Metabolic Regulatory Network Alterations in Response to Acute Cold Stress and Ginsenoside Intervention Xiaoyan Wang,†,‡,# Mingming Su,†,‡,# Yunping Qiu,†,‡,# Yan Ni,† Tie Zhao,† Mingmei Zhou,§ Aihua Zhao,† Shengli Yang,‡ Liping Zhao,‡ and Wei Jia*,†,‡,§ School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China, Shanghai Institute for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China, and Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China Received January 28, 2007
Acute stress may trigger systemic biochemical and physiological changes in living organisms, leading to a rapid loss of homeostasis, which can be gradually reinstated by self-regulatory mechanisms and/ or drug intervention strategy. However, such a sophisticated metabolic regulatory process has so far been poorly understood, especially from a holistic view. Urinary metabolite profiling of SpragueDawley rats exposed to cold temperature (-10 °C) for 2 h using GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of urinary metabolites and demonstrated the protective effect of total ginsenosides (TGs) in ginseng extracts on stressed rats. The metabonomics approach enables us to visualize significant alterations in metabolite expression patterns as a result of stress-induced metabolic responses and post-stress compensation, and drug intervention. Several major metabolic pathways including catecholamines, glucocorticoids, the tricarboxylic acid (TCA) cycle, tryptophan (nicotinate), and gut microbiota metabolites were identified to be involved in metabolic regulation and compensation required to restore homeostasis. Keywords: metabolic profiling • metabonomics • acute cold stress • ginsenosides • multivariate statistical analysis • gas chromatography/mass spectrometry
Introduction Neuropsychiatric symptoms of post-traumatic stress disorder such as depression, apathy, anxiety, cognitive impairment (a potential precursor to dementia), and others have aroused pressing awareness of the adverse impact of various acute stresses (lasting for seconds to hours) such as extreme cold or heat, toxins, panic, sadness, and tension. Most of these stressors are universally present in our lifespan and perturb the wellbalanced metabolism of living organisms by chance.1 It has been reported that cold stress, commonly occurring in clammy weather for human beings, is closely connected with cardiovascular and respiratory diseases, chilblain, diarrhea, and so forth, thereby causing higher mortality, especially among the elderly.2 Numerous works have centered on individual gene expression, protein structure, and function,3,4 as well as conventional pathophysiological studies on the hypothalamicpituitary-adrenal (HPA), neuroendocrine, circulatory systems, and immunological responses.5 * To whom correspondence should be addressed. E-mail: weijia@ sjtu.edu.cn. † School of Pharmacy, Shanghai Jiao Tong University. ‡ Shanghai Institute for Systems Biomedicine, Shanghai Jiao Tong University. # These authors contributed equally to this work. § Shanghai University of Traditional Chinese Medicine. 10.1021/pr070051w CCC: $37.00
2007 American Chemical Society
Panax ginseng is a highly favored herb restorative in many countries, particularly in China, as it possesses a variety of beneficial anti-inflammatory, antioxidant, glycolipid metabolic, and anti-cancer effects.6 The secondary metabolites of P. ginseng are ginsenosides (Rg1, Rb1, Rb2, Rc, Rd, Re) with a wide spectrum of activity, presumably because these active compounds can interact with an array of targets and tissues, and a single ginsenoside is able to initiate multiple actions in vivo. It has also been reported that ginsenosides are beneficial in preventing or alleviating the impairment of cold stress.7 Understanding the systemic effects of acute cold stress and dietary intervention with ginsenosides will undoubtedly enrich our current knowledge of cold-induced diseases and provide insights into the holistic beneficial effect of ginsenosides. However, the in-depth study of the biochemical effects induced in the metabolic regulatory network by acute stress and drug intervention has been a challenging task primarily due to limitations in analytical instrumentation and multivariate data processing techniques. Emerging global metabolic profiling represents a major leap forward, enabling qualitative and quantitative bioanalysis of very low level metabolites from in vivo samples with little prior knowledge of expected results.8 1 H NMR- based metabolic profiling of biological fluids, cells, and tissue extracts has yielded valuable information by successfully assessing drug safety, diagnosing the presence and Journal of Proteome Research 2007, 6, 3449-3455
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research articles severity of coronary heart disease, characterizing early and chronic psychological stress-induced variance and diet intervention, and exploring the intricate relationships between mammalian systems and symbiotic gut microbiota.9-11 Meanwhile, the soaring development of mass spectrometry (MS)based metabolic signature identification has widened our window of identified endogenous metabolites which are essential for an understanding of the mechanistic aspects of diseases and toxicology at the systems level.12,13 In this study, we have conducted a metabonomic study on the systemic responses to cold stress in rats by means of gas chromatography/mass spectrometry (GC/MS)-based urinary metabolite analysis. Additionally, the protective effects of ginsenosides in restoring homeostasis of the metabolic network in cold-stressed rats were investigated.
