published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 1993.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaves 128-149).

Thesis (Ph. D.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 140-173).

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

8-Cyclopentyl-3-(3-(4-fluorosulfonylbenzoyl)oxy)propyl-propylxanthine (44, FSCPX) has been reported to exhibit potent and selective irreversible antagonism of the A1 adenosine receptor when using in vitro biological preparations. However, FSCPX (44) suffers from cleavage of the ester linkage separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine pharmacophore when used in in vivo biological preparations or preparations containing significant enzyme activity, presumably by esterases. Cleavage of the ester linkage renders FSCPX (44) inactive in terms of irreversible receptor binding. In order to obtain an irreversible A1 adenosine receptor antagonist with improved stability, and to further elucidate the effects of linker structure on pharmacological characteristics, several FSCPX (44) analogues incorporating the chemoreactive 4-(fluorosulfonyl)phenyl moiety were targeted, where the labile ester linkage has been replaced by more stable functionalites. In particular, ether, alkyl, amide and ketone linkers were targeted, where the length of the alkyl chain was varied from between one to five atoms. Synthesis of the target compounds was achieved via direct attachment of the N-3 substituent to the xanthine. These compounds were then tested for their biological activity at the A1 adenosine receptor via their ability to irreversibly antagonise the binding of [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX, ( 9) to the A1 adenosine receptor of DDT1 MF-2 cells. For comparison, the xanthines were also tested for their ability to inhibit the binding of [3H]-4-(2-[7-amino-2-{furyl} {1,2,4}- triazolo{2,3-a} {1,3,5}triazin-5-ylamino-ethyl)]phenol ([3H]ZM241385, 36) to the A2A adenosine receptor of PC-12 cells. The results suggest that the length and chemical composition of the linker separating the reactive 4-(fluorosulfonyl)phenyl moiety from the xanthine ring contribute to the potency and efficacy of the irreversible A1 adenosine receptor ligands. Like FSCPX (44, IC50 A1 = 11.8 nM), all derivatives possessed IC50 values in the low nM range under in vitro conditions. Compounds 94 (IC50 A1 = 165 nM), 95 (IC50 A1 = 112 nM) and 96 (IC50 A1 = 101 nM) possessing one, three and five methylene spacers within the linkage respectively, exhibited potent and selective binding to the A1 adenosine receptor versus the A2A adenosine receptor. Compound 94 did not exhibit any irreversible binding at A1 adenosine receptors, while 95 and 96 exhibit only weak irreversible binding at A1 adenosine receptors. Those compounds containing a benzylic carbonyl separating the 4-(fluorosulfonyl)phenyl moiety from the xanthine ring in the form of an amide (119, IC50 A1 = 24.9 nM, and 120, IC50 A1 = 21 nM) or ketone (151, IC50 A1 = 14 nM) proved to be the most potent, with compound 120 exhibiting the highest selectivity of 132-fold for the A receptor over the A2A receptor. compounds 119, 120 and 151 also strongly inhibited the binding of [3H]DPCPX irreversibly (82%, 83% and 78% loss of [3H]DPCPX binding at 100 nM respectively). compounds 120 and 151 are currently being evaluated for use in in vivo studies. Structure-activity studies suggest that altering the 8-cycloalkyl group of A1 selective xanthines for a 3-substituted or 2,3-disubstituted styryl, combined with N-7 methyl substitution will produce a compound with high affinity and selectivity for the A2A adenosine receptor over the A1 adenosine receptor. Compound 167 (IC50 A2A = 264 nM) possessing 8-(m-chloro)styryl substitution and the reactive 4-(fluorosulfonyl)phenyl moiety separated from the xanthine ring via an amide linker in the 3-position (as for 119 and 120), exhibited relatively potent binding to the A2A adenosine receptor of PC-12 cells, with a 16-fold selectivity for that receptor over the A1 adenosine receptor. However, compound 167 exhibited only very weak irreversible binding at A2A adenosine receptors. Overall, at this stage of biological testing, compound 120 appears to possess the most advantageous characteristics as an irreversible antagonist for the A1 adenosine receptor. This can be attributed to its high selectivity for the A1 adenosine receptor as compared to the A2A adenosine receptor. It also has relatively high potency for the A1 adenosine receptor, a concentration-dependent and selective inactivation of A1 adenosine receptors, and unbound ligand is easily removed (washed out) from biological membranes. These characteristics mean compound 151 has the potential to be a useful tool for the further study of the structure and function of the A1 adenosine receptor.

