Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values. Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

L'objectif principal de ma thèse était de développer de nouvelles technologies et des outils pour l’étude de l’activation des récepteurs couplés aux protéines G (GPCR).À la surface de la cellule se trouve une multitude de récepteurs qui jouent un rôle critique dans la communication cellule-cellule, dont les GPCR, une famille de récepteur utilisant les protéines G intracellulaires pour transmettre leurs signaux. Le ciblage de ces récepteurs à des fins thérapeutique est innovant et très prometteur. Mais à ce jour seuls quelques médicaments ciblant les GPCR ont été mis sur le marché, en partie en raison d'un manque d'outils permettant le suivi de leur action sur les cellules natives.L’objectif de cette thèse est donc de développer des tests simples pour suivre l’activation de n’importe quel GPCR. Pour développer ce type de test, nous avons décidé d'utiliser des fragments d'anticorps appelés nanobodies. Les anticorps sont des protéines du sang produites en réponse à un antigène spécifique qui sont capable de le neutraliser. Les nanobodies correspondent au domaine variable de certains anticorps de camélidés. En raison de leur faible taille (13 kDa) et de leur site de liaison à l'antigène réduit, les nanobodies se lient souvent à des cavités et présentent une grande sensibilité aux changements de conformation de l'antigène. / The main objective of my thesis was to develop technologies and tools to study activation of G protein-coupled receptors (GPCRs).The cell surface is displaying a multitude of receptors, who play critical roles in cell-cell communication. Among them, GPCRs represent a large family relying on the use of intracellular G proteins for their signaling. Targeting these receptors for therapies is very promising and innovative. So far, only few new drugs have been put on the market, partly due to a lack of tools enabling the follow-up of their action on native cells.The aim of this thesis is thus to develop simple assays to study activation of any GPCRs. To develop this kind of test, we used antibody fragments called nanobodies. Antibodies are blood protein produced in response to and counteracting a specific antigen. Nanobodies correspond to antibody fragments derived from the variable domain of a special class of camelid antibodies. Because of their small size (13 kDa) and reduced antigen binding site, nanobodies often bind cavities and show a high sensitivity to antigen conformational changes.

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The mammalian pineal gland is mainly innervated by the sympathetic nervous system which modulates the activity of indole pathway enzymes and the secretion of pineal hormones. Recently researchers have demonstrated and characterized the presence of muscarinic cholinergic receptors in the pineal gland. However the role of these receptors remains unclear. In an attempt to investigate the role of cholinergic receptors in the pineal gland, a number of studies were carried out on the various steps in the indole metabolic pathway, using various agents which act on the cholinergic system. Investigations using pineal organ cultures showed that stimulation of these muscarinic cholinergic receptor sites with a parasympathomimetic agent, a rise in levels of aHT occurred without a concomitant increase in aMT levels. Further organ culture experiments using the cholinergic agonist acetylcholine and anticholinesterase agent physostigmine, produced a similar rise in aHT without altering aMT levels. This acetylcholine-induced rise in aHT levels were not altered by the ganglion blocking agent hexamethonium whilst the antimuscarinic agent atropine prevented the acetylcholine-induced rise in aHT levels. Thesefindings suggest that cholinergic agents may play a role in regulating indoleamine synthesis in the pineal gland. Cyclic-AMP assay studies showed that acetylcholine increases pineal cAMP levels significantly and does not influence the isoproterenol-induced cAMP rise in the pineal gland. The cAMP regulator cAMP-phosphodiesterase (cAMP-PDE) was found to increase significantly in the presence of the anticholinesterase agent physostigmine. NAT enzyme studies revealed that physostigmine does not affect