Charles A. Ducsay

Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat

Objective: Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the susceptibility of adult heart to ischemia-reperfusion (I-R) injury. Methods: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) gbroups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R10) or 25 minutes of ischemia and 3 hours of reperfusion (I-R25). Results: Prenatal hypoxia did not change basal left ventricular (LV) function. I-R10 produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R25 caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R25-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. there was a significant decrease in LV heat shock protein 70 and endothelial nitric oxice synthase levels in hypoxic hearts. Prenatal hypoxia did not change β1-adrenoreceptor levels but significantly increased β2-adrenoreceptor in the left ventricle. In addition, it increased Gsα but decreased Giα. Conclusion: Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in β2-adrenoreceptor and theGsα/Giα ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.

Effects of calcium entry blocker (nicardipine) tocolysis rhesus macaques: Fetal plasma concentrations and cardiorespiratory changes

Tocolytic doses of nicardipine, a dihydropyridine calcium entry blocker, were administered to chronically catheterized rhesus monkeys between days 128 and 132 of gestation. During periods of spontaneous uterine contractility, a 500 micrograms nicardipine bolus was injected intravenously, and this was followed by continuous infusion (6 micrograms/kg/min) to the mother for 1 hour. Uterine activity (amniotic fluid pressure) and maternal heart rate and blood pressure were monitored continuously. Paired maternal and fetal blood samples were drawn at frequent intervals to monitor pH, PO2, PCO2, and plasma nicardipine concentrations. Peak maternal nicardipine concentrations ranged from 175 to 865 ng/ml while peak fetal levels ranged from 7 to 35 ng/ml. Fetal heart rate and blood pressure were unaffected. However, fetuses became acidotic (pH 7.26 +/- 0.01 versus 7.33 +/- 0.01) and hypoxemic (PO2 16.0 +/- 3.2 versus 24.5 +/- 2.0 mm Hg) after maternal nicardipine treatment (p less than 0.01). Despite the fact that maternal nicardipine treatment exerted a significant tocolytic effect, the undesirable fetal side effects are of concern and deserve further investigation.

Long-term hypoxia modulates expression of key genes regulating adipose function in the late-gestation ovine fetus

A major function of abdominal adipose in the newborn is nonshivering thermogenesis. Uncoupling protein (UCP) UCP1 and UCP2 play major roles in thermogenesis. The present study tested the hypothesis that long-term hypoxia (LTH) modulates expression of UCP1 and UCP2, and key genes regulating expression of these genes in the late-gestation ovine fetus. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dG); perirenal adipose tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dG. Quantitative real-time PCR was used to analyze mRNA for UCP1, UCP2, 11beta hydroxysteroid dehydrogenase type 1 (HSD11B1) and 2 (HSD11B2), glucocorticoid receptor (GR), beta3 adrenergic receptor (beta3AR), deiodinase type 1 (DIO1) and DIO2, peroxisome proliferator activated receptor (PPAR) alpha and gamma and PPARgamma coactivator 1 (PGC1alpha). Concentrations of mRNA for UCP1, HSD11B1, PPARgamma, PGC1, DIO1, and DIO2 were significantly higher in perirenal adipose of LTH compared with control fetuses, while mRNA for HSD11B2, GR, or PPARalpha in perirenal adipose did not differ between control and LTH fetuses. The increased expression of UCP1 is likely an adaptive response to LTH, assuring adequate thermogenesis in the event of birth under oxygen-limiting conditions. Because both glucocorticoids and thyroid hormone regulate UCP1 expression, the increase in HSD11B1, DIO1, and DIO2 implicate increased adipose capacity for local synthesis of these hormones. PPARgamma and its coactivator may provide an underlying mechanism via which LTH alters development of the fetal adipocyte. These findings have important implications regarding fetal/neonatal adipose tissue function in response to LTH.

