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BEHAVIORAL PHARMACOLOGY

Temporal Effects of Stress by Immobilization and Sensitivity of the Isolated Rat Pacemaker to Isoproterenol: Roles of Corticosterone, Neuronal Uptake, and -Adrenergic Homogeneity

Muscle Cell Biology Group, Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio

Received April 4, 2003; accepted June 10, 2003.

	Abstract

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A number of diseases and pathological conditions are related to the long-term adaptive response to stress, in particular under conditions of chronic stress when allostasis can shift from a healthy toward a pathological state. Although a vast number of studies have focused on the effects of chronic stress on brain and the immune system, fewer studies have been performed in peripheral tissues. Here, we used the intact isolated right atrium (pacemaker) from the rat to investigate the temporal effects of stress induced by immobilization (restraint stress) on the sensitivity of the pacemaker to the chronotropic response to isoproterenol (i.e., the effect of isoproterenol to increase the frequency of contractions of pacemakers). Immobilization sessions were conducted a specific number of times (1, 3, 7, 9, 11, and 14). We found that the response to stress over time approximates a Gaussian distribution (i.e., normal standard distribution) with no significant effects being detected after either 1 or 14 immobilization sessions, whereas supersensitivity to the chronotropic effect of isoproterenol occurred after 3, 7, 9, and 11 immobilization sessions, with a peak effect occurring after seven immobilization sessions. At a cellular level, we determined that both corticosterone and neuronal uptake of catecholamines were directly involved with the observed effects, whereas no alterations in the homogeneity of -adrenoceptors were detected in pacemakers of stressed animals. We hypothesize that these adaptations are essentially beneficial in nature, as they should allow the animals to more promptly respond to the demands imposed by the stressful conditions.

McEwen and collaborators proposed that "rather than referring to everything dealing with responses to environmental and psychosocial situations as stress", the formulation of two new terms, "allostasis" and "allostatic load", was needed. They have defined allostasis as "maintaining stability (or homeostasis) through change" and propose to apply this concept "to other physiological mediators, such as the secretion of cortisol as well as catecholamines". The concept of allostatic load was proposed to refer to the wear and tear that the body experiences due to repeated cycles of allostasis as well as the inefficient turning on or shutting off of these responses (Goldstein and McEwen, 2002; McEwen, 2002).

Among many of the physiological changes that occur during the stress response, the release of adrenocorticotropic hormone is very important because it leads to an increased secretion of glucocorticoids (Gottesfeld et al., 1978; Kendall et al., 1982; Dickinson et al., 1985; Kennett et al., 1985a,b), which in turn will participate in the release of catecholamines by the sympathetic nervous system (Yamaguchi et al., 1981; Toth, 1990; Pardon et al., 2002).

Several investigators have demonstrated that chronic stress causes subsensitivity of rat brain -adrenoceptor to catecholamines and that chronic stress induced by immobilization produces a reduction of rat brain -adrenoceptor density, which mediates cAMP response to catecholamines (Stone, 1978, 1981, 1983; Nomura et al., 1981; Stone et al., 1984). In addition, it has been suggested that corticosteroids are involved in the modulation of -adrenoceptor-AMP system in brains of stressed animals (Davies and Lefkowitz, 1984; Payne and Adcock, 2001; Feltus et al., 2002; Schacke et al., 2002; Vermeer et al., 2003).

In contrast with brain tissues studies, fewer attempts have been made to study the effects of chronic stress on the sensitivity to catecholamines in peripheral tissues containing -adrenoceptors, such as the rat pacemaker. Harri et al. (1974) reported that prolonged cold exposure induces subsensitivity of the isolated rat pacemaker to the chronotropic effect of noradrenaline and phenylephrine (i.e., their intrinsic effect in increasing the frequency of contractions of pacemakers), whereas Bassani and De Moraes (1988a) demonstrated that repeated inescapable footshock stress, induced supersensitivity to isoproterenol (ISO), adrenaline and salbutamol. These authors suggested that this probably occurs due to an increase in the chronotropic function of the pacemaker -adrenoceptor (Bassani and De Moraes, 1988a,b).

Stress induced by immobilization (restraint stress) is particularly effective because it combines physical stress (i.e., increased muscular work) and emotional stress (i.e., enhanced flight reaction). Here, we used this well established model of stress to investigate for the first time the temporal effects of stress induced by immobilization at a definite number of immobilization sessions (i.e., 1, 3, 7, 9, 11, and 14 immobilization sessions).

