Joshua P. Thatcher and Dr. James P. Porter, Physiology and Developmental Biology
Introduction
Cardiovascular diseases have become among the most widespread of adult diseases in the United States. Over the last few decades, the United States has experienced a dramatic increase in the rate of diseases such as Diabetes Mellitus Type II, Obesity and Hypertension.
Various experiments have been performed to study the developmental origins (also known as fetal programming) of these adult diseases, especially hypertension. My faculty mentor, Dr. James Porter, has hypothesized that a prenatal, or before the time of birth, high-salt diet would be sufficient to produce lasting hypertension in the adult offspring, or once the pups have reached adulthood.
I have been involved in research efforts to test this hypothesis for over two years. During this time we have failed to confirm this hypothesis. However, we did find that the female offspring of dams exposed to a prenatal high-salt (HS) diet exhibited an exaggerated cardiovascular response after periods of acute stress in comparison to the female offspring of dams exposed to a normal-salt (NS) diet. We saw no difference in the male offspring. Consequently, we are now searching for a mechanism to explain the stress-hypersensitivity seen in the female offspring.
Hypothesis
It was the aim of my ORCA funded research project to look at and quantify each of the components of the hypothalamic-pituitary-adrenal axis (HPA axis), which is a primary mechanism for the regulation of stress in the body. The three specific components are corticotropin-releasing hormone (CRH) secreted by the hypothalamus, adrenocorticotropic hormone (ACTH) secreted by the anterior pituitary gland, and corticosterone, the glucocorticoid in rats, secreted by the adrenal cortex. I hypothesized that each component of the HPA-axis would be enhanced in the high-salt female offspring in response to an acute stress.
Materials and Methods
Female Sprague-Dawley rats were used from dams that were fed either a high-salt or a normal-salt diet. After several weeks, the adult offspring were put in restraining cages for an hour to induce a stress response. Immediately following this procedure the rats were killed, and the blood, the brain (including the pituitary gland) and the kidneys were harvested for further study. All protocols were approved by the Institutional Animal Care and Use Committee of Brigham Young University.
In situ hybridization was performed on cross-sections of the hypothalamus to look at the level, or expression, of corticotropin-releasing hormone (CRH). Hybridization of 20 μm sections through the hypothalamus was carried out overnight at 57˚ C with ³³ P-UTP-labeled antisense riboprobe to CRH mRNA. Visualization utilized a two day exposure of slides (including 14C-standards) to autoradiographic film. Image analysis was performed using Scion Image and scanned images of the paraventricular nucleus (PVN).
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to quantify ACTH in the pituitary. Pituitary tissue was homogenized in Trizol to collect total mRNA. Isolated mRNA from the pituitary gland was reverse transcribed into cDNA by reverse transcriptase. The cDNA was then combined with appropriate primers for proopiomelanocortin (POMC), the precursor protein for ACTH, and enzymes to allow amplification in a thermocycler for 90 minutes.
In conjunction with RT-PCR, radioimmunoassay (RIA) was carried out to quantify ACTH blood levels. RIA was carried out with dilute rat serum 1:200 in various quantities with and without ACTH calibrators used as a control. ACTH 125I tracer and anti-ACTH were added and these mixtures were allowed to incubate for 2 hours. Precipitant solution was then added and the precipitate was counted in a gamma counter. Finally, RIA was also performed to look at blood levels of corticosterone by using the same procedure.
Results
In situ hybridization of the paraventricular nucleus showed that CRH expression was significantly elevated in the HS female offspring compared to the NS female offspring. This is in compliance with my hypothesis. RT-PCR of pituitary RNA revealed that there is no difference in the expression of the ACTH precursor POMC. This finding was unexpected. RIA of ACTH showed that plasma levels of the hormone are actually decreased in the HS female offspring compared to the NS offspring. And finally, plasma corticosterone levels did not differ among the two groups.
Discussion
The purpose of this study was to examine specific components of candidate hormones responsible for the enhanced sensitivity to stress seen in the female offspring of a dam exposed to a high-salt diet during pregnancy. Although each experiment described was carried out multiple times, it seems unlikely that the hormones of the HPA-axis are responsible for the increased sensitivity to stress.
The results of the in situ hybridization experiment for CRH expression and the corticosterone RIA experiment were included in the data that were used to publish a research article by my faculty mentor, Dr. James Porter in May 2007 .
References
- Porter JP, King SH, Honeycutt AD. Prenatal high-salt diet in the Sprague-Dawley rat programs blood pressure and heart rate hypperresponsiveness to stress in adult female offspring. Am J Physiol Regul Integr Comp Physiol 293: R334-R342, 2007.