The current recommendations by the American Heart Association for health promotion are that all persons should partake in regular physical activity in order to reduce the risk of cardiovascular disease. Regular physical exercise reduces blood pressure and is an important component of the management of hypertension. It is therefore important that patients with hypertension participate in habitual physical exercise. Many hypertensive patients who exercise will require anti-hypertensive medication. However, some antihypertensive agents cause fatigue during exercise. In order for patients to gain the full benefits of an active lifestyle, it is important that the prescribed antihypertensive agent does not prevent them performing and enjoying sustained exercise. It has been well documented that β-blockers cause premature fatigue during physical exercise. The effects on exercise performance of other first line antihypertensive medications, such as calcium channel antagonists have not been extensively investigated. In particular, the effects of these agents on prolonged submaximal exercise endurance have not been well studied. The object of this thesis was to compare the effects of isradipine, a dihydropyridine calcium channel antagonist, to those of atenolol, a β₁-selective antagonist, on maximal and submaximal exercise performance and on short duration high-intensity exercise in physically active hypertensive patients. The study design was a crossover trial where drug treatments were double blinded and randomised. Physically active volunteers with mild to moderate hypertension were recruited. 11 subjects performed i) progressive exercise to exhaustion for determination of maximal oxygen consumption (VO₂max), maximal work load and cardiorespiratory responses to maximal exercise, ii) prolonged submaximal exercise for determination of exercise endurance, cardiorespiratory responses and ratings of perceived exertion (APE), and iii) short duration, high intensity exercise consisting of a 30 second maximal exercise test (Wingate test) to determine skeletal muscle power output, following 4 weeks ingestion of isradipine (2.5mg bd), atenolol (50mg bd) or placebo. Diastolic blood pressure at rest was reduced by both atenolol and isradipine, but was lowered to a greater extent by atenolol (83.3 vs 89.0 vs 96.1 mmHg, atenolol vs isradipine vs placebo, p<.0005). Systolic blood pressure at rest tended to be similarly reduced by both agents, but was significantly reduced during maximal and submaximal exercise by atenolol only (p<.001, atenolol vs isradipine, placebo). Heart rate at rest and during maximal and submaximal exercise was decreased by atenolol only (p<.0005, atenolol vs isradipine, placebo). Maximal exercise performance was reduced after atenolol ingestion compared to placebo but not after isradipine ingestion. Peak workload achieved during the maximal exercise test was decreased after atenolol but unchanged after isradipine ingestion (214 vs 243 W, atenolol vs placebo, p<.01). Similarly, VO₂max was reduced after atenolol compared to placebo but was unchanged after isradipine ingestion (33.6 vs 36.4, 33.6 vs 36.1 mlO₂/kg/min, atenolol vs placebo, atenolol vs isradipine, p<.05). Both atenolol and isradipine ingestion reduced submaximal endurance time compared to placebo (27.8 vs 46.4, 34.4 vs 46.4 min, atenolol vs placebo, isradipine vs placebo, p<.005), and increased rating of perceived exertion (APE) after 30 min of submaximal exercise (p<.05). Submaximal oxygen consumption (VO₂), ventilation, respiratory exchange ratio (REA) and blood lactate, glucose and free fatty acid concentrations were not altered after the ingestion of either agent. Neither agent influenced peak skeletal muscle power, total work done, or rate of fatigue during the Wingate test compared to placebo. The results of these studies indicate that impaired performance and increased RPE during submaximal exercise after ingestion of either atenolol or isradipine is not due to alterations of ventilation, VO₂, RER, or blood lactate, glucose and free fatty acid concentrations during prolonged submaximal exercise. Similarly, reduced submaximal exercise performance after atenolol or isradipine ingestion is not due to factors which would also limit the ability of skeletal muscle to perform short duration, high intensity exercise before a bout of prolonged exercise. This study demonstrates that prolonged submaximal exercise testing can reveal an impairment in exercise performance after ingestion of antihypertensive medication which is not evident during maximal exercise testing. This finding is important as prolonged submaximal exercise is the form of exercise which most hypertensive patients actually perform. Further research is required on the effects of anti-hypertensive medications on submaximal exercise performance before firm recommendations can be made regarding medications most suitable for the physically active hypertensive patient. The results of these and other studies indicate that it is not yet possible to make claims that the calcium channel antagonist agents are without effect on physical exercise performance in physically active hypertensive patients.