NAT enzyme levels significantly and HIOMT studies showed that this agent does not inhibit HIOMT activity. The mechanism by which acetylcholine and physostigmine are able to cause a increase in aHT and not aMT levels needs to be researched further. Acetylcholinesterase enzyme assay studies revealed that the AChE enzyme undergoes a diurnal rhythm in the pineal gland with activity being higher during the day and lower at night. Investigations using the drug reserpine showed that this rhythm is not under the control of the sympathetic nervous system. Further research needs to be done however, in determining whether or not this enzyme is present in the pineal gland to regulate the levels of acetylcholine interacting with muscarinic receptors in the gland, or for some other reason. Choline acetyltransferase studies demonstrate the presence of the enzyme in the rat brain cerebral cortex as well as showing that melatonin increases ChAT enzyme activity in this tissue. This suggests that melatonin plays a role in cholinergic transmission there. ChAT activity could not be measured in the pineal gland however. Muscarinic receptor binding studies also carried out on rat brain cerebral cortex show that melatonin enhances cholinergic receptor affinity and receptor number in this tissue. In summary, data presented herein concur with proposals that: i) the cholinergic system affects the indole metabolic pathway by causing a rise in aRT but not aMT levels. ii) cholinergic agonist acetylcholine causes cAMP levels to rise with a concomitant increase in cAMP-PDE levels. iii) the enzyme acetylcholinesterase undergoes a diurnal rhythm in the pineal gland which is not under the control of the sympathetic nervous system. iv) the activity of the enzyme choline acetyltransferase is increased by melatonin in the rat brain cerebral cortex suggesting that melatonin facilitates cholinergic transmission in this tissue. v) melatonin enhances cholinergic receptor affinity and receptor number in the cerebral cortex of rat brain.

In 1985, Mendelson and Gorzalka proposed the dual role hypothesis of serotonergic modulation of lordosis behaviour. In this hypothesis it was proposed that serotonergic activity can either inhibit or facilitate lordosis behaviour. Specifically it was suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at 5-HT₁ receptors, whereas lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ receptors. The purpose of the following series of studies was both to confirm and to extend the dual role hypothesis. The intraperitoneal administration of the 5-HT2 antagonists pizotefin (1 mg/kg), cyproheptadine (1 mg/kg), metitepine (1 mg/kg), and ketanserin (1 mg/kg) were found to inhibit lordosis behavior in ovariectomized rats that had been primed with estradiol benzoate (EB) and progesterone (P). Pipamperone was ineffective. The 5-HT₂ agonist guipazine (3 mg/kg) was ineffective alone, but it reversed the inhibitory effects of pizotefin, cyproheptadine, and ketanserin. It did not reverse the effects of metitepine. The highly selective 5-HT₂ antagonist LY53857 (0.3 mg/kg) was also found to inhibit lordosis behaviour in female rats that had been primed with EB and P. The lordosis-inhibiting effect of LY53857 (1 mg/kg) in females primed with EB and P was reversed by quipazine (3 mg/kg). The nonselective 5-HT antagonist methysergide (7 mg/kg) was found to inhibit lordosis behavior 30 min after intraperitoneal administration to females treated chronically with EB, or with EB and P. However, methysergide was found to facilitate lordosis behavior 200 and 300 min after administration to female rats treated acutely with EB. In an analysis of dose response it was found that methysergide (0.02 - 7 mg/kg) administered 30 min prior to behavioural testing produced no facilitation of lordosis in females primed with EB. However, when administered 200 min prior to testing, methysergide (1 mg/kg) produced a significant facilitation of lordosis. The administration of the 5-HT₁ A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) inhibited lordosis behavior in ovariectomized rats primed with EB. 8-OH DPAT was ineffective at 0.01 mg/kg, whereas inhibition occurred at the 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg doses. In an evaluation of the effects of 8-OH DPAT on the