Elsevier

Alcohol

Volume 37, Issue 3, November 2005, Pages 157-166
Alcohol

Article
Ethanol consumption increases rat stress hormones and adrenomedullary gene expression

https://doi.org/10.1016/j.alcohol.2005.09.007Get rights and content

Abstract

Numerous reports document altered drinking behavior following acute stressors but few describe physiological responses to acute stress of chronic ethanol consuming subjects. We tested rats' responses to 120-min foot restraint immobilization (Immo) after 1 week of liquid diet containing 5% wt/vol ethanol (ethanol-fed). Controls consumed isocaloric liquid diet ad libitum (adlib-fed) or in amounts equal to that of ethanol-fed subjects on the previous day (pair-fed). Each rat was implanted with a tail artery cannula on day 7 to allow remote blood collection before and during Immo on day 8. Plasma epinephrine (Epi); norepinephrine (NE); corticosterone (Cort); prolactin (PRL); adrenomedullary gene expression of catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine-N-methyl transferase (PNMT); and TH protein levels were measured. Ethanol-fed rats had two to threefold higher basal plasma Epi and NE and tended to have increased Cort compared to adlib-fed or pair-fed rats. Immo increased Epi and NE in ethanol-fed rats more than twofold above those observed in controls, and also increased Cort more in ethanol-fed than in control rats. PRL was marginally affected. Ethanol potentiated the normal immobilization-induced increase in adrenomedullary TH, DBH, and PNMT messenger RNA (mRNA). TH protein increased only in ethanol-fed rats. Increased plasma catecholamine levels, adrenomedullary gene expression, and TH protein concentration in nonimmobilized ethanol-fed rats strongly suggest that ethanol consumption was itself a stressor, which potentiated the subsequent response to acute Immo. Moreover, the observed interaction of ethanol and stress on plasma catecholamine levels illustrates the importance of minimizing additional stressful stimuli when investigating ethanol's physiological effects.

Introduction

Many publications document increased ethanol consumption in response to stressful stimuli. A striking example is the extensive study of increased alcoholic beverage use in New York City after the World Trade Center attacks in 2001 (Boscarino et al., 2005). However, few authors describe stress responses during prolonged ethanol consumption. This overlooked aspect of ethanol's potential physiological effects may be crucial to an individual's survival under circumstances involving acute challenge.

The interactions between psychosocial stressors and ethanol consumption in humans as well as experimental animals have been studied for many years and reviewed extensively (Gianoulakis, 1998, O'Doherty, 1991, Pohorecky, 1981, Pohorecky, 1990, Pohorecky, 1991, Sayette, 1999, Sillaber and Henniger, 2004, among many others). However, the data presented are highly complex and inconsistent, in part because of the great diversity of stressful stimuli that have been studied. Selye (1936) observed that many stressors appeared to produce the same syndrome, including adrenal enlargement, gastrointestinal ulcers, and other related pathologies. He believed that all of these were a nonspecific response to any stressful stimulus and focused primarily on the hypothalamic-pituitary-adrenal (HPA) axis as the primary regulator of these responses. Unfortunately, the stress response is not so simple as Selye believed, because more recent research has attested to the specificity of the responses resulting from exposure to various stressors. For example, Pacak et al. (1998) specifically tested Selye's hypothesis by comparing plasma epinephrine (Epi), norepinephrine (NE), and ACTH responses to a broad spectrum of stimuli. These included saline injection (±handling), hypoglycemia, pain, and tissue damage due to formalin injection, hemorrhage, cold exposure, and restraint immobilization. Not surprisingly, all stressors increased the levels of these hormones, but increases differed markedly in extent and duration. Thus, it is highly probable that the interactions of stressors and ethanol will also differ with the nature of the stressor. In their review of interactions of stressors with ethanol consumption by human subjects, Sillaber and Henniger (2004) emphasized the importance of HPA axis activation in response to stress and ethanol as well as differences between psychogenic stressors such as anticipation or anxiety and neurogenic stimuli such as bodily injury.