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A major physiological risk factor of temporomandibular disorders (TMD) is sensitization of peripheral and central nervous system pain processing pathways. Calcium channel, voltage-dependent, alpha-2/delta subunit-1 (CACNA2D1) has a crucial role in relaying nociceptive information in the spinal dorsal horn. Up-regulation of CACNA2D1 results in abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release. Blocking CACNA2D1 with gabapentinoid-class drugs relieves orofacial hypersensitivity. Drs. Foley, Horton, and Sciote previously reported that in a small sample group (n=12), CACNA2D1 expression was greater in males than females, but increased in women with TMD. The objectives of this study are to corroborate these data and investigate expression patterns of other ion channel and conducting system genes. Additionally, since the null polymorphism ACTN3-577XX associates with muscle fiber microdamage during eccentric contraction, we tested for possible gene associations with ACTN3-R577XX genotypes. Masseter muscle samples came from human subjects (n=23 male; 48 female) with malocclusions undergoing orthognathic surgery. This population had skeletal disharmony of the jaws and thus was prone to eccentric contraction. Three males and eighteen females were diagnosed with localized masticatory myalgia. Muscle total RNA was isolated and CACNA2D1, CACNA1S, GABARAP, and TRPM7 expression was quantified using RT-PCR. Expression of these genes were compared based on TMD status and various characteristics that may influence TMD including: sex, age, facial symmetry, sagittal dimension, vertical dimension, ACTN3-577 genotype and fiber type. CACNA2D1 expression differed significantly between sexes, overall (p&lt;0.02), and without TMD (p=0.001). Women with (n=13) and without (n=23) TMD differed significantly (p&lt;0.03). CACNA2D1 expression was also significantly higher (p=0.031) in subjects below age 25. Similarly, GABARAP expression was significantly higher (p=0.001) for patients younger than 25 and for patients less than or equal to age 18 (p=0.013). Otherwise, CACNA1S, TRPM7 and GABARAP differences were not significant. GABARAP expression differed, but not significantly by sex and for the ACTN3-577XX-null genotype. In a population of malocclusion patients, masseter muscle CACNA2D1 expression is significantly higher than CACNA1S, TRPM7, and GABARAP. CACNA2D1 expression is greater in males than females without TMD. However, CACNA2D1 expression increases significantly in females with TMD-associated myalgia. This may support evidence for calcium channel regulation of nociception differences seen between sexes in TMD. It was also found that expression of CACNA2D1 and GABARAP is significantly higher in younger subjects. Additionally, observations presented here suggest potential influence of ACTN3-null condition on function of GABARAP. / Oral Biology

Kattaya-Annappa-Seema. / Thesis submitted in: December 2006. / "2+" and "+" in the title are superscripts. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-188). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.viii / Abstracts published based on work in this thesis --- p.ix / Table of contents --- p.x / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Pulmonary hypertension / Chapter 1.1.1 --- Pulmonary circulation and its functions --- p.1 / Chapter 1.1.2 --- Pulmonary vascular diseases and symptoms --- p.3 / Chapter 1.2 --- Muscarinic Receptor functions --- p.5 / Chapter 1.3 --- Acetylcholine (ACh) and its function --- p.7 / Chapter 1.4 --- ACh receptors in pulmonary vascular bed --- p.11 / Chapter 1.5 --- Potassium channel classification and functions --- p.12 / Chapter 1.5.1 --- "Importance of High-conductance, Ca2+ activated potassium channel (BKca) in vascular smooth muscle functions" --- p.15 / Chapter 1.5.2 --- Modulation of BKca channel by various cations --- p.18 / Chapter 1.6 --- Calcium signaling and homeostasis --- p.20 / Chapter 1.7 --- Role of sodium in hypertension --- p.22 / Chapter 1.8 --- Na+-H+ exchanger (NHE) functions --- p.25 / Chapter 1.9 --- Na+-Ca2+ exchanger (NCX) in vascular smooth muscle cells --- p.29 / Chapter 1.10 --- Spontaneously hypertensive rat (SHR) / Chapter 1.10.1 --- Hypertension in SHR --- p.32 / Chapter 1.10.2 --- BKca in smooth muscle vasculature of SHR --- p.33 / OBJECTIVES OF THE STUDY --- p.34 / Chapter Chapter 2: --- Material and methods / Chapter 2.1 --- Material / Chapter 2.1.1 --- Solutions and Drugs --- p.35 / Chapter 2.1.2 --- Chemicals and Enzymes --- p.39 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Isolation of single pulmonary arterial smooth muscle cells --- p.40 / Chapter 2.2.2 --- Electrophysiological measurement --- p.42 / Chapter 2.2.3 --- Data analysis --- p.44 / Chapter Chapter 3: --- Receptor-mediated activation of BKca Channel / Chapter 3.1 --- BKCa activation by ACh/ Carbachol (CCh) --- p.45 / Chapter 3.2 --- Role of extracellular