Accepted for publication February 10, 1986

Transcription

1 /86/ $OO.OO/O THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright (C) 1986 by The American Society for Pharmacology and Experimental Therapeutics Vol 237, No.3 Printed in USA Effect of Repeated Restraint Stress, Desmethylimipramine or Adrenocorticotropin on the Alpha and Beta Adrenergic Components of the cyclic AMP Response to Norepinephrine in Rat Brain Slices1 ERIC A. STONE, JANE E. PLATT, ARIEL S. HERRERA and KENNETH L. KIRK Department of Psychiatry (E.A.S., J.A.P., ASH.), New York University School of Medicine and Nationalinstitute of Arthritis, Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland Accepted for publication February 1, 1986 ABSTRACT NE is known to stimulate the formation of the second messenger camp in rat brain slices (Daly, 1977). This nucleotide may -mediate the electrophysiological and metabolic effects of beta adrenergic receptor stimulation by NE and other catecholamines in the brain (Bloom, 1975). The NE-cAMP response has been shown to be influenced by prior exposure of rats to stress. Repeated footshock, restraint or prolonged isolation stress reduces the ability of NE to elevate camp levels in slices of the cerebral cortex, hypothalamus and brainstem of rats (Stone, 1979a; Stone et al., 1985; Kraechi et al., 1981). The occurrence of this change has been shown to be temporally correlated with the development of resistance to stress and has therefore been hypothesized to have a role in adaptation to chronic stress (Stone, 1979b, 1983b). A similar reduction of the NE-cAMP response also occurs during treatment with most antidepressant drugs (Sulser, 1982). These findings have prompted speculation that the latter response is a possible Received for publication September 17, I This work was supported in part by Grants MH22768 and MH8618. The cyclic AMP response to catecholamines in rat cortical slices is mediated by a beta adrenergic receptor which is coupled to adenylate cyclase and an alpha adrenergic receptor which potentiates the response to beta receptor stimulation. The present studies examined the effects of repeated restraint stress, adrenocorticotropin or desmethylimipramine administration on the beta and alpha adrenergic components of this response. Restraint was found to produce a small nonsignificant decrease of the beta receptor response accompanied by a significant reduction of the alpha receptor-induced potentiation of the beta response. Desmethylimipramine was found to lower the cyclic AMP response to beta receptor stimulation but not to alter the alphainduced potentiation of the beta response. Adrenocorticotropin, like restraint stress, was found to reduce only the alpha-induced potentiation of the beta response. Experiments with adenosine and histamine showed that restraint stress lowered the alphainduced potentiation of cydic AMP responses to these neurohormones also. It is concluded that restraint stress acts primarily to reduce the response to stimulation of central a/pha adrenergic receptors whereas desmethylimipramine acts primarily to reduce the response to stimulation of beta adrenergic receptors. Adrenocorticotropin has the same effect as restraint stress suggesting that pituitary adrenal hormones mediate the stress effect. neurochemical locus for the interaction of stress and antidepressant agents (Stone, 1979b). The mechanism by which stress alters the camp response has not yet been established. However, it does not appear to involve an increase in phosphodiesterase activity because the inclusion of a phosphodiesterase inhibitor in the incubation medium does not abolish stress-induced subsensitivity or is there an increase of PDE activity in brain homogenates of stressed rats (Stone, 1981). Alterations in NE uptake and metabolism in brain slices also do not appear to play important roles because the reduction in the camp response is greatest at high concentrations of NE (1 M) which saturate uptake systems and catecholamine metabolizing enzymes. The stress effect may involve a change in an adrenergic receptor or in the adenylate cyclase system to which it is coupled. However, from existing studies it is not clear which receptor alteration is responsible for the effect. The camp response to NE in the rat brain is known to be mediated by two adrenergic receptors, a beta and an alpha receptor (Perkins and Moore, 1973). The beta adrenergic receptor acts to stimu- ABBREVIATIONS: NE, norepinephrine; camp, cyclic AMP; 6-FNE, 6-fluoronorepinephnne; DM1, desmethylimipramine; ACTH, adrenocorticotropic hormone; ISO, isoproterenol. 72