Circadian myometrial and endocrine rhythms in the pregnant rhesus macaque: Effects of constant light and timed melatonin infusion

Six chronically catheterized rhesus macaques maintained on a 12-hour-light/dark cycle (lights on from 7 AM to 7 PM) showed a nocturnal uterine activity rhythm with peak contractile events between 9 and 11 PM (p less than 0.05). In blood samples collected at 3-hour intervals over a 24-hour period, we determined that plasma melatonin and progesterone concentrations were elevated at night whereas estradiol, estrone, and cortisol reached peak concentrations in the early morning (p less than 0.05). Lights were then left on for the remainder of the study. After 12 days in constant light, daily rhythms in uterine activity and plasma steroid levels were relatively unchanged, whereas melatonin concentrations were suppressed. Animals then received a timed infusion of melatonin (0.2 mg/kg/hr each day from 7 PM to 6 AM daily until delivery). The nocturnal uterine activity rhythm and the rhythms in plasma steroid concentrations were maintained. We conclude that the 24-hour patterns in maternal uterine activity and plasma steroid hormone levels are circadian rhythms generated by an endogenous biologic clock and do not appear to be driven by the pattern of melatonin in circulation.

eNOS activation and NO function: Differential control of steroidogenesis by nitric oxide and its adaptation with hypoxia

Nitric oxide (NO) plays a role in a wide range of physiological processes. Aside from its widely studied function in the regulation of vascular function, NO has been shown to impact steroidogenesis in a number of different tissues. The goal of this review is to explore the effects of NO on steroid production and further, to discern its source(s) and mechanism of action. Attention will be given to the regulation of NO synthases in specific endocrine tissues including ovaries, testes, and adrenal glands. The effects of hypoxia on generation of NO and subsequent effects on steroid biosynthesis will also be examined. Finally, a potential model for the interaction of hypoxia on NO synthesis and steroid production is proposed.

Chronic Hypoxia and Developmental Regulation of Cytochrome C Expression in Rats

Objective: To test the hypothesis that chronic hypoxia upregulates cytochrome c expression in heart, brain, and liver of fetal and maternal rats. Methods: Time-dated pregnant Sprague-Dawley rats were divided into normoxic and hypoxic (48 hours of 10.5% oxygen from days 19 to 21) groups, and were killed on day 21. Tissue levels of cytochrome c in heart, brain, and liver were determined by using monoclonal antiserum for cytochrome c. Results: Chronic hypoxia caused a decrease in fetal body weight (5.3 ± 0.1 to 4.7 ± 0.1 g) and an increase in heart/body weight ratio (0.0048 ± 0.0001 to 0.0061 ± 0.0002). Cytochrome c levels were 4-, 2.6-, and 13-fold higher in heart, liver, and brain, respectively, of the mother than of the fetus. Chronic hypoxia did not change cytochrome c levels in maternal tissues but caused a 70% increase and 54% decrease in cytochrome c levels in the fetal heart and liver, respectively. No difference was observed in the fetal brain. Conclusions: The results suggest that expression of cytochrome c is tissue specific and developmentally regulated. Chronic hypoxia showed differential regulation of cytochrome c levels both developmentally and tissue specifically. The increased sensitivity of cytochrome c in fetal tissue to chronic hypoxia is likely to represent a fetal adaptive mechanism to the stress of chronic hypoxia.

Long-Term Hypoxia Alters Endocrine and Physiologic Responses to Umbilical Cord Occlusion in the Ovine Fetus

Objective: This study was designed to determine the effect of umbilical cord occlusion (UCO) on fetal endocrine responses in the long-term hypoxemic (LTH) ovine fetus. Methods: Pregnant ewes were maintained at high altitude (3820 m) from day 30 of gestation. Normoxic control and LTH fetuses were catheterized, and an inflatable occluder was placed on the umbilical cord at day 132 of gestation. In the LTH group, maternal oxygen tension was maintained at approximately 60 mmHg by nitrogen infusion through a maternal tracheal catheter. On day 137, two 5-minute UCOs were performed. On day 139, the study was repeated with a 10-minute UCO. Results: Basal adrenocorticotropic hormone (ACTH) levels and peak responses to the first 5-minute UCO were not different between control and LTH fetuses (17.6 ± 4.0 to 418.8 ± 41.3 in controls, 25.7 ± 4.0 to 530.0 ± 93.0 pg/mL in LTH fetuses). A similar pattern was observed during the second UCO. Basal cortisol levels were similar in both groups. In response to UCO, a significant increase in cortisol was observed in both groups, but peak concentrations in the LTH group were significantly higher than those in the control group (23.9 ± 4.8 versus 14.8 ± 2.9 mg/mL, respectively, P < .05). The second occlusion also increased cortisol concentrations, but no differences were observed between groups. After the 10-minute UCO, the ACTH and cortisol responses were similar to the first 5-minute occlusion, with higher cortisol levels in the LTH fetuses. Conclusion: Despite similar ACTH responses to UCO, the cortisol response was greater in the LTH fetuses than in normoxic controls. LTH appears to result in enhanced adrenal sensitivity to a secondary stressor or altered cortisol metabolism.

