MECHANISMS OF STEROID-INDUCED HYPERTENSION IN MAN AND RAT George Jack Mangos MB BS Thesis submitted for the degree of Doctor of Medicine University of New South Wales Supervisors: Judith A Whitworth John J Kelly Department of Medicine, St George Hospital, University of New South Wales, Kogarah, NSW, Australia 1999 Mangos GJ, MD TABLE OF CONTENTS Declaration. iv Foreword. .v Acknowledgements. vii Publications. .ix Abstract. .xii Abbreviations. .xiv Errata. xvii Chapter 1 The role of cortisol in human hypertension. 1 Chapter 2 General methods. 73 Chapter 3 Endothelial function in cortisol-induced hypertension in man . 117 Chapter 4 Endothelial function in dexamethasone-induced hypertension in man . 135 Chapter 5 Endothelial function in fludrocortisone-induced hypertension in man . 155 ii Mangos GJ, MD Chapter 6 Can the features of exogenous adrenocorticotrophin excess in the rat be explained by the adrenal production of corticosterone ? . .172 Chapter 7 The role of the parathyroid glands in adrenocorticotrophin - induced hypertension in the rat. 212 Chapter 8 Rat bioassay for the detection of parathyroid hypertensive factor. .230 Chapter 9 Summary. 251 Bibliography. .256 iii Mangos GJ, MD Declaration The work in this thesis contains no material submitted for the award of any other degree or diploma in any university. The work described in this thesis is directly attributable to my own research. Acknowledgement has been made where I have benefitted from collaboration with individuals other than my supervisors. George J Mangos June 1999 iv Mangos GJ, MD Foreword The work presented in this thesis is concerned with mechanisms of steroid- induced hypertension in man and rat. Each chapter has its own introduction and conclusions and can be read independently. The aim of this thesis is to examine mechanisms by which adrenal steroids raise blood pressure in man and rat. There is large literature describing the physiology of steroid excess in experimental models in rat, sheep and man in ACTH-induced hypertension, dating back to work which commenced in the 1970s at the Howard Florey Institute of Medical Research in Melbourne (Whitworth, 1977). A key feature of this body of research is the dissociation of sodium and water retention from the blood pressure raising effects of ACTH and cortisol in man (Connell, Whitworth et al, 1988). Some of the mechanisms of ACTH- and steroid-induced hypertension which have been explored during this time include the role of the SNS (Sudhir, Jennings et al, 1989, Tam, Williamson et al, 1997, Macefield, Williamson et al 1998), delineation of the haemodynamic profiles (Connell, Whitworth et al, 1987) and the actions of a variety of vasoactive hormones (Whitworth, van Leeuwen et al, 1984, Connell, Whitworth et al, 1987, Connor, Whitworth et al, 1987). Since the 1970s, significant discoveries have occurred which have further increased our understanding of cardiovascular pathophysiology. These v Mangos GJ, MD include; the understanding of endothelial function - the Nobel prize winning discovery of the endothelial derived relaxing factor in 1980 (Furchgott and Zawadzki, 1980) and the subsequent description of nitric oxide accounting for this factor in 1987 (Palmer, Ferrige et al, 1987), the discovery of endothelin (Yanagisawa, Kurihara et al, 1988), the description of the various natriuretic peptides, the introduction of new classes of antihypertensive agents such as the angiotensin converting enzyme inhibitors and more recently the angiotensin II receptor antagonists and the description of new vasoactive hormones such as parathyroid hypertensive factor (Lewanczuk, Wang et al, 1989) and the dilator adrenomedullin (Kitamura, Kangawa et al, 1993). In this thesis, I have examined some of the mechanisms by which ACTH and steroid hormones raise blood pressure. In man, I have examined the role of endothelial dilator function in cortisol-induced hypertension and whether the endothelial dysfunction observed in this model of hypertension can be attributed to GR- or MR-mediated effects. In the rat, I have examined whether ACTH-induced hypertension is explicable in terms of ACTH-induced glucocorticoid production (corticosterone) as it is in man (cortisol) (Whitworth, Saines et al, 1984). Further, I have examined the role of the parathyroid glands (and the putative parathyroid hypertensive factor) in ACTH-induced hypertension in the rat. vi Mangos GJ, MD Acknowledgements These studies were performed at the Department of Medicine, St George Hospital and were generously assisted by travel grants to Edmonton from the Australia and New Zealand Society of Nephrology (COBE Haemodialysis Travel Award) and ASTRA Pharmaceuticals. Travels grants to national and international meetings were also kindly provided by the