Animal models make it somewhat easier to study the responses to stressors and/or ethanol consumption, given that one can control the genetic background and environmental conditions to a greater extent than when studying human subjects. Hormonal responses of rats to stress include both those of the HPA axis and also those of the sympathetic neural system. For example, many have documented the importance of the former, reporting activation by ethanol of hypothalamic production of corticotropin releasing hormone leading to increased ACTH and ultimately corticosterone (Cort) (Fahlke et al., 1994, Fahlke et al, 1996, Ogilvie et al., 1997, Rivier, 1996). Hypothalamic control is also seen in the prolactin (PRL) response to both stress and ethanol (Dave et al., 2000, De et al., 2002, Emanuele et al., 1992, Jurcovicova et al., 1990, Pohorecky et al., 2004).

The hormonal responses to stressors under the influence of prior ethanol consumption may vary with the type of stressor, the mode and duration of administration of ethanol, and the parameter measured. For example, Patterson-Buckendahl et al. (2004) reported that adrenomedullary gene expression for enzymes of the catecholamine synthetic pathway (tyrosine hydroxylase, TH; dopamine beta-hydroxylase, DBH; and phenylethanolamine-N-methyl transferase, PNMT) was increased by 7 weeks consumption of 6% wt/vol ethanol in drinking water, but significantly decreased in ethanol consuming rats subjected to 2-h daily restraint in wire mesh cylinders. However, plasma Cort levels in that experiment were not significantly affected by either ethanol or restraint (Macho et al., 2003). In another study, rats given acute intraperitoneal injections of ethanol showed lower concentrations of Cort than saline-treated rats after foot shock or tail pinch, but not after restraint in wire mesh cylinders (Brick & Pohorecky, 1984). Later experiments showed that acute injection of ethanol also dampened the response of plasma Epi and NE levels to the same restraint compared with saline-injected rats (Patel & Pohorecky, 1988).

We hypothesized that the hormonal response to stress, which normally adapts to chronic repetition of the same stimulus, would be further adapted in ethanol consuming animals. To test this hypothesis, we fed rats a liquid diet, with or without the addition of 5% wt/vol ethanol, as the sole source of food and fluid, for 8 days. Rats were cannulated on day 7, stressed by Immo on day 8, and blood was collected. We measured plasma concentrations of Epi, NE, Cort, and PRL and quantified adrenomedullary gene expression of TH, DBH, and PNMT and also TH protein levels in relation to the diet and stressor imposed.

Section snippets

Animal care and experimental treatment

All protocols were reviewed and approved by Institutional Animal Care and Use Committees of Rutgers University and the Institute for Experimental Endocrinology, Bratislava. Male Sprague–Dawley derived rats, obtained from Charles River Laboratories, Wiga, Germany, were housed in the vivarium of the Institute for Experimental Endocrinology, 4 per cage for 1 week prior to beginning the experiment.

Experimental housing

Rats were transferred to polypropylene cages (44 × 21 × 21 cm) fitted with raised wire floor grids to allow

Animal weight and food consumption

Rate of weight gain by rats is shown in Table 1. ANOVA indicated a significant effect of diet on weight gain [F(2, 37) = 74.5, P < .001]. Adlib-fed rats gained more weight than either ethanol-fed or pair-fed rats (P < .0001), whereas ethanol-fed and pair-fed rats did not differ from each other (P = .74). Adlib-fed rats' food consumption was approximately 85% greater than that of ethanol-fed and pair-fed rats (P < .0001), resulting in the greatly increased weight gain shown for these rats. Pair-fed rats'

Discussion

The hormonal influences reported here are interactive and confirm the complexity of ethanol influence. The data demonstrated that chronic ethanol consumption (a) increased adrenomedullary gene expression for catecholamine synthesizing enzymes, (b) increased basal plasma levels of Epi and NE, and (c) especially potentiated plasma catecholamine response to Immo as compared with adlib-fed controls. That ethanol's influence is apparently specific for catecholamine synthesis and is reflected in the

Acknowledgments

The authors would like to thank Dale Buckendahl for fabricating and adapting the cage dividers to hold feeding tubes.

Research was conducted with support from National Institutes of Health; National Institute for Alcoholism and Alcohol Abuse, R21 AA 12705-01 (PP-B), Slovak Grant Agency for Science #5125 and SP Grant 51/0280800-2004 (R.K.); and funds from the Center of Alcohol Studies, Rutgers, The State University of New Jersey (L.A.P.).

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