2 1986 Stress on Brain Adrenoreceptors 73 late adenylate cyclase activity whereas the alpha adrenergic receptor acts to potentiate the beta response (Daly et al., 1981; Leblanc and Ciaranello, 1984). Previous studies have shown that stress can produce changes in the density and affinity of both beta and alpha receptors in the brain. Decreases in beta receptor density (U Prichard and Kvetnansky, 198; Stone and Platt, 1982) increases and decreases in alpha-2 receptor density and affinity (U Prichard and Kvetnansky, 198; Lynch et al., 1983) as well as increases in alpha-i receptor density (Lopez de Ceballo et al., 1983) have each been reported to follow stressors of various types. As far as the stress-induced reduction in beta receptors is concerned it is unlikely that this change plays an important role in the camp response reduction because the number of beta receptors returns to control values within 24 hr after stress whereas the NE-cAMP response remains depressed at this time (Stone et al., 1985). Persistent changes in beta receptor coupling to adenylate cyclase, however, could be a factor. As for the stress-induced increase in alpha-2 receptor density it is also unlikely that this plays a role in the camp response change because it occurs already after only one exposure to stress whereas the reduction in the camp response occurs only after repeated exposure to stress. A significant problem in interpreting the latter studies on receptor binding changes after stress has to do with the fact that the identity of the alpha receptor which mediates the potentiation of the camp response is not yet established unambiguously. Although the potentiation effect is reduced by alpha-i antagonists (Daly et al., 1981; Johnson and Minneman, 1985) it is not blocked completely by these agents (Pilc and Enna, 1986). Furthermore the effect is unlike other alpha-i responses in the brain because it is not stimulated by the selective alpha-i agonists, phenylephrine, methoxamine or cirazoline (Vetulani and Sulser, 1975; Mobley and Sulser, 1979; Pilc and Enna, 1986) although it is stimulated by the nonselective alpha agonists, NE, epinephrine, 6-FNE and a-methylnorepinephrine (Johnson and Minneman, 1985). Pilc and Enna (1986) have shown that alpha-2 blockers also inhibit the potentiation effect and therefore have suggested that both alpha-2 and alpha-i receptors participate in the response. Clonidine, however, which is an agonist at other brain alpha-2 receptors is not an agonist at this receptor (Skolnick and Daly, 1975). Therefore although they appear to be similar to alpha-i and alpha-2 adrenergic receptors in their inhibition by antagonists the alpha receptor(s) which mediate the potentiation effect are different from these receptors in their agonist characteristics and may represent subgroups of the latter. It is not clear therefore how to interpret the above changes in alpha-i and alpha-2 receptor binding with respect to the effects of stress on the alpha adrenergic component of the camp response to NE. The present experiments were undertaken to clarify how the alpha and beta adrenergic components of the camp response are altered as a result of stress. Because of uncertainties concerning the expected site of action of stress on the receptoradenylate cyclase complex we chose to examine only the camp response to receptor stimulation. Also because the respective roles of alpha-i and alpha-2 receptors are as yet unresolved we chose to use only nonselective alpha agonists and antagonists. The experimental design was based on the fact that the response to alpha stimulation is a potentiation of the response to beta stimulation. Therefore, the beta response was examined in the presence and absence of alpha receptor stimulation and the degree of potentiation was calculated from the ratio of potentiated and nonpotentiated responses. For purposes of comparison we also examined