Alterations in renal and myocardial adrenoceptors associated with ethynyloestradiol- and levonorgestrel-induced hypertension in the rat

Hypertension is one of many side effects of oral contraceptive use in a small percentage of women. Although the underlying pathology has yet to be fully resolved, alterations in the renin-angiotensin-aldosterone axis, sympathetic nervous system/ renal and cardiac function have been implicated. In the thesis to be presented, the possible involvement of alterations in renal and myocardial adrenoceptor characteristics in the pathogenesis of steroid contraceptive-induced hypertension in rats was examined by radioligand binding techniques. In Chapter 2, a rat model of OC hypertension is described. Chronic low-dose administration of ethynyloestradiol (EE2), levonorgestrel (NG) or a combination of both steroids (EE2/NG) to female Sprague-Dawley rats was shown to significantly increase systolic blood pressure (SBP). Renal and cardiac hypertrophy developed in association with EE2-, EE2/NG- but not NG-induced hypertension. Moreover, whereas administration of NG alone attenuated body weight gain, combined EE2/NG administration increased body weight gain from the second week of treatment onwards. Based on the above observations, it is proposed that EE2 and NG induce hypertension in rats via different mechanisms. Although SBP was elevated to a similar maximum in all steroid-treated groups (+ 20 mmHg compared to controls), only with EE2 administration did SBP remain elevated for the duration of the 17 week treatment regimen. NG may therefore have a protective effect on blood pressure with long-term combined steroid contraceptive treatment. In Chapter 4, renal adrenoceptors were characterized using radioactively labelled adrenocephor antagonists. Under appropriate conditions, binding of [3H]-prazosin and [3H]-rauwolscine to membrane preparations of whole rat kidney displayed the kinetics, saturability and specificity of α1- and α2 -adrenoceptors respectively, which were present in a ratio 3:1. In contrast, [3H]-dihydroergocryptine ([3H]-DHE) apparently bound to both α1 and α2-adrenoceptors. Binding sites identified by [125I] –iodocyanopindolol (ICYP) had the recognition characteristics of β-adrenoceptors. In drug competition studies using the subtype-selective antagonists practolol (β1) and ICI 118,551 (β2)/ the ratio of β1- to β2 -adrenoceptors was found to be approximately 2:1. Subsequently, renal adrenoceptors were investigated at various stages during the development of hypertension with the different steroid contraceptive treatments (Chapters 5 and 6). Preliminary binding studies with [3H]-DHE and [3H]-prazosin suggested that the number of renal α2 - but not α1-adrenoceptors was reduced in rats with established EE2-induced hypertension (17 weeks treatment). This was subsequently confirmed using [3H]-rauwolscine, which in addition showed that the reduction in renal α2 -adrenoceptor number occurred during the developmental stage of EE2/NG~induced hypertension (6 weeks treatment) and established EE2-induced hypertension (12 weeks treatment). NG induced hypertension was unassociated with changes in renal α1- and α2-adrenoceptor characteristics. Renal β-adrenoceptor affinity was reduced in established EE2-, but not NG- or EE2/NG- induced hypertension. Moreover, the β-adrenoceptor agonist (-)-isoprenaline bound to renal β-adrenoceptors with reduced affinity following EE2 administration. Several endogenous and synthetic steroids were found to be ineffective inhibitors of [3H] –prazosin, [3H] –rauwolscine and ICYP binding excluding a direct interaction of these steroids with renal α1-, α2- and β -adrenoceptors. In Chapter 7, myocardial adrenoceptors were characterized and investigated in steroid-treated rats. In membrane preparations of whole myocardium, [3H]-prazosin binding was characteristically to α1- adrenoceptors, whereas there was a notable absence of [3H]-rauwolscine binding. Using ICYP, β-adrenoceptors were also detected, the ratio of β1- to β2~adrenoceptors being 3:1. Steroid contraceptive-induced hypertension was not associated with myocardial α1-adrenoceptor changes. Similarly, myocardial β-adrenoceptors were unchanged in established EE2-, NG- and EE2/NG-induced hypertension (12 weeks treatment). The affinity of (-)-isoprenaline for myocardial β-adrenoceptors was unaffected by EE2 aditiinistration. These studies suggest that established EE2- but not NG-induced hypertension in rats is associated with selective alterations in renal α2- and (β-adrenoceptors. These adrenoceptor changes may help to maintain elevated blood pressure by affecting the control of renal function by the sympathetic nervous system, catecholamines and several hormones which affect renin release and the transport of fluid and electrolytes in the nephron.

ß1/ß2/ß3-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β1/β2/β3). To test this hypothesis, we generated β1/β2/β3-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.