Benzodiazepines are frequently used not only as a part of general anesthesia but also for the purpose of sedation during regional anesthesia. Effects of these drugs on the hypothalamic-pituitary-adrenal axis activity have been studied, but are still controversial. It is not known whether benzodiazepines affect expression of proopiomelanocortin, precursor protein of adrenocorticotropic hormone and related peptides.
AtT20PL cell line, a clone of AtT20/D16v mouse corticotroph tumor cells stably transfected with approximately 0.7 kilobases (kb) of the rat proopiomelanocortin 5' promoter-luciferase fusion gene, was used. In the presence or absence of diazepam or midazolam, cells were stimulated by corticotropin-releasing hormone (CRH) or forskolin. Proopiomelanocortin gene expression was estimated by measurement of luciferase activity. Furthermore, to study the mechanism of benzodiazepine effects, cyclic adenosine 3',5'-monophosphate (cyclic AMP) efflux was measured by enzyme immunoassay.
Diazepam and midazolam dose-dependently increased the proopiomelanocortin gene expression induced by CRH or forskolin. The potentiating effect was not affected by benzodiazepine receptor antagonists flumazenil and PK11195, but was abolished by a cyclic AMP-dependent protein kinase inhibitor H89. Cyclic AMP efflux induced by CRH or forskolin was also enhanced by diazepam and midazolam. In the presence of isobutylmethylxanthine, a nonspecific phosphodiesterase inhibitor, potentiation of proopiomelanocortin gene expression and enhancement of cyclic AMP efflux by benzodiazepines were not observed.
Benzodiazepines potentiate the effect of CRH or forskolin on proopiomelanocortin gene expression. The potentiating effect is not mediated by the benzodiazepine receptors, but its mechanism probably involves inhibition of phosphodiesterase.
THE hypothalamic-pituitary-adrenal (HPA) axis plays a central role in maintaining the homeostasis of an organism against various stressful stimuli, including surgical noxious stimulations. 1,2When an organism suffers stressful stimuli, corticotropin-releasing hormone (CRH) is released from the hypothalamus to stimulate the corticotrophs in the anterior pituitary, resulting in secretion of adrenocorticotropic hormone (ACTH). The ACTH, in turn, induces adrenal glucocorticoid release, which exhibits diverse systemic actions against stresses. Among the HPA axis, ACTH secretion is controlled by a variety of secretagogues, and the control mechanism has been extensively studied. 3At the cellular level, ACTH, stored in intracellular pool is secreted by elevation of cytosolic Ca2+concentration induced by activation of voltage-gated Ca2+channels or intracellular Ca2+release. 4Moreover, most of the secretagogues are supposed to be involved in the regulation of ACTH production. 5The ACTH production is regulated by CRH, vasopressin, catecholamines, and cytokines through the modulation of the gene expression of proopiomelanocortin a precursor protein of ACTH and related peptides. 6–9
Control of excessive responses to surgical stresses is one of the objectives of anesthesia. Effects of general and regional anesthesia on the surgical stress responses, including those mediated by HPA axis, have been investigated. 10,11Benzodiazepines, including diazepam and midazolam, are widely used in clinical anesthesia for the purpose of premedication, induction and maintenance of general anesthesia, and sedation during regional anesthesia. However, the effect of benzodiazepines on stress responses has been controversial. It was reported that benzodiazepines significantly inhibited the ACTH and cortisol secretion after CRH administration in normal subjects, 12and attenuated the stress-induced release of corticosterone and catecholamine. 13In contrast, Vargas et al. showed that diazepam can stimulate basal HPA axis activity. 14Whether benzodiazepines affect ACTH synthesis or secretion in anterior pituitary cells has not been examined at the cellular or molecular level.
In this investigation, to elucidate the effects of benzodiazepines on ACTH synthesis in anterior pituitary cells, we have analyzed the effect of benzodiazepines, diazepam and midazolam, on the proopiomelanocortin gene expression in AtT20PL cells, a clone of AtT20/D16v mouse corticotroph tumor cells stably transfected with approximately 0.7 kilobases (kb) of the rat proopiomelanocortin 5′-promoter-luciferase fusion gene. By the use of these cells, we could analyze the level of proopiomelanocortin gene expression and elucidate the mechanism of its regulation.