Prenatal cocaine exposure increases heart susceptibility to ischaemia–reperfusion injury in adult male but not female rats

The present study tested the hypothesis that prenatal cocaine exposure differentially regulates heart susceptibility to ischaemia–reperfusion (I/R) injury in adult offspring male and female rats. Pregnant rats were administered intraperitoneally either saline or cocaine (15 mg kg−1) twice daily from day 15 to day 21 of gestational age. There were no differences in maternal weight gain and birth weight between the two groups. Hearts were isolated from 2‐month‐old male and female offspring and were subjected to I/R (25 min/60 min) in a Langendorff preparation. Preischaemic values of left ventricular (LV) function were the same between the saline control and cocaine‐treated hearts for both male and female rats. Prenatal cocaine exposure significantly increased I/R‐induced myocardial apoptosis and infarct size, and significantly attenuated the postischaemic recovery of LV function in adult male offspring. In contrast, cocaine did not affect I/R‐induced injury and postischaemic recovery of LV function in the female hearts. There was a significant decrease in PKCɛ and phospho‐PKCɛ levels in LV in the male, but not female, offspring exposed to cocaine before birth. These results suggest that prenatal cocaine exposure causes a sex‐specific increase in heart susceptibility to I/R injury in adult male offspring, and the decreased PKCɛ gene expression in the male heart may play an important role.

Long-term Hypoxia Increases Endothelial Nitric Oxide Synthase Expression in the Ovine Fetal Adrenal

This study was designed to test the hypothesis that fetal adrenal nitric oxide synthase (NOS) is elevated in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for approximately the last 100 days of gestation. Between days 138 and 141 of gestation, adrenal glands were collected from LTH fetuses and age-matched normoxic controls. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western analysis were used to quantify NOS expression, and NOS distribution was examined by immunohistochemistry and double-staining immunofluorescence for endothelial NOS (eNOS) and 17α-hydroxylase (CYP17). Neuronal NOS (nNOS) was expressed at very low levels and with no differences between groups. Expression of eNOS was significantly greater in the LTH group compared with control. Neuronal NOS was distributed throughout the cortex while the greatest density of eNOS was observed in the zona fasciculata/reticularis area and eNOS co-localized with CYP17. We conclude that LTH enhances eNOS expression in the inner adrenal cortex which may play a role in regulation of cortisol biosynthesis in the LTH fetus.

Nitric oxide inhibits ACTH-induced cortisol production in near-term, long-term hypoxic ovine fetal adrenocortical cells.

We previously reported that in the sheep fetus, long-term hypoxia (LTH) resulted in elevated basal plasma adrenocorticotropic hormone (ACTH1- 39) whereas the cortisol levels were not different from normoxic controls. We also showed that LTH enhances endothelial nitric oxide synthase (eNOS) expression in the fetal adrenal. This study was designed to determine the effect of NO on cortisol production in adrenocortical cells from LTH fetal sheep. Ewes were maintained at high altitude (3820 m) from ∼40 days’ gestation (dG) to near term. Between 138 and 141 dG, fetal adrenal glands were collected from LTH and age-matched normoxic control fetuses. Adrenal cortical cells were pretreated with sodium nitroprusside (SNP), nitro-L-arginine methyl ester (L-NAME), L-arginine, or diethyleneamine NO (DETA-NO) and then challenged with 10 nmol/L ACTH. Cortisol responses were compared after 1 hour. Adrenocorticotropic hormone -induced cortisol secretion was significantly higher in LTH versus control (P < .01). Enhancement of NO with L-arginine resulted in a significant reduction of ACTH-mediated cortisol production in the LTH group. DETA-NO also caused a significant decrease in ACTH-mediated cortisol production (P < .05). Inhibition of NOS with L-NAME significantly increased cortisol production in the LTH group (P < .05 compared to ACTH alone), whereas the effect on the control group was not significant. Nitric oxide synthase activity was significantly higher in the LTH group compared to control, but this difference was eliminated following ACTH treatment. These data indicate that LTH enhances adrenal cortical sensitivity to the inhibitory effects of NO on cortisol production. Nitric oxide may, therefore, play an important role in regulating ACTH-induced cortisol production in the LTH fetal adrenal.