The Chikaraishi laboratory investigates the role of catecholamine neurotransmitters during development using pharmacological, physiological and genetic approaches. The prevailing view had been that catecholamines (dopamine, norepinephrine and epinephrine) played no role during development until late in gestation. However, this view has been challenged by the finding that 90% of catecholamine-deficient mice (tyrosine hydroxylase (TH) and dopamine _ hydroxylase (DBH) knockouts) die before birth, most die at midgestation. Some catecholamine-deficient fetus show symptoms of congestive heart failure with pooled blood in organs and in the cardiovascular system (Slides 1 and 2). The few catecholamine-deficient mice that survive to birth, die before weaning because they fail to eat and are severely runted within two weeks after birth (Slide 3).
Based on physiological studies in late gestation mammals and the well-described role for norepinephrine in the “fight or flight” response, we hypothesize that catecholamines enable the fetus to survive stress in utero. We use hypoxia (reduced oxygenation) as the experimental stressor, since hypoxia is known to occur throughout gestation in all placental mammals, even in healthy pregnancies.
We hypothesize that hypoxia elicits two systemic responses at midgestation: a reduction in heart rate, and a release of norepinephrine (NE). Using Doppler echocardiography (ultrasound) on midgestational fetuses in utero, hypoxia reduces fetal heart rate (Slide 4). Hypoxia also reduces heart rate in cultured fetuses. To ask whether norepinephrine plays a role in maintaining heart rate, we pharmacologically block norepinephrine receptors and show that heart rate is further reduced and that arrhythmias (irregular contractions) develop. If you click on the photo of the fetus (Slide 5, play movie), the first clip is a fetus incubated under normoxic conditions, the second is the same fetus under hypoxia where heart rate is regular, but slowed, and the last clip is the same fetus after β adrenergic receptor blockade, where heart rate is even slower and irregular. These data suggest that catecholamines, particularly norepinephrine, act via β adrenergic receptors to reduce the extent of and effects of hypoxia. A corollary is that catecholamine-deficient mice die in utero because they cannot survive hypoxia. Our recent data are consistent with the prediction that pharmacological activation of β1ARs rescues catecholamine-deficient fetuses to birth, while pharmacological blockade of βARs in wild type animals mimics catecholamine deficiency (diagonal arrows in box). Our work proposes that sympathetic responses are essential in the early fetus, significantly before the sympathetic nervous system matures and innervation is established.
Current work in the laboratory is focused on 1) finding which receptor and signal transduction system mediates fetal heart rate and survival; 2) which cells make the essential catecholamines (Slide 6) and 3) how catecholamines are released in response to hypoxia. Based on various knockout animals, it is unclear if classical neurotransmitter release via Ca+2-dependent, vesicular exocytosis can account for release of neurotransmitters at midgestation.
This work explores how the fetus adapts to hypoxia and extends work on late term fetuses to an earlier stage of development. Understanding fetal responses to hypoxia has significant clinical importance. Fetal hypoxia/asphyxia is among the ten leading causes of infant mortality. It is also a contributing factor for preterm birth, a major cause of infant mortality worldwide. Our work may have direct clinical relevance. Adrenergic antagonists are prescribed to pregnant women as second line therapy for hypertension. They cross the placenta and could elicit varying degrees of receptor blockade in the fetus. If NE acts in the human as it does in the mouse, blocking adrenergic action could exacerbate acute hypoxic/anoxia damage.
Our working hypothesis is that catecholamines, particularly norepinephrine, enable the early fetus to survive hypoxic stress in utero. A corollary of this hypothesis is that catecholamine-deficient (tyrosine hydroxylase null) fetuses will suffer greater hypoxia and transcriptionally induce genes associated with hypoxia. We tested the effect of hypoxia and catecholamines in the early (E12.5) mouse fetus by interrogating Operon v.3 spotted arrays at the Duke Microarray Facility (http://mgm.duke.edu/genome/dna_micro/core/data.htm). Using whole fetal RNA for microarrays, we assessed global genetic changes after maternal exposure to hypoxia (8% O2 plus balanced N2 for 6 hr) using GeneSpring software.
About 20-25% of the genes (4403 oligonucleotides) showed a statistically significant change (2-way ANOVA) with hypoxia (data) and several hundred genes (626 oligonucleotides) changed by more than 2 fold (data). Changes greater than 2 fold with hypoxia were seen in the Th+/+ fetus (data) and in the Th-/- fetus (data). We also assessed the influence of genotype within the same oxygen condition. A small number of genes were 2 fold or greater different between Th+/+ and Th-/- in normoxia (data) while in hypoxia, several hundred genes were different between the genotypes (data). Several genes associated with hypoxia were higher in Th-/- fetuses compared to Th+/+ fetuses during hypoxia (data).
|
Contact |
