Introduction
β2 adrenergic receptor (ADRB2) agonists are currently widely used in the treatment of childhood and adult asthma and chronic obstructive pulmonary disease. ADRB2 agonists are mainly used to treat neonatal wet lung syndrome [
1], bronchopulmonary dysplasia (BPD), and wheezing in premature infants [
2,
3]; however, the indications are not unified. These agonists directly act on ADRB2 to activate ADRB2–G protein–adenylyl cyclase (AC) signaling associated with regulation of airway function. Therefore, receptor-density distribution and intensity are key factors affecting drug effects; however, the key genes associated with this signaling pathway and the spatiotemporal changes in the expression spectrum of some of their subtypes remain unclear. In particular, there are few studies on neonates at the perinatal stage, resulting in an insufficient theoretical basis for formulating the dose and method of drug administration for neonates.
ADRB2 is the main subtype of βAR in human lung. ADRB2 is a downstream effector of the adrenergic signaling pathway and specifically binds adrenaline synthesized by phenylethanolamine N-methyltransferase (PNMT) catalysis of noradrenaline [
4]. The binding of β2AR agonist with ADRB2 on cell membrane can activate adenylate cyclase(AC), and AC catalyzes the conversion of ATP into cAMP, increasing the level of cyclic adenosine monophosphate (cAMP) to induce airway smooth muscle (ASM) relaxation [
5]. There are currently at least nine AC subtypes identified in humans [
6]. cAMP performs its biological actions by activating various effectors, including protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), which has two isoforms, EPAC1 and EPAC2 [
7]. cAMP and its effectors are strictly spatiotemporal controlled by a scaffold protein family of more than 50 members called A-kinase anchoring proteins (AKAPs) [
8]. Meanwhile, phosphodiesterase (PDE) is a key enzyme involved in cAMP hydrolysis, resulting in a decrease in its concentration [
9].
There are 11 families and 30 subtypes of PDE in humans, of which PDE4(A, B, C, and D), PDE7(A and B), and PDE8 (A and B) have high specificity for cAMP [
10,
11]. PDE3(A and B) and PDE4 are the two major cAMP-hydrolyzing enzymes [
12].
In this study, we measured and analyzed spatiotemporal changes in key genes intimately associated with the ADRB2–Gs–AC signaling pathway and some of their subtypes during lung development in rhesus monkeys, particularly during the neonatal stage. The findings provide a theoretical basis for rational drug use in the neonatal stage and references for the development and utilization of some key gene subtypes in this pathway.
Discussion
PNMT is a rate-limiting and essential enzyme that catalyzes the methylation of noradrenaline to adrenaline [
16], and the only N-methyltransferase that can synthesize adrenaline [
17]. Human PNMT is mainly expressed in the adrenal medulla and also present in human lung tissues [
17,
18]. Previous studies report that the lungs can synthesize adrenaline locally and regulate adrenaline. Moreover, PNMT in human lungs and bronchial epithelial cells exhibit high substrate affinity and specificity similar to that of adrenal PNMT [
17]. In the present study, we found persistent high expression of
PNMT at the late gestational and neonatal stages in rhesus monkeys, with its peak observed at the neonatal stage. Moreover,
PNMT expression in the lungs at the neonatal stage was significantly higher than that in other tissues (brain, intestine, and liver), suggesting that the respiratory tract is connected to the external environment after birth, and that various natural stimuli require dynamic adaptations (such as airway relaxation). Adrenaline expression is an important method of bodily adaptation; therefore,
PNMT expression might represent a preparatory mechanism for increasing adrenaline. These findings suggest that a role of elevated
PNMT expression might be to prepare the fetus for birth maintaining its health at the neonatal stage.
Both
ADRB2 and
PNMT expression showed persistent and progressive increases in the late gestational and neonatal stages; however, in contrast to
PNMT,
ADRB2 expression increased with age and showed differences in expression only in the liver before and after delivery. Moreover,
ADRB2 expression in the lungs at the neonatal stage was not significantly different, whereas significant differences were present at the adolescence stage. This confirms that the density of ADRB2 receptor is related to age, reaching adult levels at school age [
19]. ADRB2 is mainly located in airway smooth muscle (ASM) cells, type II pneumocytes, mast cells, small blood vessels in the bronchi, and epithelial cells, among which ASM cell density is the highest. The main function of ADRB2 in ASM cells is to relax the airway [
4]. These findings suggest that PNMT and ADRB2 activate the Gs–AC–PKA signaling pathway to cause airway dilation, thereby ensuring optimal ventilation in the lungs [
20]. Furthermore, we found elevated expression of both PNMT and ADRB2 in the late gestational stage; therefore, we speculate that this adrenergic mechanism also applies to premature infants.