Foundation for High Blood Pressure Research and the High Blood Pressure Research Council of Australia. I also graciously acknowledge the financial support of Associate Professor MA Brown and Professor JA Whitworth in my travel to Edmonton. My supervisors, Professor JA Whitworth and Dr JJ Kelly have provided invaluable support and guidance in the planning, execution and analysis of these studies and to them I am very grateful. I am also indebted to Associate Professor Mark Brown for his supervision of the earlier experiments and his expert advice. Dr Brian Walker and Professor David Webb collaborated with our group in the human studies and provided expertise in the technical aspects of establishing bilateral strain gauge venous plethysmography in the laboratory at St George Hospital. The human studies described were performed entirely by myself at St George Hospital. I am grateful to Johneen Tierney and Anthony Galuzzo of the Pharmacy, St George Hospital, for their assistance in randomization of the human studies and preparation of drugs. I am also vii Mangos GJ, MD grateful for technical assistance provided by Jane Lawson, Arlene Robinson, Allison Martin and Paula Williamson in these studies. I performed the rat studies at St George Hospital. In 1995, I travelled to the University of Alberta, Edmonton, Canada, where I performed a small series of experiments in the laboratory of Dr RZ Lewanczuk, to whom I am grateful (Chapter 8). I am particularly indebted to Steven Turner for his generous assistance with the series of corticosterone experiments (Chapter 6), where he provided assistance with blood pressure and metabolic measurements and analysis of plasma nitrate/nitrite concentration. I am also grateful for the advice and technical assistance provided by Dr Cheng Wen, Dr Ming Li, Tafline Fraser and John Ramunni in these experiments. Novartis generously provided ACTH for use in these experiments. In the human and rat experiments, The South Eastern Area Laboratory Service kindly provided pathology services. I would also like to thank Dr Therese Jacques for allowing me to use the Intensive Care blood gas analyser and Dr Peter Varelis, Nuclear Medicine, St George Hospital, for his assistance with peptides. Finally, I would like to thank my wife Nella for her patient support during these years. viii Mangos GJ, MD Publications & presentations arising from this thesis PUBLICATIONS Mangos GJ, Brown MA, Whitworth JA. Difficulties in detection of parathyroid hypertensive factor in the rat. Clinical and Experimental Pharmacology and Physiology 1998. 25: 936 - 938. Kelly JJ, Mangos GJ, Williamson PW, Whitworth JA. Cortisol and hypertension. Clinical and Experimental Pharmacology and Physiology 1998. 25 (suppl): S51 - S56. IN PRESS Mangos GJ, Fraser TB, Turner SW, Whitworth JA. The role of the parathyroid glands in adrenocorticotrophin-induced hypertension in the rat. Clinical and Experimental Hypertension 1999. SUBMITTED Mangos GJ, Walker BRW, Kelly JJ, Lawson JA, Webb D, Whitworth JA. Cortisol inhibits cholinergic vasodilatation in the human forearm (1999). IN PREPARATION Mangos GJ, Walker BRW, Kelly JJ, Lawson JA, Whitworth JA. The effects of fludrocortisone and dexamethasone on forearm blood flow in man (1999). ix Mangos GJ, MD Mangos GJ, Turner SW, Fraser TB, Whitworth JA. Are the features of adrenocorticotrophin-induced hypertension in the rat explained by the adrenal production of corticosterone ? (in preparation) 1999. PRESENTATIONS “Endothelial function in steroid-induced hypertension.” Oral presentation at the Annual Scientific Meeting of the Australian and New Zealand Society of Nephrology, Brisbane, March 1999. “Forearm vascular responses to acetylcholine in glucocorticoid-treated subjects.” Oral presentation at the International Society of Hypertension Scientific Meeting in Amsterdam, June 1998. “Inhibition of cholinergic vasodilatation in the human forearm is not mediated by mineralocorticoid or glucocorticoid receptors.” Poster presentations at the St George Hospital Annual Symposium and the High Blood Pressure Research Council Annual Scientific Meeting, Melbourne, November and December 1998. “Parathyroid hypertensive factor in the SHR.” Poster presentation at the International Society of Nephrology Scientific Meeting, Sydney, May 1997. x Mangos GJ, MD “Corticosterone administration mimics ACTH-induced hypertension in the rat and is prevented by L-arginine.” Poster and oral presentations at the St George Hospital Medical Symposium and the Annual Scientific Meeting of the High Blood Pressure Research Council of Australia, Fremantle, November and December, 1997. “The role of the parathyroid glands in adrenocorticotrophin-induced hypertension in the rat.” Oral presentation at the scientific meeting of the NSW branch