We found that the response to stress over time approximates a normal (i.e., Gaussian) distribution with no significant effects being detected when animals were immobilized for either 1 or 14 sessions, whereas supersensitivity to the chronotropic effect of ISO occurred after 3, 7, 9, and 11 immobilization sessions, with a peak effect being detected after seven immobilization sessions. At a cellular level, we determined that both serum corticosterone and neuronal uptake of catecholamines (referred hereafter as neuronal uptake) were involved with the observed effects, whereas no alterations in the homogeneity of -adrenoceptors were detected in pacemakers of stressed animals.

	Materials and Methods

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Animals. Rats (Rattus norvergicus Berkenhout, var. albina Wistar, males, 230 -260 g) were kept at room temperature (25°C) in a controlled dark/light environmental cycle of 12 h. Animals were provided with food and water ad libitum. Animals were kept for at least 7 days before any experimental intervention to avoid the effects of shipment stress (Nosek et al., 2000; Brotto et al., 2002). All procedures regarding the care and killing method of animals was in accordance with the Guiding Principles of the National Institutes of Health and as approved by Case Western Reserve University-Medical School Animal Care Committee.

Immobilization. Rats were immobilized for 150 min for a definite number of immobilization sessions: 1 daily session (IMO-1), 3 daily sessions (IMO-3), 7 daily sessions (IMO-7), 9 daily sessions (IMO-9), 11 daily sessions (IMO-11), and 14 daily sessions (IMO-14). Animals were immobilized by being gently inserted into a flexible nylon screen that was closed and secured with nonallergic adhesive tape. When the animal was firmly inserted into the screen, it was supported horizontally in a wood surface, its tail was placed outside the screen and secured to the wood surface with adhesive tape, ensuring that the animal could not move during the immobilization sessions (we have found that this procedure is more stressful than allowing tails to remain free; unpublished observations). Immobilization sessions lasted for 150 min and were always performed from 1:00 to 3:30 PM. Animals were killed by cervical dislocation after 0 (control, not immobilized), 1, 3, 7, 9, 11, and 14 daily sessions of immobilization.

Isolated Right Atrium: The Pacemaker. After killing pairs of animals (one from control and one from the experimental group being tested), hearts were removed, and right atria were isolated and mounted for isometric recording of spontaneous contractions in a temperature-controlled 35-ml organ bath containing a Krebs-Henseleit solution with the following composition: 115.0 mmol/l NaCl, 4.2 mmol/l KCl, 2.5 mmol/l CaCl₂ · 2 H₂O, 1.2 mmol/l KH₂PO₄, 2.5 mmol/l MgSO₄ · 7 H₂O, 25.0 mmol/l NaHCO₃, and 11.0 mmol/l glucose, at 37 ± 0.5°C. To reduce ISO oxidation, 0.15 mM ascorbic acid was added to this solution. This solution was continuously bubbled with a mixture of 95% O₂ and 5% CO₂ to provide for an optimal level of oxygenation for the pacemakers and also to maintain the pH at 7.4 ± 0.1, because a physiological bicarbonate pH buffer system was used. Diastolic tension (0.5 g) was adjusted to permit recording of the spontaneous beating (0.5 cm/beat) of the pacemakers. Isometric contractions were monitored and registered by a force transducer (F-50; Narco-Bio-Systems, Inc., Houston, TX) attached to a chart recorder (Narco-Bio-DMP-4). Isolated pacemakers were incubated until a stable frequency was achieved (60 min). The bathing solution was changed every 15 min during the equilibration period before the onset of the concentration-effect curves. As previously reported (Bassani and De Moraes, 1988a,b), pacemakers with an equilibrating frequency of less than 260 contractions/min or more than 320 contractions/min were discarded, as well as pacemakers that had any type of rhythmic irregularities, which occurred in <10% of the pacemakers used in this study. Thus, pacemakers showed a high level of stability and reproducibility and we believe that the discarded preparation better reflects experimental dissecting mistakes than intrinsic problems with the preparation itself.