Experimental Methods Animal Handling and Sampling. All of the animal studies followed the relevant national legislation and local guidelines, and were performed at the Centre of Laboratory Animals, Shanghai University of Traditional Chinese Medicine, Shanghai, P. R. China. Eight-week-old male Sprague-Dawley rats (200 ( 20 g) were purchased from the Shanghai Laboratory Animal Co. Ltd. (SLAC, Shanghai, China), individually housed in stainless steel wire-mesh cages, and fed with a certified standard rat chow and tap water ad libitum. Room temperature and humidity were regulated at 24 ( 1 °C and 45 ( 15%, respectively. A light cycle of 12 h on 12 h off was set, with lights on at 08:00 a.m. After 2 weeks of acclimatization, rats were randomly divided into two groups as follows: Ginsenoside Group (n ) 7), received TGs (Total ginsenosides of P. Ginseng root, Hangzhou Greensky Biological Tech. Co., Ltd., China. Details available in Supporting Information Table 1 and Figure 1) orally at a daily dose of 100 mg/kg of body weight from day 1 to day 14; Control Group (n ) 7), received the same volume of vehicle daily. Drug or vehicle was administrated by oral gavage between 8:30 and 9:30 a.m. to minimize any effects of circadian rhythm. On day 14, all the rats were exposed to -10 °C for 2 h, and immediately returned to metabolic cages at room temperature. Twenty-four hours urine samples from each animal were collected pre- and post-cold exposure, and centrifuged at 5000 rpm for 10 min at room temperature to remove particle contaminants. The resultant supernatants were stored at -80 °C pending GC/MS analysis. The urine volume and body weight of each rat were recorded during the experimental period. GC/MS Sample Preparation, Derivatization, and Spectral Acquisition. The urine samples were prepared for GC/MS, and relevant spectral acquisition was obtained according to published methods with minor modifications.14 Briefly, rat urine samples were prepared by using a 600-µL aliquot of diluted urine sample (urine/water ) 1:1, v/v) for ethyl chloroformate (ECF) derivatization, with L-2-chlorophenylalanine employed as an internal standard to monitor the batch reproducibility in parallel. A 1-µL aliquot of analyte was injected into a DB5MS capillary column coated with 5% diphenyl cross-linked 95% dimethylpolysiloxane (30 m × 250 µm i.d., 0.25-µm film thickness; Agilent J&W Scientific, Folsom, CA) and conducted on a hyphenated Perkin-Elmer gas chromatograph and TurboMass-Autosystem XL mass spectrometer (Perkin-Elmer Inc.) as described.14 Data Reduction and Pattern Recognition. Non-processed GC/MS files were converted into NetCDF format via DataBridge 3450
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(Perkin-Elmer Inc.) and directly processed by custom scripts in MATLAB (The MathWorks, Inc.), where baseline correction, peak discrimination and alignment, internal standard exclusion, and normalization to the total sum of the chromatogram were carried out. The resulting three-dimensional matrix, including arbitrary compound index (paired retention time-m/z), sample names (observations), and normalized peak areas (variables) were introduced into the SIMCA-P 11.0 Software package (Umetrics, Umeå, Sweden) for multivariate statistical analysis. Orthogonal signal correction (OSC),15,16 a data filtering method, was utilized to remove systematic variation from GC/MS spectra prior to multivariate statistical analysis. The auto-scaled, normalized spectral data were conducted by principal component analysis (PCA) to visualize general clustering, trends, or outliers among the observations. Partial least-square-discriminant analysis (PLS-DA) was utilized to validate the OSCPCA model and identify the differential metabolites accountable for cold exposure (details provided in Supporting Information Table 2). Univariate Statistical Analysis. On the basis of the thresholds on the fold change rank and p-values from Kruskal-Wallis test, the differentially expressed metabolites from multivariate statistical analyses were validated at a univariate analysis level. In addition to the nonparametric test, classical one-way analysis of variance (ANOVA) was also utilized to judge whether the results were statistically significant. The critical p-value of both tests was set to 0.05 in this study.