Vasopressin, a hormone released from the neurohypophysis, contributes to the regulation of body fluid balance through its known actions on the kidney and the vasculature. Release of vasopressin is influenced by plasma osmolality and by afferent activity from sensory receptors in the high and low pressure vascular systems. Previous studies have not defined the relative importance of the carotid sinus baroreceptors, aortic baroreceptors and atrial receptors in the control of the plasma concentration of vasopressin in the rabbit. Experiments were carried out in anaesthetized rabbits to define the quantitative relationship between stimulation of the carotid sinus baroreceptors and the plasma concentration of vasopressin. This relationship was examined in the presence and absence of afferent input from the aortic and atrial receptors. Changes in blood volume were induced to produce a change in the stimulus to the aortic baroreceptors and atrial receptors at high or low, constant carotid sinus pressure. Section, of the aortic depressor nerves and the vagus nerves allowed examination of the individual contributions of atrial receptors or aortic baroreceptors on the plasma concentration of vasopressin. It was also possible to examine the interaction between the carotid sinus baroreceptors and the aortic and atrial receptors. The results showed that plasma concentration of vasopressin was reduced by minimal stimulation of carotid sinus baroreceptors and that maximal inhibition of the release of vasopressin was achieved with a relatively low total arterial baroreceptor input. No influence of carotid sinus baroreceptors on vasopressin release was seen in the presence of intact aortic baroreceptors demonstrating the important interaction between the effects of stimulation of these two sets of receptors. It was not possible to demonstrate, in the rabbits used in this study, a significant contribution of atrial receptors to the control of vasopressin release either in response to changes in carotid sinus pressure or in response to changes in blood volume. To minimize the inhibitory effect of arterial baroreceptors on the release of vasopressin the aortic depressor nerves were cut and carotid sinus pressure was set at a low level. It was still not possible to demonstrate an effect of a reduction in blood volume on vasopressin release, confirming the absence of a contribution from atrial receptors in the anaesthetized rabbit. There appears to be considerable variation between species in the contribution of the different receptor groups to the release of vasopressin. The results suggest that in the normal rabbit there is likely to be significant tonic inhibition of the release of vasopressin by stimuli arising from arterial baroreceptors. The absence of a demonstrable influence of atrial receptors in these rabbits is consistent with findings in primates but differs from those in dogs. It is unlikely that changes in plasma vasopressin concentration induced by small changes in blood volume contribute to the control of arterial pressure through direct effects on vascular resistance and capacitance. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

When dabbling ducks are involuntarily submerged, arterial vasoconstriction produces a large increase in the peripheral resistance to blood flow which is balanced by a decrease in output of the heart, and arterial blood pressure is maintained. Arterial baroreceptors sense systemic blood pressure, and provide the afferent information to the baroreflex for pressure regulation. The effector limbs of the baroreflex are the same as those involved in the diving responses, and the baroreceptors are likely to be important in the integration of the cardiovascular responses to diving. The purpose of this study was to investigate the role of the arterial baroreceptors in maintaining blood pressure during diving, and in the initiation and maintenance of the diving responses. Baroreceptor function was studied by diving ducks at various times after barodenervation, and by electrically stimulating the central end of one baroreceptor nerve in baroreceptor-denervated animals to simulate a controlled baroreceptor input before and during submersion. Intact baroreceptor innervation was not necessary for the development of peripheral vasoconstriction, but loss of the baroreceptors modified the cardiac response to submersion by impairing the vagally mediated bradycardia. There was no effect of baroreceptor nerve stimulation on peripheral resistance during diving, and the baroreflex operated via the heart rate in modulating blood pressure early in the dive. Later in the dive, stimulation was ineffective in altering either heart rate or blood pressue. Strong chemoreceptor drive results from decreased blood oxygen and increased carbon dioxide levels in the dive, and the results of experiments to determine the mechanism of baroreflex attenuation showed that an interaction between chemoreceptor and baroreceptor inputs may be at least partly responsible for reducing baroreflex effectiveness. The main conclusion from this work is that the arterial baroreceptors contribute to the diving responses through modulation of heart rate, to help balance the fall in cardiac output against the baroreceptor-independent peripheral vasoconstriction in the first minute of forced dives. / Science, Faculty of / Zoology, Department of / Graduate