sodium ([Na+]o）on BKca activation --- p.49 / Chapter 3.3 --- Receptor-mediated activation of BKca in a [Na+]o-containing solution --- p.51 / Chapter 3.4 --- Receptor-mediated activation of BKca in a [Na+]o-free solution --- p.55 / Chapter Chapter 4: --- Non-receptor mediated activation of BKCa Channel / Chapter 4.1 --- Effect of different concentrations of sodium nitroprusside (SNP) on BKCa activation --- p.60 / Chapter 4.2 --- Effect of SNP on BKca activation in a [Na+]o-containing and [Na+]o-free solutions --- p.62 / Chapter Chapter 5: --- Role of NHE in modulating activation of BKCa Channel / Chapter 5.1 --- Effect of Monensin on BKca activation / Chapter 5.1.1 --- Effect of monensin on CCh-mediated activation of BKca in a [Na+]o-containing solution --- p.70 / Chapter 5.1.2 --- Effect of monensin on CCh-mediated activation of BKca in a [Na+]o-free solution --- p.74 / Chapter 5.1.3 --- Effect of monensin on SNP- mediated activation of BKca in [Na+]o-containing and [Na+]o-free solutions --- p.78 / Chapter 5.2 --- Effect of 5-(N-ethyl-N-isopropyI) amiloride (EIPA) on BKCa activation / Chapter 5.2.1 --- Effect of EIPA on CCh-mediated activation of BKca in a [Na+]o-containing solution --- p.85 / Chapter 5.2.2 --- Effect of EIPA on CCh-mediated activation of BKca in a [Na+]。-free solution --- p.89 / Chapter 5.2.3 --- Effect of EIPA on SNP-mediated activation of BKCa in [Na+]o-containing and [Na+]o-free solutions --- p.93 / Chapter Chapter 6: --- Role of NCX in modulating activation of BKCa Channel / Chapter 6.1 --- Effect of KB-R7943 on CCh-mediated activation of BKCa in a [Na+]o-containing solution --- p.100 / Chapter 6.2 --- Effect of KB-R7943 on CCh-mediated activation of BKCa in a [Na+]o-free solution --- p.104 / Chapter 6.3 --- Effect of KB-R7943 on SNP-mediated activation of BKca in [Na+]o-containing and [Na+]o-free solutions --- p.109 / Chapter Chapter 7: --- Effect of intracellular sodium ([Na+]i) on BKCa channel activation / Chapter 7.1 --- Effect of CCh on BKCa channel activation with elevated [Na+]i pipette solution --- p.117 / Chapter 7.2 --- Effect of SNP on BKca channel activation with elevated [Na+]j pipette solution --- p.130 / Chapter Chapter 8: --- Discussion / Chapter 8.1 --- Modulatory effect of ACh and SNP --- p.138 / Chapter 8.2 --- Role of ion exchangers: NHE and NCX in modulating BKca channel function --- p.144 / Chapter 8.3 --- Modulatory effect of elevated [Na+]i on BKca activation --- p.153 / CONCLUSION --- p.161 / References --- p.162

The Rem, Rem2, Rad, and Gem/Kir GTPases, comprise a novel subfamily of the small Ras-related GTP-binding proteins known as the RGK GTPases, and have been shown to function as potent negative regulators of high voltage-activated (HVA) Ca2+ channels upon overexpression. HVA Ca2+ channels modulate Ca2+ influx in response to membrane depolarization to regulate a wide variety of cellular functions and they minimally consist of a pore-forming α1 subunit, an intracellular β subunit, and a transmembrane complex α2/δ subunit. While the mechanisms underlying RGK-mediated Ca2+ channel regulation remain poorly defined, it appears that both membrane localization and the binding of accessory Ca2+ channel β subunits (CaVβ) are required for suppression of Ca2+ channel currents. We identified a direct interaction between Rem and the L-type Cavα1 C-terminus (CCT), but not the CCT from CaV3.2 T-type channels. Deletion mapping studies suggest that the conserved CB-IQ domain is required for Rem:CCT association, a region known to contribute to both Ca2+-dependent channel inactivation and facilitation through interactions of Ca2+-bound calmodulin (CaM) with the proximal CCT. Furthermore, both Rem2 and Rad GTPases display similar patterns of CCT binding, suggesting that CCT represents a common binding partner for all RGK proteins. While previous studies have found that association of the Rem C-terminus with the plasma membrane is required for channel inhibition, it is not required for CaVβ- subunit binding. However, Rem:CCT association is well correlated with the plasma membrane localization of Rem and more importantly, Rem-mediated channel inhibition upon overexpression. Moreover, co-expression of the proximal CB-IQ containing region of CCT (residues 1507-1669) in HIT-T15 cells partially relieves Rem blockade of ionic current. Interestingly, Ca2+/CaM disrupts Rem:CCT association in vitro. Moreover, CaM overexpression partially relieves Rem-mediated L-type Ca2+ channel inhibition and Rem overexpression alters the kinetics of calcium-dependent inactivation. Together, these data suggest that the association of Rem with the CCT represents a crucial molecular determinant for Rem-mediated L-type Ca2+ channel regulation and provides new insights into this novel channel regulatory process. These studies also suggest that instead of acting as complete Ca2+ channel blockers, RGK proteins may function as endogenous regulators for the channel inactivation machinery.