Results Metabolic Variation Induced by Cold Stress. A typical GC/ MS total ion current (TIC) chromatogram of urine samples from pre- and post-cold stress exposure is illustrated in Figure 1A,B. Visual inspection of the spectra revealed some obvious differences between the two groups. To remove the unwanted disturbance from GC/MS spectra of the two groups, meancentered and auto-scaled (scaling factor is standard deviation) data were analyzed by OSC in the subjects before exposure (class 1) and after exposure (class 2). A three-dimensional PCA scores plot of two-component OSC-filtered data was subsequently utilized to depict the general variation between the two states (Figure 1C). Clear separation was observed in the first principal component (PC), implying that exposure to cold stress may lead to a systemic variation of living systems. As such, the correlation coefficient of each OSC-filtered variable was further calculated by a cross-validated PLS-DA model so as to characterize the major metabolites contributing to the variation. A number of important urinary metabolites contributing to the deviated metabolic profile from cold-stressed animals were identified and are summarized in Table 1. Compound identification was performed by comparing mass spectra and retention time with those obtained with commercially available reference compounds. Variation in these compounds was confirmed by a Kruskal-Wallis test and classical one-way ANOVA (Table 1). Metabolic Effects of Cold Stress with Ginsenoside Intervention. The metabolites of ginsenosides in urine are not detectable by GC/MS analysis because their molecular structures are not suitable for ECF derivatization;17 therefore, the interference from exogenous chemical compounds and metabolites in GC/ MS determination of endogenous metabolites is minimal. Visual comparison of the GC/MS TIC of urine samples reflected little variance between pre-exposure (Figure 2A) and postexposure (Figure 2B) to cold stress in the ginsenoside group. A
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Figure 1. Visualization of biochemical effects of cold stress in rats using a metabolic profiling approach. Typical GC/MS spectra of urine samples from pre-exposure (A) and postexposure (B) to cold stress rats. The key is provided in Table 1. (C) Metabolic profiles depicted by 3D-PCA sores plot of GC/MS spectral data from urine samples at these two states (n ) 7; pre-exposure to cold, dark blue dot; postexposure to cold, red dot; each dot denotes an individual rat.). Table 1. Univariate Statistical Analysis of Metabolites Identified by PLS-DA Model To Be Accountable for the Separation between Pre- and Postexposure to Cold in Control Group, and of These Metabolites in the Ginsenoside-Treated Groupa control group (n ) 7)
key
metabolites
P (KruskalWallis)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
glycine 4-methyl-Phenol glutamine isocitrate aconitate nicotinate aspartate 4-hydroxyphenylacetate citrate glutamate hippurate homovanillate dopamine 5-hydroxy-indole-3-acetate tyrosine tryptophan
0.0351 0.0075 0.0001 0.0339 0.0295 0.0268 0.1547 0.0066 0.0174 N.S. 0.0323 0.0135 0.0261 0.0156 0.0427 0.0153
ginsenosides group (n ) 7)
P (one-way ANOVA)
fold change (pre vs post)a
P (KruskalWallis)
P (one-way ANOVA)
fold change (pre vs post)
0.0350 0.0127 0.0027 0.1102 0.0127 0.0476 0.0476 0.0060 0.0127 0.0476 0.0127 0.0127 0.0253 0.0027 0.0476 0.0253
-1.9 2.2 -2.6 -1.6 -2.2 1.8 1.8 -2.4 -2.2 1.8 -2.2 2.2 -2.0 2.6 -1.8 -2.0
N.S. N.S. 0.0080 N.S. 0.0441 N.S. 0.0354 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
N.S.b N.S. 0.0060 N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S. N.S.