animals treated chronically with the tricyclic antidepressant, DM1, or the stress hormone, ACTH. Chronic treatment with DM1 is believed to selectively reduce brain beta adrenergic receptor function (Sulser, 1982) whereas ACTH treatment may have a similar selective action on brain alpha receptor function (Kendall et al, 1982; Mobley et al., 1983). Materials and Methods Animals. Male Sprague-Dawley rats weighing 15 to 2 g at the start of the experiments were used. The rats were housed in groups of four to five and maintained on a 12 hr light/dark cycle (lights on 7: A.M.). Food and water were freely available. Stress, drug and hormone treatment. Restraint stress accompanied by mild intermittent tactile stimulation was administered twice daily as described previously (Stone et al., 1985). The duration of each stress period was 75 mm as this has been found to represent the minimum time necessary to induce a significant reduction of the camp response to NE in the rat brain (Stone et al, 1985). DM1 (kindly donated by Merrell Dow Pharmaceuticals, Inc., Cincinnati, OH) was administered at 1 mg/kg i.p., twice daily for 1 days. Controls were injected with saline. ACTH124 (kindly donated by Organon Laboratories, Inc., West Orange, NJ) was administered at 5 lu/kg s.c., once daily for 12 days. The hormone was prepared as the long-acting zinc phosphate complex (DeWied, 1966). Control rats were injected with vehicle. Animals were sacrificed 15 to 2 hr (9: A.M.-2: P.M.) after the last stress or injection. All animals given ACTH were found upon autopsy to have marked adrenal hypertrophy (wet weight of adrenals 3-5% of vehicle-injected controls). Catecholamine-stimulated camp formation in brain slices. Cerebral cortical slices were prepared, incubated with various agents and assayed for camp and protein by methods described previously (Stone, 1979a). In all cases antagonists were added to the incubation medium 1 mm before agonists. Agonists were allowed to stimulate tissues for 1 mm before boiling and centrifugation. camp in the supernatant was determined according to Brown et al. (1971) and protein in the pellet was assayed according to Lowry et al. (1951) using bovine albumin as the standard. The camp response to a catecholamine was defined as the camp content obtained in the presence of the catecholamine divided by the content obtained in its absence (basal level). The camp response to beta adrenergic stimulation was taken as the response either to the beta agonist, ISO, 1 M; or to NE, i M, in the presence of the alpha antagonist, phentolamine, 5.5 x io M. The response to alpha adrenergic stimulation was measured as that either to the alpha agonist, 6-FNE, io M, or to NE, i M, in the presence ofthe beta antagonist, timolol, 1 M. The response to combined alpha and beta stimulation was taken as that either to NE, io M, or to ISO, io- M, in the presence of 6-FNE, io M. Responses to histamine, i- M, and to adenosine, i M, were measured in the presence or absence of 6-FNE, i M. The concentrations used for agonists represent maximally effective ones as determined from dose-response curves as previous experiments have not found significant effects of stress on EC values for NE or ISO (Stone et a!., 1985). Concentrations of antagonists were those estimated to block more than 95% of receptors as determined in competition experiments. To obtain a quantitative estimate of the potentiating effect of alpha stimulation on the beta adrenergic response, the response to combined alpha and beta stimulation was divided by the response to beta stimulation after first subtracting the alpha component from the total. Potentiation values so obtained from 4 nontreated rats were found to be distributed normally. The same procedure was used to calculate the potentiating effects of alpha stimulation on histamine and adenosine responses (formulas in table 1). The necessity for subtracting the alpha compo-

