Time course of inhibition of hypertension by antisense oligonucleotides targeted to AT1 angiotensin receptor mRNA in spontaneously hypertensive rats

Abstract

Antisense oligodeoxynucleotides (AS-ODN) can be designed to provide inhibition of a specific protein. Since angiotensin receptors are involved in blood pressure regulation we constructed AS-ODN to angiotensin II type-1 receptor (AT1) mRNA. When given centrally, the AS-ODN reduces blood pressure in spontaneously hypertensive rats (SHR) 24 h after injection. To study the time course of a single AS-ODN injection on blood pressure and heart rate, groups of SHR were injected intracerebroventricularly (icv) with either single dose of AS-ODN or scrambled (SC) ODN and blood pressure was recorded through implanted catheters daily for up to 9 days. Blood pressure decreased significantly in the AS-ODN treated rats compared to the SC-ODN rats for up to 7 days. The maximum decrease (38 mm Hg) occurred at 3 days. There appeared to be no toxic reaction or side effects and the blood pressure level had recovered by days 8 and 9. Heart rate was not altered by AS-ODN treatment. To test that the ODN was entering the brain tissue, fluorescein-isothiocyanate labelled (FITC) ODN was injected in Sprague-Dawley rats and the fluorescence detected 1 h later by confocal microscopy. Within 1 h there was rapid uptake into cells close to the site of injection and into brain parenchyma around the third and lateral ventricles. To test that the AS-ODN had reduced AT1 receptors, binding studies were carried out on membranes from hypothalamic tissue. There was a modest (approximately 20%) but significant (P < .05) decrease in the AT1 receptor binding after 25 microm or 50 microm AS-ODN. AT2 receptors were not altered by the AS-ODN, indicating its specificity for the AT1 receptor. The small decrease in receptor binding, relative to its large effect on blood pressure, is discussed in terms of the AT1 receptor life cycle. The mechanism for the long action of a single AS-ODN injection is hypothesized as resulting from the persistence of AS-ODN in the nucleus, preventing transport of the mRNA into the cytoplasm.