-1.6 1.2 -2.4 -1.3 -1.8 1.4 1.2 -1.3 -1.4 1.1 -1.1 1.3 -1.3 1.4 -1.1 -1.4
a Fold changes were obtained by calculating the relative concentration between pre- and postexposure to the cold stress in model group, or the relative concentration between pre- and postexposure to cold condition in the ginsenoside-treated group. b N.S. ) Non-significant.
two-component OSC-filtered 3D-PCA scores plot (Figure 2C) shows that these animals, as compared to Figure 1C, exhibit little deviation in systemic metabolic profiles between pre- and post-cold stress exposure. Univariate statistical methods, in-
cluding classical one-way ANOVA and nonparametric KruskalWallis test (Table 1), were utilized to verify the multivariate statistical strategy. The result also suggests that the relative content of the key metabolites accountable for stress was Journal of Proteome Research • Vol. 6, No. 9, 2007 3451
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Figure 2. Visualization of biochemical effects of acute cold stress in rats pretreated with ginsenosides using a metabolic profiling approach. Typical GC/MS spectra of urine samples from pre-exposure (A) and postexposure (B) to cold stress rats pretreated with ginsensodies. The key is provided in Table 1. (C) Metabolic profiles are depicted by a 3D-PCA sores plot of GC/MS spectral data from urine samples at these two states (n ) 7; pre-exposure to cold, dark blue square; postexposure to cold, red square; each square denotes an individual rat.).
comparable between pre- and postexposure to cold stress in the ginsenoside group. Taken together, pretreatment or longterm nutrition with ginsenosides may strengthen their physiological capability of resisting abrupt environmental changes such as extreme cold temperature.
Discussion The primary aim of this study was twofold: first, to investigate the effects of single acute cold exposure and define the metabolic changes responsible for the differences between preand postexposure; second, to verify the postulation that ginsenoside intervention can holistically protect individuals against the adverse influences of cold exposure, thereby validating the systemic alteration of metabolic profiles in response to cold stress. Considering the basic objective of assessing metabolic changes in rats in response to cold stress, collecting blood samples may lead to agitation, and the process itself may serve as a source of stimulation which may cause another stress. In contrast, urine sampling is noninvasive and commonly used for in vivo metabolic profiling. After the period of cold exposure, a number of important urinary metabolites contributing to the deviated metabolic profile from cold-stressed animals were identified and discussed in the following paragraphs (Figure 1 and Table 1). Catecholamine and Glucocorticoid Variation. Tyrosine, the precursor of catecholamines (the sympathetic nervous system 3452
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(SNS) neurotransmitters such as dopamine, noradrenaline, and adrenaline), was significantly decreased, whereas homovanillate, a catecholamine product, was increased in the urine samples of animals exposed to cold stress. These alterations indicate enhanced SNS activity, leading to an up-regulated catecholamine metabolic pathway (Figure 3A), and is in agreement with previous reports of increased levels of plasma catecholamines (noradrenaline and adrenaline) and activated tyrosine hydroxylase (which catalyzes tyrosine to dopa) in tissues of animals exposed to cold stress.4 Increased SNS activity during cold stress contributes to a number of instant physiologic effects such as rapid heart rate, tensed muscles, constricted peripheral vasculature, and increased alertness. Higher urinary excretion of glutamate and aspartate, and a lower urinary excretion of glutamine and glycine, were observed in urine samples of animals exposed to cold stress and is indicative of elevated glucocorticoid levels, which, in turn, upregulate excitatory amino acids such as aspartate and glutamate, and down-regulate inhibitory amino acids such as gamma aminobutyric acid (GABA) and glycine in the brain.18 More glutamine is biotransformed into glutamate during the stressinduced biological response, thereby resulting in a low level of glutamine and high level of glutamate in urine (Figure 3B). It has also been reported that elevated glucocorticoid excretion has the ability to activate tyrosine aminotransferase to facilitate
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Figure 3. The perturbed metabolic pathways in response to cold exposure. (A) Tyrosine-related metabolite changes in response to cold exposure; (B) variation of glucocorticoid secretion-related amino acids in response to cold exposure; (C) TCA cycle self-regulation against cold temperature; (D) variation in tryptophan pathway upon cold stimulus. Red square denotes an elevated concentration of metabolites present in the urine from rats postexposed to cold temperature, whereas blue square means a reduced level of metabolites.