3 74 Stone et al. Vol. 237 nent from the combined response is due to the fact that alpha stimulation by itself produces a small increase in camp formation due to potentiation of responses to endogenous factors, primarily adenosine, released from the slices during incubation (Daly et al., 198). Statistics. Planned comparisons between two groups were evaluated by independent or dependent t tests. In cases in which variances differed significantly the t test modified for heterogenous variance was used (Edwards, 1962). Results Effects of restraint, DM1 or ACTH treatment on the response to alpha, beta and alpha + beta stimulation. The effects of restraint stress on camp responses to noradrenergic receptor stimulation are shown in figure i. Restraint significantly reduced the camp response to combined alpha and beta stimulation produced either by NE (-22.6%) or by 6- FNE in the presence of ISO (-2.9%). A much smaller nonsignificant reduction was seen in the response to selective beta stimulation produced either by ISO (-6.5%) or by NE in the presence of phentolamine (-9.4%). No effect of stress was observed on the response to selective alpha stimulation produced either by 6-FNE or by NE in the presence of timolol. The magnitude ofthe potentiation of the beta response by alpha stimulation was significantly reduced in the stressed animals (table i; experiment I). This decrease was similar whether the alpha, beta and combined alpha + beta responses were estimated using NE + timolol, NE + phentolamine and NE alone (-i8.i%) or 6-FNE, ISO and 6-FNE + ISO (-i9.4%), respectively. The effects of DM1 treatment on camp responses are shown in figure 2. The drug was found to significantly reduce the camp response to combined alpha and beta stimulation elicited either by NE (-25.9%) or by ISO in the presence of 6-FNE (-28.5%). It also significantly reduced the response to selective beta stimulation produced either by ISO (-24.%) or by NE in the presence of phentolamine (-23.3%). There was no reduction in the response to selective alpha stimulation produced ALPHA BETA ALPHA.BETA TABLE 1 EffeCt of restraint stress, DM1 or ACTh administration on the magnitude of the alpha adrenergic-induced potentiation of cortical camp responses to beta adrenergic, adenosine and histamine receptor activation Values are means and SE. for 1 to 2 rats. b Calculated from: C Calculated from: S Calculated from: * <.5 vs. control group. U (1).. Beta AdrenergicI#{149} Beta Mrenergic ll Adenosine Histnine5 Exp. I Control 2.21 ± ±.15 Restraint 1.81 ± ±.15 Exp. II Control 1.81 ± ±.12 DM ± ±.28 Exp. Ill Control 2.67± ±.22 ACTH 2. ± ±.1 1 * Exp. IV Control 2.81 ± ± ±.44 Restraint 2.22 ±.14* 2.6 ±.1k 2.6 ±.18* a Calculated as described under methods from formula: (response to NE) - (response to NE + timolol) (response to NE + phentolamine - 1) (response to 6-FNE + ISO) - (response to 6-FNE) (response to ISO - 1) (response to 6-FNE + adenosine) - (response to 6-FNE) (response to adenosine - 1) (response to 6-FNE + histamine) - (response to 6-FNE) (response to histamine - 1) ALPHA BETA ALPHA #BETA, - I ( I U). 5% NE+ 6-FNE NE+ ISO timolol phentol. Fig. 1. Effect of repeated restraint stress, twice daily for 1 days, on the camp response to adrenergic receptor stimulation in cortical slices. Control group is shown by open bars, restraint group by solid bars. Selective alpha, beta and alpha + beta adrenergic stimulation was accomplished by incubation with the compounds listed under the abscissa. Each bar and vertical line represents the mean and S.E.M. of 1 rats. Mean basal camp values (picomoles per milligram of protein): control group, 9.3 ±.5; restraint group, 9.7 ±.5 P <.1 vs. control. phentol, phentolamine. NE+ 6FNE NE+ ISO NE 6-FNE timolol phentol. +ISO Fig. 2. Effect of repeated injection of DM1, 2 mg/kg daily, for 1 days on the CAMP response to adrenergic receptor stimulation in cortical slices. Control group is shown by open bars, DM1 group by solid bars. See legend to figure 1 for other details. Each bar and vertical line represents the mean and S.E.M. of 1 rats. Mean basal CAMP ValueS N E 6-FN E per milligram of protein): control group, ± 1.3; DM1 fiso group, P <.1 vs. control. phentol, phentolamine. either by 6-FNE or by NE in the presence of timolol. DM1 was not found to have any effect on the magnitude of the potentiation of the beta response by alpha adrenergic stimulation (table i, experiment II). This was true whether NE + timolol, NE + phentolamine and NE alone or 6-FNE, ISO or 6-FNE + ISO were used as estimates of the alpha, beta and alpha + beta responses, respectively.