expression of male sexual behaviour by females treated chronically with testosterone, 8-OH DPAT ( 1 mg/kg) increased the number of females mounting and significantly increased mount frequency. The 5-HT₁ A agonists ipsapirone (0.1 mg/kg) and gepirone (0.3 mg/kg) facilitated lordosis in females treated with EB. When administered at higher doses, ipsapirone (3.0 mg/kg) and buspirone (3.0 mg/kg) inhibited lordosis in rats treated with EB. In females treated with EB and P, ipsapirone (> 1.0 mg/kg), gepirone (> 0.3), and buspirone (> 0.3) inhibited lordosis behaviour. The newly developed 5-HT₁ A antagonist BMY 7378 (0.2 mg/kg) facilitated lordosis behaviour in females treated with EB. However, this facilitation was no longer apparent at the 5 mg/kg dose. BMY 7378 (0.04 - 5 mg/kg) was ineffective in females primed with EB and P. The 5-HTTB agonist 1 -(3-trifluoromethylphenyl)piperazine (TFMPP, 0.2 -5 mg/kg) was found to facilitate lordosis in females treated with EB. In females primed with EB and P, TFMPP (5 mg/kg) produced a significant inhibition of lordosis. The 5-HT₁ B agonist m-chlorophenylpiperazine (MCPP, 0.04 - 5 mg/kg) was ineffective in females primed either with EB or with EB and P. The 5-HT₃ Antagonist ICS 205-930 (5 mg/kg) was found to facilitate lordosis behaviour, whereas the 5-HT₃ Antagonist MDL 72222 (0.05 - 5 mg/kg ) was found to be ineffective in females primed with EB. The results of these studies tend to confirm that serotonergic activity can either inhibit or facilitate lordosis behaviour. It is suggested that the lordosis-inhibiting effects of serotonin are mediated by activity at postsynaptic 5-HTTA and possibly 5-HT₃ Receptors. The lordosis-facilitating effects of serotonin are mediated by activity at 5-HT₂ and possibly presynaptic 5-HT₁ B receptors. Finally, it is suggested that activity at somato-dendritic 5-HT₁ A autoreceptors may mediate facilitatory effects of low doses of 5-HT₁ A agonists. In closing, there is a discussion of the implications these results might hold for the understanding of the effects of serotonergic drugs on human behaviour. / Arts, Faculty of / Psychology, Department of / Graduate

Three major systems participate in the control of the peripheral circulation: the renin-angiotensin, the arginine vasopressin (AVP) and the sympathetic nervous systems. These studies examined the roles of the AVP and the sympathetic nervous systems in the regulation of blood pressure at both the central and the peripheral level. Anatomical studies have revealed that hypothalamic neurons containing AVP extend to the nucleus tractus solitarius (NTS) in the medulla. Since the NTS is the primary site of termination of the afferent neurons of the baroreceptor reflex arc, it suggests that AVP may be involved in central cardiovascular regulation. The effect of central AVP on mean arterial pressure (MAP) and sympathetic nerve activity, estimated from plasma catecholamine levels, was investigated. The injection of AVP into the fourth cerebroventricle and NTS of conscious, unrestrained rats increased MAP and plasma noradrenaline and adrenaline levels, suggesting that AVP may act centrally at the NTS to modulate sympathoadrenal outflow. However, the injection of a selective vascular antagonist of AVP, d(CH₂)₅Tyr(Me)AVP, into the fourth ventricle or NTS did not affect MAP or plasma catecholamine levels, either in normotensive rats, in rats subjected to hypotensive stress, or in neurogenically-stressed rats. This suggests that endogenously-released AVP may not have a tonic influence on central cardiovascular regulation. The role of AVP in the control of MAP, cardiac output (CO) and its distribution was investigated in anesthetized, surgically-stressed rats. The i.v. injection of d(CH₂)₅Tyr(Me)AVP decreased MAP and total peripheral resistance (TPR), did not alter CO, and increased the distribution of blood flow (BF) to the stomach and skin. The vascular role of AVP was found to be greater in the absence of influence from the renin-angiotensin and the sympathetic nervous systems. After blockade of the renin-angiotensin system by the infusion of saralasin the AVP antagonist increased BF to the skin and muscle, while after blockade of the α-adrenergic system with the infusion of phentolamine, the AVP antagonist markedly increased BF to the