Actions of hypoxia on catecholamine synthetic enzyme mRNA expression before and after development of adrenal innervation in the sheep fetus

Abstract

We have investigated adrenal mRNA expression of the catecholamine synthetic enzymes tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) following acute hypoxia in fetal sheep before (< 105 days gestation, n = 20) and after (> 125 days gestation, n = 20) the development of adrenal innervation and following pretreatment with the nicotinic receptor anatgonist hexamethonium (n = 12). Total RNA was extracted from fetal adrenal glands collected at specific time points at 3-20 h after the onset of either hypoxia ( approximately 50% reduction in fetal arterial oxygen saturation (SO2) for 30 min), or normoxia. Before 105 days, there was a decrease in adrenal TH mRNA expression at 20 h after hypoxia and adrenal TH mRNA expression was directly related to the changes in arterial PO2 measured during normoxia and hypoxia. After 125 days, adrenal TH mRNA levels were suppressed for up to 12 h following hypoxia. In both age groups, adrenal PNMT mRNA expression increased at 3-5 h after hypoxia and was inversely related to the changes in fetal arterial PO2 during normoxia or hypoxia. After 125 days, the administration of hexamethonium (25 mg kg(-1), I.V.) reduced TH mRNA but not PNMT mRNA expression after normoxia. After hexamethonium pretreatment, there was no significant change in either adrenal TH or PNMT mRNA expression following hypoxia. We conclude that acute hypoxia differentially regulates adrenal TH and PNMT mRNA expression in the fetal sheep both before and after the development of adrenal innervation. After the development of adrenal innervation, however, the effect of acute hypoxia upon adrenal TH and PNMT mRNA expression is dependent upon neurogenic input acting via nicotinic receptors.

Figure 1. Effects of experimental hypoxia and normoxia upon fetal arterial blood gas parameters

Average arterial P_O2 (A, B), P_CO2 (C, D), and pH (E, F) values for 96-105 days (left-hand panel; A, C, E) and 129-144 days (right-hand panel; B, D, F) fetal sheep exposed to a 30 min period of experimental hypoxia (•; 96-105 days, n = 14; 129-144 days, n = 15) or normoxia (○; 96-105 days, n = 5; 129-144 days, n = 4) as indicated by the shaded areas on the graphs. Values from 129-144 days fetal sheep treated with hexamethonium (Hex) and exposed to either normoxia (▿, n = 6) or hypoxia (▾, n = 6) are also shown. *P < 0.05, significant difference from basal values for hypoxia group; †P < 0.05, significant difference from basal values for hypoxia + Hex group; ‡P < 0.05, significant difference from basal values for normoxia + Hex group.

Figure 2. Time course of acute hypoxia induced changes in adrenal TH and PNMT mRNA expression in the fetal sheep

The ratios of adrenal TH mRNA:18 S rRNA (A, B) and PNMT mRNA:18 S rRNA (C, D) expression in 96-105 days (left-hand panel; A, C) and 129-144 days (right-hand panel; B, D) fetal sheep 3-5 h (96-105 days, n = 5; 129-144 days, n = 5), 12 h (96-105 days, n = 5; 129-144 days, n = 5), and 20 h (96-105 days, n = 4; 129-144 days, n = 5) after the onset of a 30 min period of experimental hypoxia or normoxia (control; 96-105 days, n = 5; 129-144 days, n = 5). *P < 0.05, significant difference from control value.

Figure 3. Autoradiographs of Northern blots of total adrenal RNA from fetal sheep exposed to acute hypoxia

Representative autoradiographs of a Northern blot after hybridisation of radiolabelled TH cDNA, PNMT and 18 S antisense oligonucleotide probes with total RNA (20 μg lane⁻¹) extracted from adrenal glands collected from fetal sheep at 129-144 days gestation after experimental normoxia (Control, indicated by area under open triangle, n = 5) or 3-5 h (n = 5), 12 h (n = 5), and 20 h (n = 5) after 30 min of experimental hypoxia (indicated by the areas under the filled triangles). The approximate sizes of the relevant transcripts are indicated on the right-hand side of the panels.

Figure 4. Relationships of adrenal TH and PNMT mRNA expression with experimentally induced changes of arterial P_O2 in fetal sheep

The expression of adrenal TH mRNA:18 S rRNA (A) and PNMT mRNA:18 S rRNA (B) plotted against the experimentally induced changes in arterial P_O2 (ΔP_O2) for 96-105 days (○; A, control and 20 h values; B, control and 3-5 h values) and 129-144 days fetal sheep (•; A, control and 3-5 h values; B, control and 3-5 h values). Dotted and continuous lines represent the lines of best fit derived by regression analysis for the 96-105 days (TH mRNA = 3.1ΔP_O2+ 54.1, r²= 0.55, P < 0.05; PNMT mRNA = -19.0ΔP_O2+ 202.9, r²= 0.42, P < 0.05) and the 129-144 days groups (TH mRNA = -0.5(ΔP_O2)³– 4.1(ΔP_O2)²+ 8.3(ΔP_O2) + 117.9, r²= 0.82, P < 0.05; PNMT mRNA = -9.4 ΔP_O2+ 75.6, r²= 0.69, P < 0.01), respectively.

Figure 5. The effect of ganglionic blockade upon the hypoxia-induced changes in adrenal TH and PNMT mRNA expression in 129-144 days fetal sheep

Adrenal TH (A) and PNMT mRNA (C) expression in 129-144 days fetal sheep following exposure to experimental hypoxia (filled columns, 3-5 h post hypoxia) or normoxia (open columns) following the administration of either hexamethonium (25 mg kg⁻¹) or saline. Different superscript letters denote significant differences (P < 0.05) in the average adrenal expression of TH mRNA or PNMT mRNA between groups. The right-hand panel shows the expression of adrenal TH mRNA (B) and PNMT mRNA (D) plotted against the experimentally induced changes in arterial P_O2 (ΔP_O2) for hexamethonium-treated 129-144 days fetal sheep.