4 1986 Stress on Brain Adrenoreceptors 75 ACTH administration was not found to alter significantly the responses to combined alpha + beta, selective beta or selective alpha adrenergic stimulation (results not shown). There was, however, a small nonsignificant reduction in the response to combined alpha + beta stimulation (NE, -8.%; 6- FNE in the presence of ISO, -8.%) and a small nonsignificant increase in the response to selective beta stimulation (ISO, +14.i%; NE in the presence ofphentolamine, +9.%). Because these nonsignificant effects were in opposite directions, however, the hormone significantly reduced the magnitude of the potentiation of the beta response by alpha stimulation (table I, experiment III). This was true using either NE + timolol, NE + phentolamine and NE alone (-32.9%) or 6-FNE, ISO and ISO + 6-FNE (-25.3%) as estimates of the aipha, beta and alpha + beta responses, respectively. Effect of restraint stress on alpha adrenergic potentiation of camp responses to adenosine, histamine and ISO. camp responses to adenosine, histamine and ISO and their potentiation by 6-FNE in stressed and control rats are shown in table 2. The responses to these agonists either in the presence or absence of 6-FNE were not significantly different between stressed and control rats. The potentiating effect of alpha stimulation, however, was significantly reduced in the stressed group for all three responses: potentiation of ISO (-2i.%), of adenosine (-i7.5%) and of histamine (-3i.2%) (table i, experiment IV). To visualize the reduction in a phainduced potentiation more readily the results are presented in figure 3 as percentage of control response for both the nonpotentiated and potentiated responses. It can be seen that the potentiated responses are significantly lower than the corresponding nonpotentiated responses in all three cases. Discussion The present results indicate that repeated restraint stress lowers the camp response to NE in the cerebral cortex by producing a significant reduction in the function of alpha adrenergic receptors accompanied by a smaller decrease of the function of beta adrenergic receptors. As discussed above alpha adrenergic receptors in the rat brain function to potentiate the camp response to stimulation of other neurotransmitter receptors, such as the beta adrenergic, which are coupled with adenylate cyclase. A quantitative measure of this potentiation was obtained by dividing the response to combined alpha and beta stimulation by the response to beta stimulation. This measure was found to be significantly reduced 2 to 25% in the cortex of repeatedly restrained rats. The response to beta stimulation was also reduced but to a much smaller extent (5-iO%). The overall reduction of the response to NE represents the summation of the two effects and therefore is greater than each. Stress was not found to reduce the response to alpha stimulation alone, however. It is not clear why no effect on the latter was observed, however, as noted above, the response to selective alpha stimulation is complex in that it represents the alphainduced potentiation ofthe response to endogenous factors that are released from the slices during incubation. Thus, it is possible that stress may have caused alterations in the release of these substances which obscured the decrease in potentiation. It may be argued that the decrease in the alpha-induced potentiation of the beta response does not result from an alteration in the response to alpha receptor stimulation but rather from some change in the beta receptor response which makes the latter less able to be potentiated. To test this possibility we examined the effects of stress on the alphainduced potentiation of camp responses to adenosine and histamine. It was found that stress produced similar reductions in the potentiation of these responses despite the fact that it did not significantly alter the nonpotentiated response to either compound. It is likely therefore that it is the change in the alpha and not beta response that accounts for the decrease in the alpha-induced potentiation of the beta response. Although the above fmdings indicate an effect on the alpha response they do not provide information as to the type of alpha response that is affected. As discussed in the introduction there is evidence that the population of alpha receptors that mediates the potentiation effect includes members of both the alpha-i and alpha-2 types each having somewhat unusual agonist properties. Although this issue is not yet resolved clearly we performed one experiment using prazosin to block the alphai component of the potentiation effect. Prazosin at io M was found to reduce the potentiation effect caused by NE in control rats from 2.21 ±.i2 to i.59 ±.9 and in stressed rats from i.8i ±.io to 1.58 ±.9. The difference in potentiation effect between control and stressed groups in the presence of prazosin is no longer significant. These data would suggest therefore that the alpha-i receptor response is the site ofthe stress effect. Further studies using selective alpha-2 blockers, however, are required to support this conclusion. The effects of stress on alpha receptor function found in the present study agree with the effects of stress hormones found in previous studies. It has been shown that repeated treatment with ACTH produces a lowering of the camp response to NE with no change in beta adrenergic receptor