Sensitivity of the Isolated Pacemaker to the Chronotropic Effects of ISO. All experiments described here were paired (controls versus experimental groups). After the equilibration period, concentration-effect curves were obtained to the chronotropic effects of ISO by using the cumulative dose-response method (van Rossum et al., 1983). When three consecutive and increasing concentrations of the agonist did not significantly alter the functional response of the pacemakers, this effect was considered maximal (i.e., equal to 100%) and the experiment was concluded. The concentration of the agonist that caused an effect equal to 50% of the maximal response was termed EC₅₀. All data were expressed as geometric averages and their respective 95% confidence intervals (Westfall et al., 1972). Sensitivity variations were analyzed by the ratios of the EC₅₀ (DR) of control/IMO or control/experimental groups.

pA₂ Value and Constant of Dissociation for Metoprolol. The Schild method (Poch et al., 1992; Ghosh et al., 1999) was applied as modified here. In brief, isolated pacemakers were subjected to the following treatment. First, in vitro chemical denervation: after the initial equilibration period in normal Krebs-Henseleit solution, 6-hydroxydopamine (6-OHDA, 300 mg · ml^-1) was added to a modified Krebs-Henseleit solution (NaHCO₃ was replaced with glutathione to prevent oxidation of 6-OHDA) for 10 min. Second, after 10 min, this bathing medium was replaced with normal Krebs-Henseleit solution. Third, the bathing solution (normal Krebs') was then changed two additional times (every 15 min) to remove 6-OHDA and glutathione. Fourth, phenoxybenzamine (PBZ; 20 µM) was added to the bathing solution (normal Krebs') for 15 min. Fifth, the bathing solution (normal Krebs') was then changed every 15 min to remove PBZ and until a stable pacemaker frequency was achieved (30-45 min). Sixth, ISO concentration-effect curves were performed in the absence and in the presence of three increasing concentrations of metoprolol (10, 100, and 1,000 nM). For a proper equilibration of the antagonist (metoprolol) with the isolated pacemakers, an incubation time of 30 min was observed before the onset of each concentration-effect curve to ISO. Seventh, results were accepted only when the angular regression coefficients of Schild plots did not differ from 1.0 (Poch et al., 1992; Lazareno and Birdsall, 1993; Ghosh et al., 1999), and values of pA₂ (i.e., pA₂ = -log K_B, where K_B is the constant of dissociation), were independent from the concentrations of metoprolol (MacKay, 1978). 8) The constant of dissociation K_B (pA₂ = -log K_B) was determined as in Besse and Furchgott (1976): K_B = [B]/(DR - 1), where [B] is the molar concentration of metoprolol, (DR - 1) is the quotient of the EC₅₀ value for ISO in the presence and in the absence of metoprolol (-1).

Measurement of Serum Corticosterone Levels. When each animal was killed, trunk blood was collected, centrifuged, and separated serum was frozen. Serum from each subject was later assayed in duplicate samples using a corticosterone radioimmunoassay kit (ICN Pharmaceuticals, Costa Mesa, CA). Corticosterone values were expressed in micrograms per milliliter serum. The antibody anticorticosterone is highly specific for corticosterone with <2% cross-reactivity for 11-deoxycorticosterone and <1% cross-reactivity for 18-hydroxydeoxy-corticosterone, cortisol, progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, aldosterone, testosterone, and estradiol. The lower limit of sensitivity of the assay was 20 ng/ml, and the standard curve was linear over the range of 20 ng/ml to 6.0 µg/ml. Intra-assay and interassay variabilities were <5%.

Statistical Analysis. SigmaStat (SPSS Science, Chicago, IL) was used for all statistical analyses. Fisher-Snedecor Analysis of variance followed by Tukey's post hoc test was used for statistical analysis of data containing parametric variables. If data were not normally distributed, the Wilcoxon sign rank test for nonparametric variables was applied. Bertullini's test was used for multiple comparisons of data sets. Significance was accepted at the 0.05 level of probability (Margenau and Murphy, 1965; Sokal and Rohlf, 1969). Results of EC₅₀, pA₂, and K_B are expressed as geometric averages and 95% confidence intervals (Westfall et al., 1972). Other results are expressed as means ± S.E.M.

Drugs and Solutions. All drugs and salts were American Chemical Society standard and obtained from Sigma-Aldrich (St. Louis, MO). Krebs-Henseleit was prepared daily with nanopure water. PBZ was dissolved in acidified ethanol and diluted to the appropriate concentration in Krebs-Henseleit solution. 6-OHDA was diluted in a 20% ascorbic acid solution. ISO, metoprolol, and glutatione were diluted in Krebs-Henseleit immediately before use.