muscle. Thus, the amount of vasoconstriction produced by AVP in different vascular beds was found to depend on the endogenous vasomotor tone from the renin-angiotensin and α-adrenergic systems. Cross-circulation studies were conducted to concurrently observe the peripheral and central effects of α-agonists in two anesthetized rats, designated rat A and B, respectively. The i.v. injection of clonidine into rat A was found to increase MAP and decrease HR in rat A, and reduce MAP and HR in rat B. Since the stimulation of peripheral α-adrenoceptors in rat A by clonidine increased MAP, it suggests that the effects of peripheral post-junctional α₂-adrenoceptors predominate over those of peripheral pre-junctional α₂-adrenoceptors. In contrast, the i.v. injection of the α₁-agonist, methoxamine, in rat A increased MAP and decreased HR in rat A, and increased both MAP and HR in rat B. This suggests that central α₁-adrenoceptors may mediate responses in the opposite direction to those produced by α₂-adrenoceptors. To verify the results of the cross-circulation studies in animals free of the influence of surgery and anesthesia, and to determine whether the responses to a-agonists were mediated by changes in sympathoadrenal outflow, clonidine and a more selective α₂-agonist, B-HT 920, were injected centrally in conscious rats. The i.e.v. injection of clonidine (1 µg) significantly decreased MAP and HR and slightly decreased plasma noradrenaline and adrenaline levels; however, contrary to expectations, the i.c.v. injection of B-HT 920 (1, 10 µg) increased MAP, decreased HR and slightly increased plasma noradrenaline and adrenaline levels. To determine whether the responses to central injection of clonidine or B-HT 920 were due to the stimulation of α₂-adrenoceptors, i.c.v. injections of these drugs were given after pretreatment with rauwolseine, a selective α₂-antagonist. The i.c.v. injection of rauwolscine in conscious rats increased MAP and plasma noradrenaline and adrenaline levels, suggesting that central α₂-adrenoceptors may mediate tonic inhibition of the cardiovascular system. However, i.c.v. injections of clonidine or B-HT 920 produced the same responses in the absence or presence of rauwolscine. Further studies with different α-adrenergic agonists and antagonists with various selectivities are necessary before we can explain the differential effects of central clonidine and B-HT 920. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

In order to characterize the types of histamine receptors (H₁ or H₂) in rabbit atria, isolated atrial preparations were exposed to selective histamine receptor agonists, alone and in the presence of selective histamine receptor antagonists. The rate of isolated right atria was increased by histamine (H₁ and H₂), 4-methylhistamine (H₂), and impromidine (H₂). 2-Pyridylethylamine (PEA, H₁) had very little effect on atrial rate. Cimetidine pretreatment (selective H₂ receptor blockade) competitively antagonized the positive chronotropic effects of histamine, 4-methylhistamine, and impromidine. Promethazine pretreatment (selective H₁ receptor blockade) competitively blocked the chronotropic effects of histamine, but had no effect on the responses to 4-methylhistamine or impromidine. The force of isolated left atria was increased by all four agonists. Cimetidine pretreatment competitively blocked the inotropic effects of histamine, 4-methylhistamine, and impromidine, and had no effect on the response to PEA. Promethazine pretreatment competitively blocked the inotropic responses to histamine and to PEA, and had no effect on the responses to 4-methylhistamine or impromidine. The phosphodiesterase inhibitor theophylline (3 x 10⁻⁴M) potentiated the inotropic but not the chronotropic effects of 4-methylhistamine. (1 x 10⁻⁴M , impromidine (1 x 10⁻⁸M) and PEA (1 x 10⁻⁴M) . Histamine (1 x 10⁻⁵M) exposure for 20 seconds did not alter the cyclic AMP levels of isolated rabbit right and left atria. We report that both types (H₁ and H₂) of histamine receptors are present in rabbit right and left atria. However, I cannot confirm reports (Hughes,1978) that both H₁ and H₂ histamine receptor agonists act through the generation of cyclic AMP. / Pharmaceutical Sciences, Faculty of / Graduate

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