density in the rat cortex (Kendall et ci., i982). Moreover, adrenalectomy has been found to produce an increased camp response to NE but not to ISO (Mobley et at., i983). This suggests that these hormones produce selective alterations in the function of alpha adrenergic receptors. To test this in the present study the effects of repeated administration of ACTH on alpha, beta and combined alpha + beta stimulation were determined. In agreement it was found that the hormone selectively reduces the alpha-induced potentiation effect without significantly altering the response to selective alpha or beta receptor stimulation. After the present TABLE 2 Effect of restraint stress on cortical camp responses to ISO, adenosine and histamine In the presence and absence of 6-FNE Basal values (pkomoles per mifligram of protewi) were 6.22 ±.34 for control and 6.66 ±.84 for restraint groups. Values are means and S.E.M. of 1 rats. Control 185. ±12.3 Restraint ±7.8 6.FNE ISO ISO + 6FNE Adenosine Menos* + &FNE Histarine IistaTine + &FNE ± ± ± ±23.6 % basa!c.4mp vakie 129 ± ± ± ± ± ± ± ±15.8

5 76 Stone et al. Vol J L) o Unpotentioled R Potentioted by 6-FNE ISO Adeno Hist. Fig. 3. Effect of repeated restraint stress on 6-FNE-induced potentiation of camp responses to isoproterenol, 1 M, adenosine, 1 M, and histamine, io- M. The means and S.E.M. of the restraint group (n = 1 ) are shown expressed as a percentage of the corresponding control group means. Mean basal CAMP levels (picomoles per milligram of protein):control group, 6.2 ±.3; restraintgroup, 6.7 ±.5. <.5; ** P <.1 vs. unpotentiated response. Adeno, adenosine; Hist, histamine. experiments were completed a paper by Duman et a!. (i985) was published showing identical effects of ACTH on the alpha receptor component. These authors also found that the effect of ACTH on the camp response could be abolished if alpha-i receptors were blocked by prazosin during stimulation by NE. This indicates that the effect of ACTH treatment is similar to that of stress and suggests that pituitary hormones mediate the latter s effect on alpha receptor function. The present study also compared the mechanism of subsensitivity after stress with that produced by repeated administration of the tricyclic antidepressant, DM1. It was found that unlike stress DM1 did not alter the potentiation of the beta response by alpha receptor stimulation but did significantly reduce the response to selective beta stimulation and to alpha + beta stimulation. The decreased response to alpha + beta stimulation after DM1 can therefore be accounted for entirely on the basis of the reduced function of beta adrenergic receptors. These results agree with previous findings that have indicated that DM1 produces a fairly selective reduction in beta as compared with alpha receptor density in the rat brain (reviewed by Reisine, 1981). Duman et al. (1985) have reported essentially identical results using the antidepressant, imipramine. Previous findings have suggested that the beta and alpha adrenergic receptors that control camp production in the rat brain are modulated through different physiological pathways (Sulser et al., i983). The alpha adrenergic response seems to be sensitive primarily to changes in pituitary and/or adrenal cortical hormones (Mobley et al., i983) whereas the beta response appears to be affected primarily by agents that affect the availability of brain NE (Sulser, 1982). The present results are in agreement with these findings in that ACTH treatment was found to affect primarily the alpha-induced potentiation effect whereas the NE reuptake inhibitor, DM1, affected only the beta response. We have confirmed these findings in more recent experiments which have shown that hypophysectomy selectively increases the alpha component whereas lesion of central noradrenergic neurons selectively increases the beta component of the cortical and hippocampal NE-cAMP response (Stone et al., i986). However, the separation of these systems does not appear to be complete as adrenal cortical hormones have been shown to produce marked changes in beta adrenergic receptor density both in the brain (Roberts and Bloom, i98i; Duman et at., i984) and periphery (reviewed by Davies and Lefkowtiz, i984) whereas central noradrenergic lesions can produce dramatic alterations in the density of brain alpha-i and alpha-2 adrenergic receptors (U Prichard et at, i98; Menkes et al., i983). In the present study an effect of pituitary-adrenal hormones on beta receptor function was suggested by the finding that ACTH treatment, although it did not have a significant effect on beta receptor function, nevertheless produced an increase in the beta response that was large enough to offset the reduced alpha potentiation effect so that the net response to combined alpha and beta stimulation by NE was not significantly reduced from control values. Therefore, further research is necessary to delineate the conditions under which the pituitary-adrenal and central noradrenergic systems modulate the functions of brain beta and alpha adrenergic receptors. Acknowledgments The authors thank Stuart Schwam for his expert advice and assistance in computer techniques. References BLOOM, F.: The role of cyclic nucleotides in central synaptic function. Rev. Physiol. Biochem. Pharmacol. 74: 1-14, BROWN, B. L., ALBANO, J. D. M., EKIN5, R. P., SQHERZI, A. M. 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