	Results

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Corticosterone levels were determined as the biochemical index of stress levels in all experimental groups. Serum corticosterone levels were increased after three immobilization sessions and remained increased throughout the 11th immobilization session compared with control (nonimmobilized). In rats immobilized for either 1 or 14 sessions, corticosterone levels were not significantly increased (Table 1). There were no significant differences in resting frequency rates or maximal frequency rates of pacemakers isolated from control (nonimmobilized, NI) and immobilized groups (Table 1).

Chronic stress induced by immobilization resulted in a leftward displacement (i.e., supersensitivity) of the concentration-effect curve to ISO in groups IMO-3, IMO-7, IMO-9, and IMO-11. The data are summarized in Table 1 and illustrated in Fig. 1.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Mean concentration-effect curves for the chronotropic effect of ISO in pacemakers isolated from control (i.e., rats not subjected to immobilization) and from rats subjected to immobilization as shown in the legends. Each point is the mean of 6 to 32 experiments. Vertical bars represent S.E.M. A significant leftward shift (i.e., supersensitivity to the chronotropic effect of ISO) is indicated by *, p < 0.01). a, a significant (p < 0.05) leftward shift. b, no significant differences were detected (p > 0.05). Levels of significance were not indicated for each concentration simply to avoid clutter (see Table 1 for details).

Because the peak of the supersensitivity effect to ISO was observed in the IMO-7 group, additional studies were performed in this group. Figure 2 shows that bilateral adrenalectomy (ADX) carried out 3 days before the onset of seven immobilization sessions abolished the supersensitivity to the chronotropic effect of ISO (see data summary in Table 2). Furthermore, there were no significant changes in pacemaker resting, maximal frequency rates, and ISO sensitivity in control-adrenalectomized rats (Table 2).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Mean concentration-effect curves for the chronotropic effect of ISO in pacemakers from rats subjected to IMO-7, rats subjected to bilateral adrenalectomy (ADX-control), and from rats subjected to ADX and immobilized for seven sessions (ADX + IMO-7). Each point is the mean of seven to eight experiments. Vertical bars represent S.E.M. A significant leftward shift (i.e., supersensitivity to the chronotropic effect of ISO) is indicated by *, p < 0.01.

As previously reported (Bassani and De Moraes, 1988a,b), adrenalectomy did not eliminate corticosterone plasma levels, but did significantly decrease them. Furthermore, corticosterone levels were unaltered in rats subjected to seven sessions of immobilization that were also adrenalectomized, demonstrating the significant effect of ADX in preventing both the increase in corticosterone and supersensitivity to ISO (Fig. 2; Table 2).

It has been previously demonstrated that stress can induce a conformational alteration of -adrenergic receptors (Bassani and De Moraes, 1988b) that in turn may lead to supersensitivity to catecholamines. To investigate this possibility, we applied the Schild method (Poch et al., 1992; Ghosh et al., 1999) for the determination of pA₂ value and the constant of dissociation for metoprolol (i.e., antagonist) using ISO as the agonist. These results are shown in Fig. 3 and summarized in Table 3. We found that under the experimental conditions used here, receptor populations were unchanged, because pA₂ values and constant of dissociation for metoprolol were not significantly different in pacemakers from either control or IMO-7 groups.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Schild plots for pA2 and KB for metoprolol, using ISO as the agonist. Paired Experiments performed in isolated pacemakers from NI rats and rats subjected to IMO-7. Three consecutive concentration-effect curves to ISO were sequentially performed in the presence of increasing concentrations of metoprolol. Before the first curve, pacemakers were treated in vitro with 6-OHDA + PBZ. b, no significant differences were detected (p > 0.05).

To further investigate the cellular mechanisms of ISO's supersensitivity in pacemakers of chronically stressed rats; we performed a series of experiments designed to test the involvement of neuronal uptake processes. To this end, pacemakers from the NI group and from the IMO-7 group were treated "in vitro" with 6-OHDA and PBZ to chemically block both neuronal uptake 1 (i.e., major uptake process of catecholamines in the pacemaker, also called uptake-1; blocked by cocaine and tricyclic antidepressants) and, uptake 2 (i.e., an extraneuronal uptake of catecholamines can occur; so- called uptake-2. This uptake is into the parenchymal cells of the organ. It is not blocked by cocaine or tricyclic antidepressants).