The results showed that all subtypes (except AC8, AKAP4/5) associated with the ADRB2–Gs–AC signaling pathway were expressed in the lungs at the neonatal stage. These results indicated that this signaling pathway might play a role in airway dilation during the neonatal stage.
We speculated that AC6 might play an important role in this signaling pathway. Previous studies report that transcripts of all AC subtypes, except
AC2, are detected in human ASM, and western blot results and functional testing show that AC5/6 exhibit important functions in hASM [
21]. Xu et al. detected mRNA for three AC subtypes (
AC2,
AC4, and
AC6) in cultured hASM cells [
22], and Shailesh et al. reported that the ADRB2 response in hASM is mainly associated with AC6 in lipid rafts [
23]. These findings indicated that specific expression of AC subtypes in hASM remains unclear.
PDE3 and PDE4 are the two major cAMP-hydrolyzing enzymes in ASM. PDE3 is an enzyme hydrolyzing both cAMP and cGMP, but the rate of hydrolyzing cAMP is 10 times that of hydrolyzing cGMP. PDE3 and PDE4 can regulate different cAMP pools because they are located in different parts of the ASM [
24,
25]. PDE3 is located in a compartment more closely associated to regulation of Ca
2+ fluxes affecting contractility. PDE3 inhibitor is important in preventing mast cells predominantly in the ASM layer degranulation. Therefore, it is an acute bronchodilator in humans [
26‐
29]. However, PDE4 inhibitor cannot induce acute bronchodilator, which is consistent with its lack of mast cell or ASM function [
24,
28]. Despite this, PDE4 inhibitors have been found to reduce the pro-inflammatory activity of hASM cells and thus increase airway relaxation, and PDE4 inhibitors show some efficacy against the late asthmatic response [
30‐
32]. Some scholars have proposed that PDE3/4 combined inhibitors have better bronchodilation and anti-inflammatory activities [
29,
33]. In this paper,
PDE3 and
PDE4 were expressed during the entire lung-development process. We speculate that the application of double PDE3/4 inhibitors may be feasible. Moreover, we found that
PDE4B was highly expressed during the entire lung-development process. Previous studies proposed that PDE4B and PDE4D played critical roles in airway cells [
34,
35], PDE4B performed many beneficial anti-inflammatory effects without the side effects, whereas PDE4D had vomiting effects related to central nervous system (CNS) [
36,
37]. We should pay more attention to PDE4B in ASM. However, PDE4B is also highly expressed in brain tissues and might be involved in CNS-related side effects.
AKAP has been shown to regulate intracellular cAMP localization and regulate ADRB2 signaling in human ASM [
38]. We found that
AKAP(1/2/
8/9/12/13, and
EZR) were highly expressed during the entire lung-development process. Previous studies have shown that there are protein and/or mRNA expressions of
AKAP(
1/2/3/5/8/9/10/11/12/13,
EZR, and
MAP2B) subtypes in human ASM. Especially,
AKAP12 and
EZR were highly expressed [
8,
39]. EZR(also known as Ezrin) is considered to be a key regulator of airway membrane receptor complex and its signal transduction pathway [
40]. We speculate that AKAP(1/2/
8/9/12/13, and EZR) play an important role in this pathway, especially EZR. Further studies are needed to confirm the expression of AKAPs subtypes in ASM.
However, our study has some limitations. First, because our samples were limited, we did not perform reverse transcription polymerase chain reaction analysis to confirm expression levels, western blotting analysis to detect protein levels, or immunohistochemical analysis to determine protein localization. We focused solely on the expression of key genes in this signaling pathway in the entire lung and did not measure the expression and activity in specific cell types in ASM; therefore, this requires further study. Second, the sample size of rhesus monkeys in this study was low. Moreover, there might be species-specific differences in the expression of various subtypes. Therefore, it remains unclear whether these results truly reflect human lung development.
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