Vessel-Specific Regulation of Angiotensin II Receptor Subtypes During Ovine Development

Abstract

Umbilical and systemic responses to angiotensin II differ in term fetal sheep, and peripheral vascular responses are attenuated or absent before and after birth. These observations may reflect developmental differences in angiotensin II receptor (AT) subtypes in vascular smooth muscle (VSM). Studies of AT subtype ontogeny and regulation are generally limited to the aorta, which may not be extrapolated to other arteries, and neither is completely described during ovine development. We therefore characterized VSM AT subtype expression and regulation throughout an extended period of development in umbilical and carotid artery and aorta from fetal (85-146 d gestation), postnatal (5-23 d), and adult sheep, measuring AT 1 and AT 2 mRNA and protein and performing immunohistochemistry. Parallel increases in umbilical AT 1 mRNA and protein began early in gestation and continued to term, and although AT 2 mRNA was unchanged, protein levels decreased Ͼ90% at term. Fetal carotid AT 1 mRNA was Ͻ40% of adult values and unchanged before birth; however, AT 1 protein rose Ͼ2-fold at term. After birth, AT 1 mRNA increased to 85% of adult values and was associated with another 2-fold rise in protein. In contrast, carotid AT 2 mRNA and protein fell in parallel throughout development and were barely detectable in the newborn and the adult. Immunostaining was consistent with observations in both arteries. A third pattern occurred in aortic VSM. The ontogeny of AT subtype expression and regulation is vessel specific, with changes in umbilical VSM beginning very early in development. Although the mechanisms that regulate mRNA and protein expression are unclear, these changes parallel differences in VSM maturation and function and local blood flow. The renin-angiotensin system (RAS) is expressed early in gestation and considered an important modulator of cardiovascular development, adaptation, and blood pressure control before and after birth (1-4). In fetal and neonatal sheep, hemorrhage and hypovolemia increase circulating angiotensin II (Ang II) (5-7). Although inhibition of Ang II receptors (AT) or converting enzyme accentuates hypovolemic episodes (3,7), their effects on basal arterial pressure are inconsistent (8,9). AT blockade also modifies the baroreflex and reflex control of renal sympathetic nerve activity after birth (10,11). Recent evidence suggests the RAS also contributes to the differentiation, maturation, and/or growth of vascular smooth muscle (VSM) (12-15). Thus, prevailing evidence suggests the RAS contributes to the regulation of vascular function, maturation, and growth during development.The effects of the RAS are mediated primarily by Ang II activation of ATs, which belong to the superfamily of seven transmembrane receptors (12,16,17). They are present in mammalian fetal and adult VSM and demonstrate similar binding characteristics during development and in the adult (18 -20). In fetal sheep, AT binding density and affinity in aorta and placental arteries are unchanged in the last third of gestation and resemble ...

Meconium Increases Type 1 Angiotensin II Receptor Expression and Alveolar Cell Death

“…At the time of tissue collection, pieces of left lung were frozen on dry ice and stored at Ϫ20°C. At the time of assay, sodium dodecyl sulfate (SDS) homogenates were prepared from 15-20 mg samples (23). The homogenate was centrifuged at 10,000 ϫ g for 2 min, and the supernatant containing soluble protein was used to determine protein contents (BCA reagent, Pierce, Rockford, IL); 30 g were subjected to electrophoresis in 7.5% polyacrylamide gels and transferred to nitrocellulose paper (Amersham Pharmacia Biotech Inc., Piscataway, NJ).…”

“…Blots were blocked in buffer containing powdered milk (5% wt/vol) and incubated overnight at 4°C with antiserum against AT 1 R (1:1500; N-10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or AT 2 R (1:3000). Both have been validated (23)(24)(25). The nitrocellulose paper was incubated with donkey anti-rabbit IgG conjugated with affinity purified horseradish peroxidase diluted at 1:5000 with TTBS.…”

“…Densitometry was performed, and values were expressed as arbitrary units. Ovine umbilical artery smooth muscle at 145 and 116 d of ovine gestation served as positive controls for AT 1 R and AT 2 R, respectively (23).…”

“…Additional lung segments were fixed in 4% paraformaldehyde and embedded in paraffin (23). Sections were mounted on positive slides, deparaffinized, hydrated, incubated with avidin-biotin blocking agent, and incubated overnight with 1:200 AT 1 R antibody, 1:3000 AT 2 R antibody or diluent.…”

“…We performed semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays to examine subtype mRNA in lung tissue. Samples stored at Ϫ80°C had total RNA extracted and concentration and purity measured as described (23). RT was performed with 2 g total RNA in 50 L of reaction solution (23).…”