Figure 4 compares the concentration-effect curves of controls-NI, IMO-7, controls-NI treated in vitro with 6-OHDA + PBZ, and IMO-7 treated in vitro with 6-OHDA + PBZ. As shown, the abolishment of the neuronal uptake processes by chemically treating pacemakers in vitro with 6-OHDA + PBZ effectively shifted the concentration-effect curve to the left. In addition, in pacemakers from the IMO-7 group treated in vitro with 6-OHDA + PBZ the leftward shift is essentially identical to that displayed by both IMO-7 and IMO-7 treated in vitro, demonstrating that the effect induced by chronic stress could be mimicked by eliminating the involvement of neuronal uptake processes. The data are summarized in Table 4.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Mean concentration-effect curves for the chronotropic effect of ISO in isolated pacemakers of rats from NI rats, rats subjected to IMO-7, rats with pacemakers pretreated in vitro with 300 µg ml-1 hydroxydopamine and 20 µM phenoxybenzamine before commencement of concentration-effect curves [NI + (6-OHDA + PBZ)], and rats with pacemakers subjected to seven immobilization sessions that were pretreated in vitro with 6-OHDA + PBZ [IMO-7 + (6-OHDA + PBZ)] before commencement of concentration-effect curves. Each point is the mean of 6 to 32 experiments. Vertical bars represent S.E.M. A significant leftward shift for all three experimental groups compared with NI is indicated by *, p < 0.01). b, no significant differences were detected between the three experimental groups (p > 0.05).

	Discussion

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Stress has been induced in animal models by a number of methods such as cold, electrical shock, forced swimming, dehydration, and immobilization. In rats, it has been demonstrated that stress induced by immobilization (restraint) is particularly effective because it combines physical stress (i.e., increased muscular work) and emotional stress (i.e., enhanced flight reaction). Few mammals have adapted as well as rats to stressful conditions and environments. Therefore, studying the effects of stress in rats is relevant. Despite this, most studies on chronic stress are focused on the brain and the immune system and fewer attempts have been made to investigate the effects of chronic stress on other peripheral tissues.

The rat cardiac pacemaker offers a relevant physiological system for such studies inasmuch as this preparation is expected to be an impact tissue for the effects of stress and physiologically and pharmacologically relevant studies can be performed in these preparations. To achieve our goals, we investigated, for the first time, the temporal effects of chronic stress induced by immobilization on the sensitivity of pacemakers to isoproterenol.

We found that the sensitivity of the rat pacemaker to ISO was unaltered from control at the extremes of the number of restraint sessions (i.e., 1 and 14 sessions). Our interpretation of these findings is that the IMO-1 group did not show any significant effects because the stress was acute and too short to produce significant changes. Furthermore, because the effects of acute stress can be beneficial and/or neutral in healthy subjects (Henry, 1996; McEwen, 2002), it is possible that the effects of one single session of immobilization in rats may reflect these possibilities. It is possible that a single session of immobilization is only identified by the system as an acute form of allostatic load, which is then quickly normalized by the system, which in otherwise healthy animals, is able to quickly restore allostasis (Goldstein and McEwen, 2002; McEwen, 2002).

On the other hand, when rats were chronically stressed by immobilization for 3, 7, 9, and 11 sessions, a significant supersensitivity to the chronotropic effect of ISO was detected. This effect is demonstrated by a leftward shift in the concentration-effect curves. Thus, a significant lower concentration of ISO is required to produce its chronotropic effect. Supersensitivity to ISO and other catecholamines has been previously reported in pacemakers of rats chronically stressed with inescapable foot shocks (Bassani and De Moraes, 1988a,b; Zanesco and De Moraes, 1992). The common link between stress modalities and supersensitivity to catecholamines seems to be the increased level of plasma corticosterone. Previous studies have suggested that chronic stress caused by inescapable foot shock induced a conformational alteration of -adrenergic receptors that in turn led to supersensitivity to the chronotropic effect of catecholamines (Bassani and De Moraes, 1988a,b; Zanesco and De Moraes, 1992).

We investigated this possibility by determining both pA₂ values and constant of dissociation for metoprolol in pacemakers isolated from control and IMO-7 groups. Our findings did not support a modification in the population of -adrenergic receptors under our experimental conditions. It is possible that different stress modalities will lead to slightly different adaptation processes (Torres et al., 2002; Viau and Sawchenko, 2002). For example, in the studies where inescapable foot shock was used as the stress modality (Bassani and De Moraes, 1988a,b; Zanesco and De Moraes, 1992), an extra component may have been the additional involvement of opioid receptors directly involved with nociperception. We strongly believe that these mechanisms were not evoked under our experimental conditions, which may account for some of the observed differences.