the biosynthesis of glutamate from tyrosine and 2-oxo-glutarate, a participant in the TCA cycle.19 Similar to previous findings with different types of stress,20 we believe that increased glucocorticoid secretion and enhanced SNS activity are two major upstream metabolic regulatory pathways activated as a fundamental response to cold stress. These important physiological effects could trigger a cascade of reactions to facilitate protection of living organisms from cold or other stress-induced damage. Such a metabolic compensatory regulation can be exemplified by the thermoregulatory system, which not only responds directly to cold temperature, but is also modulated by SNS activity, thereby affecting systemic energy metabolism in different ways.21 Cold stress-induced elevations in noradrenaline and glucocorticoid provide two opposing impacts on the body; one effect is to mobilize the whole body to resist the physical insult; the other can restrain the immune system and result in damage to the neuroendocrine system, hippocampus, and blood-brain barrier.22 Therefore, protective reactions, including endogenous triggered compensatory regulation and exogenous medical intervention such as ginsenosides, are required to counteract these adverse effects. Self-Compensatory Regulation. The time-dependent fluctuation in urinary metabolites is presumably an indication of a series of well-organized and interactive biochemical changes as a result of self-compensatory regulation in response to cold stress. Regression coefficients of PLS-DA indicated that a number of important members of the tricarboxylic acid (TCA) cycle were altered in rat urine post-cold stress (Figure 3C). The urinary excretion levels of citrate, isocitrate, and aconitate were decreased, which seems contradictory to our expectation of increased energy consumption during cold exposure. Our
explanation is that the drastically perturbed biochemical process may encompass two different stages. Initially, the TCA cycle is accelerated due to enhanced adrenergic nerve activity, as adrenergic activity is reported to activate key TCA cycle enzymes such as isocitrate transhydrogenase, dehydrogenase, and succinate dehydrogenase.23 After rats were transferred back to metabolic cages at room temperature, however, they appeared less active or responsive, suggesting that their metabolic regulatory network entered into a much slower energy consumption period. The short-term exposure to acute cold stress followed by prolonged room-temperature recovery ultimately leads to an overall lower level of tricarboxylic acids in 24 h urine samples. Herein, we conceive that the alteration of the TCA cycle is an important part of a metabolic regulatory and compensatory mechanism in response to exposure to cold stress. Nicotinate and 5-hydroxyindole-3-acetate, two metabolites of tryptophan detected in this study, were significantly increased, indicating that their biosynthesis from tryptophan was elevated in response to cold exposure (Figure 3D). During cold exposure, there was an increased need for nicotinamide, an important precursor of the coenzymes NADH and NADPH which are indispensable electron transporters involved in the TCA cycle.24 Under low temperature, augmented energy metabolism calls for elevated electron transporters facilitating increased synthesis of nicotinamide. After the cold stress process, as the TCA cycle slows down, overexpressed nicotinamide is bio-transformed into nicotinate, resulting in a high excretion level in urine. On the other hand, the enhanced SNS activity and high glucocorticoid levels in stressed animals may stimulate lipolysis, increasing the release of fatty acids from white adipose tissue.25 Since nicotinate is able to inhibit plasma Journal of Proteome Research • Vol. 6, No. 9, 2007 3453
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levels of free fatty acids in vivo,26 a relatively high plasma level of nicotinate is thus necessary, after environmental temperature is reinstated, to eliminate the excessive free fatty acids and restore the biochemical balance in the metabolic system. Thus, we conceive that the nicotinate pathway also represents a selfcompensatory action in cold-stress-induced metabolic changes. Ginsenoside Intervention. Preadministration of TGs was hypothesized to be protective in cold-stressed rats, and was another objective of the current study. From the PCA analysis, the response to acute cold stress could be attenuated to some extent, as evidenced by the overlapping distribution of spatial boxes in TGs-treated rats (Figure 2C), versus distinct separation in control rats (Figure 1C). Ginseng and its products have been used to fight the effects of stress for thousands of years. In modern pharmacology, ginseng is reported to mediate bidirectional regulation in glucocorticoid, sympathetic nervous system, and immunomodulatory system disorders induced by stress.27 Ginsenoside Rg1, an important ingredient of TGs, down-regulates the glucocorticoid receptor in the brain and other tissues, thereby reducing