to a combination of μ- and δ-opioid agonists for 7 days resulted in the naloxone-sensitive upregulation of Kir6.2 subunit and the downregulation of CAII. Similar to chronic nicotine exposure, the effects of chronic opioids on the upregulation of Kir6.2 and downregulation of CAII were HIF-2α-dependent. Western blot analysis revealed that HIF-2α accumulation in opioid-treated MAH cells occurred along a time-course that paralleled the upregulation of Kir6.2 subunit. ChIP assays demonstrated the binding of HIF-2α to the promoter region of the Kir6.2 subunit gene in opioid-treated MAH cells. Moreover, PKA activity (but not PKC or CaMK) was found to be required for the effects of opioids on Kir6.2 and CAII expression, but not HIF-2α accumulation. In complementary <em>in vivo</em> studies, adrenomedullary tissues from morphine-exposed rat pups showed an increased expression of both HIF-2α and Kir6.2, and decreased expression of CA1 and II protein. These findings have uncovered novel mechanisms by which postnatal innervation contributes to the ontogeny of O₂- and CO₂-chemosensitivity in rat adrenal chromaffin cells. They also suggest mechanisms by which exposure of the fetus to nicotine in cigarette smoke or opioids from drug abuse might contribute to abnormal arousal reflexes, and pathophysiological conditions such as Sudden Infant Death Syndrome (SIDS).<strong></strong></p> / Doctor of Philosophy (PhD)

Hair disorders cause significant distress. The main, but limited, treatment for hair loss is minoxidil, an ATP-sensitive potassium (KATP) channel opener whose mechanism of stimulation is unclear. The regulatory component of KATP channels has three forms: SUR1, SUR2A and SUR2B which all respond to different molecules. Minoxidil only opens SUR2B channels, though SUR1 and SUR2B are present in human hair follicles. To expand our understanding, the red deer hair follicle model was used initially. Deer follicles expressed the same KATP channel genes as human follicles when growing (anagen), but no channels were detected in resting follicles. This reinforces the importance of KATP channels in active hair growth and the usefulness of the deer model. To assess whether SUR1 KATP channels are actually involved in human hair growth, the effects of a selective SUR1 channel opener, NNC55-9216, on scalp follicle growth in organ culture was examined. NNC55-9216 stimulated anagen; its effect was augmented by minoxidil. This creates the potential for more effective pharmaceuticals to treat hair loss via SUR1 channels, either alone or in combination with minoxidil. The dermal papilla plays a crucial regulatory role in hair follicle activity determining the type of hair produced. Minoxidil had no effect on dermal papilla cell proliferation, but altered the profile of proteins produced when assessed by proteomics. Further research into the roles of KATP channels and greater understanding of the significance of these protein changes should enhance our knowledge of hair biology and help the development of new, improved therapies for hair pathologies.