To further investigate the cellular mechanisms of ISO's supersensitivity in pacemakers of chronically stressed rats, we pretreated pacemakers from NI and from IMO-7 group in vitro with 6-OHDA and PBZ to eliminate the participation of neuronal uptake processes. We found that this treatment effectively shifted the concentration-effect curve to the left. This is expected because with the blockade of neuronal uptake processes, ISO effectiveness should be enhanced because the major mechanism for terminating its effects is blocked.

Furthermore, in pacemakers from the IMO-7 group treated in vitro with 6-OHDA + PBZ, the leftward shift was essentially identical to that displayed by both IMO-7 not treated in vitro and IMO-7 treated in vitro with 6-OHDA + PBZ, demonstrating that the effect induced by chronic stress could be mimicked by eliminating the involvement of neuronal uptake processes. Our interpretation for these results is that by blocking neuronal uptake processes, the supersensitivity to the chronotropic effect of ISO induced by chronic stress was in fact abolished, because the concentration-effect curves for all three groups essentially overlap. These results suggest that at the cellular level, neuronal uptake is the main mechanism involved with the supersensitivity to ISO induced by chronic stress induced by repeated immobilizations (Fig. 4; Table 4).

The control of adrenoceptors by steroid hormones is very complex, but it is now accepted that steroid hormones promote a direct modulation of target cell gene transcription (Davies and Lefkowitz, 1984; Collins et al., 1991). Collins et al. (1991) have demonstrated that steroid hormones can induce an increase in the rate of -2 adrenoceptor gene transcription and a resulting increase in the relative density of these receptors. More recently, Zhang et al. (2002) demonstrated that in the midbrain, acute stress had no effect on the -1-adrenoceptor mRNA level, but 2 days of stress significantly increased it. Thus, it is possible that the elevated levels of corticosterone observed in the groups IMO-3, 7, 9, and 11 enhanced the density of -1 and/or -2 receptors, causing supersensitivity to the chronotropic effect of ISO, a nonselective -agonist. Furthermore, it is possible that corticosterone may directly inhibit neuronal uptake processes. As a matter of fact, we have observed that when concentration-effect curves to ISO are performed in rat pacemakers exposed to 1 to 3 mM corticosterone, concentration-effect curves are significantly shifted to the left (our unpublished observations). It is important to note that the Gaussian distribution for the observed supersensitivity after 3, 7, 9, and 11 immobilization sessions is correlated with increased levels of serum corticosterone. In fact, statistical significance levels for both the leftward shift in the concentration-effect curves and corticosterone levels in the pacemakers from animals immobilized were higher for groups immobilized for 3 and 7 sessions compared with 9 and 11, indicating that the cellular trigger for the observed supersensitivity is indeed linked to increased serum corticosterone levels.

Together, our data demonstrate the key roles played by corticosterone and neuronal uptake processes on the supersensitivity to the chronotropic effect of ISO detected in pacemakers of chronically stressed rats.

We speculate that the observed supersensitivity is a beneficial adaptation, because it should allow the animal to more rapidly raise its heart rate and respond to a stressful and/or threatening condition. Thus, the allostatic load caused by the restraint stress (3-11 immobilizations), in otherwise healthy animals, is followed by allostasis. Interestingly, as the insult (i.e., allostatic load) is maintained, levels of corticosterone and the leftward shift in the concentration-effect curves gradually begin to decrease and after 14 immobilization sessions), yet another adaptation takes place, because the initial adaptation (i.e., supersensitivity) disappears. It is plausible to speculate that the state of sustained supersensitivity to catecholamines could eventually lead to "wear and tear" and the system adapts again (i.e., allostasis) by identifying this condition (supersensitivity) as a either a normal condition or a threatening condition, and, the supersensitivity is no longer necessary and is, therefore, lost or somehow compensated.

	Acknowledgements

 
I am grateful to my friend and mentor Prof. Dr. Thomas Nosek for continuous support and insightful discussions on the preparation of this manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grant HL60304 to Thomas Nosek and M.A.P.B.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.052670.

ABBREVIATIONS: ISO, isoproterenol; IMO, immobilization; 6-OHDA, 6-hydroxydopamine; PBZ, phenoxybenzamine; NI, control; ADX, adrenalectomy.

Address correspondence to: Dr. Marco A. P. Brotto, Muscle Cell Biology Group, Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106. E-mail: mab51{at}po.cwru.edu

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