tissue damage.28 Another main component, ginsenoside Rb1, completely antagonized the inhibition of immunological function caused by cold stress and down-regulated plasma corticosterone in stressed rats.29 Ginsenosides can also regulate excitatory and inhibitory neurotransmitters such as glutamate and GABA, and prevent injury induced by glutamate.30 We believe that the protective effect is at least partially due to the regulation of glucocorticoids and the SNS system, the two major upstream metabolic pathways. Therefore, downstream pathways are affected via changes in endogenous metabolites, contrary to those in normal rats in response to cold stress. It could be inferred that after 14 days of administration, TGs provides the body a strong defense to environmental insult, thereby reducing the response to cold stress. Gut Microbiota Variation. We observed that acute exposure to cold stress altered the urinary excretion levels of 4-methy phenol, 4-hydroxyphenylacetate, and hippurate, all of which are believed to be metabolized by the gut microbial community (microbiota).31 This suggested that there is a significant involvement of gut microbiota in the response to cold exposure. The gastrointestinal tract (GIT) represents a unique area where neuroendocrine hormones such as noradrenaline coexist with indigenous microflora,32 thereby, stress-induced variations of catecholamines and noradrenaline in particular, are consistently observed to affect gut microbiota.33 Likewise, changes in these endogenous metabolites may be a result of GIT motility and secretions in response to stress.34 It was interesting that in the TGs-treated group, these gut microbiota-related metabolites did not indicate significant variation pre- and postcold stress. Since TGs was administrated orally, and most ginsenosides are absorbed via degradation of gut flora,35 we presume that TGs could affect gut microbiota and further change related endogenous metabolites,36 and that ginsenosides themselves are hydrolyzed simultaneously. Thus, it could be implied that the protective action of ginseng extends to gastrointestinal flora from the host itself. On the basis of these results, it appears that there exists a close interrelationship between host and symbiotic bacterial metabolic regulatory responses, which is consistent with previous observations.9, 31 The fact that this symbiotic microbial community would rapidly mobilize to respond to cold stress suggests that gut microbiota somehow functions as part of the host’s metabolic regulatory 3454
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Figure 4. Scheme of the perturbed metabolic pathways as a selfregulatory process in response to cold temperature.
‘organ’ and co-regulates certain metabolic pathways, as anticipated in several recent publications.36,37 In summary, a series of well-organized and interactive biochemical changes, as indicated by time-dependent fluctuation in metabolites, form the backbone of the underlying metabolic regulatory mechanism against cold stimulus. Urinary metabolite profiling using high-resolution analytical means such as GC/MS in conjunction with modern multivariate statistical techniques permits noninvasive and simultaneous monitoring of entire metabolic pathways (precursors, intermediates, and products), and reveals the subtle interplay of functional metabolites and pathways, leading to an understanding of the systemic response to external stimuli such as cold stress (Figure 4). Our results provide new insight into mechanisms of metabolic regulation at a holistic level in response to cold stress, highlight the roles of metabolic signaling pathways, including the interrelationship between host and symbiotic bacterial metabolic regulatory responses, and reveal that the TCA cycle and nicotinate pathways involve endogenous metabolic compensatory action and ginsenosides involve exogenous protective regulation associated with cold stress. Abbreviations: GC/MS, gas chromatography/mass spectrometry; TGs, total ginsenosides; PCA, principal component analysis; OSC, orthogonal signal correction; PLS-DA, Partial least-square-discriminant analysis.
Acknowledgment. This study was financially supported by Shanghai Leading Academic Discipline Project, (Project Number: T0301), and by the Key Basic Research Project of Shanghai Science and Technology Commission (Project Number: 05DJ14009). Supporting Information Available: Table showing the chemical structure of ginsenosides and composition of the total ginsenosides, and table summarizing the GC/MS urine data sets used in PCA and PLS-DA modeling. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Pacak, K.; Palkovits, M.; Kopin, I. J.; Goldstein, D. S. Stressinduced norepinephrine release in the hypothalamic paraventricular nucleus and pituitary-adrenocortical and sympathoadrenal activity: in vivo microdialysis studies. Front. Neuroendocrinol. 1995, 16 (2), 89-150.
research articles
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PR070051W
Journal of Proteome Research • Vol. 6, No. 9, 2007 3455