Materials and Methods
Culture medium was purchased from Sigma (St. Louis, MO), and fetal bovine serum was from ICN Biomedicals (Aurora, OH). Rat CRH was purchased from Peptide Institute (Osaka, Japan). Luciferase Assay System and Reporter Lysis Buffer were from Promega (Pittsburgh, PA). Midazolam and flumazenil were purchased from Yamanouchi (Osaka, Japan), and diazepam was from Takeda (Osaka, Japan). PK11195 was from Calbiochem (San Diego, CA). Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) assay kit was from Amersham Pharmacia (Buckinghamshire, England). All the other reagents were obtained from Wako (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), and Sigma.
Isobutylmethylxanthine was dissolved in ethanol (final concentration 0.1%), and the other drugs were diluted with the culture medium to adjust adding volume to 20 μl.
Establishment of AtT20PL cell line was previously described. 6Briefly, AtT20/D16v mouse corticotroph cells were stably cotransfected with the plasmid pA3Luc containing a fragment (approximately 0.7 kb) of the rat proopiomelanocortin gene 5′-promoter fused with luciferase gene and the plasmid pRSV-Neo. Neomycin-resistant clones were screened in the medium containing G418, and a representative clone AtT20PL was selected.
AtT20PL cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, in the humidified atmosphere of 95% air and 5% carbon dioxide at 37°C. Culture medium was changed every two days. For all experiments, AtT20PL cells were plated in six-well culture dishes (Becton Dickinson, Franklin Lakes, NJ) with approximately 60% confluency and were cultured in DMEM supplemented with 1% fetal bovine serum (low-serum medium) for four days.
In each experiment, two wells were similarly treated, luciferase activity or cyclic AMP efflux was determined separately from the two wells, and the two values were averaged. Independent experiments, the numbers of which are indicated in the figure legends, were repeated using separately prepared cell cultures.
After 4-day incubation in the low-serum medium, incubation medium was replaced with 2 ml/well of fresh low-serum medium, and the cells were stimulated with CRH or forskolin for 5 h with or without simultaneous addition of benzodiazepines and inhibitors. After stimulation, cells were washed twice with ice-cold phosphate-buffered saline (PBS), and cell lysate was prepared with 200 μl/well of Reporter Lysis Buffer. The luciferase activity in the cell lysate was measured by the use of the Luciferase Assay System and the luminometer Lumat LB9507 (Berthold, Victoria, Australia), and was shown in relative luciferase unit (RLU).
Cyclic AMP Assay
After 4-day incubation in the low-serum medium, incubation medium was replaced with 2 ml/well of fresh serum-free DMEM, and then the cells were stimulated with CRH or forskolin for 3 h with or without simultaneous addition of benzodiazepines. After stimulation, culture medium in each well was collected. The cyclic AMP concentration in the collected medium was determined by the use of the cyclic AMP enzymeimmunoassay system 15(Amersham Pharmacia), which combines the use of a peroxidase-labeled cyclic AMP conjugate and a specific anticyclic AMP antiserum immobilized to microtiter plates.
Data are expressed as means ± SD. Statistical analyses of data were performed by one-way analysis of variance, followed by Fisher's protected least significance difference test. P values < 0.05 were considered statistically significant.
Potentiation of Proopiomelanocortin Gene Expression by Benzodiazepines
As representative secretagogues, we chose CRH, a physiologic stimulus for ACTH secretion, and forskolin, a direct activator of adenylate cyclase. As described previously, 6,16CRH and forskolin significantly induced proopiomelanocortin gene expression, detected as luciferase activity, in AtT20PL cells (fig. 1A). Stimulation of the cells with 1 μm CRH and 10 μm forskolin for 5 h resulted in elevation of proopiomelanocortin expression by 50 ± 9% (n = 8) and 45 ± 23% (n = 8) of the basal level, respectively. Previous reports 6,16indicated that these concentrations of the secretagogues produce nearly maximal responses. Therefore, we used CRH and forskolin of these concentrations in the following experiments.
Figure 1Bdemonstrates the effect of diazepam and midazolam on the proopiomelanocortin expression in AtT20PL cells. In the absence of CRH or forskolin, the effect of diazepam and midazolam on the proopiomelanocortin expression level was not significant. However, CRH-induced proopiomelanocortin expression was significantly increased by diazepam and midazolam at 10 and 100 μm. Forskolin-induced proopiomelanocortin expression was also enhanced by diazepam (10 and 100 μm) and midazolam (100 μm). Thus, benzodiazepines exhibited dose-dependent potentiating effects on the CRH- or forskolin-stimulated proopiomelanocortin gene expression in AtT20PL cells.
Effects of Benzodiazepine Antagonists
A number of pharmacological effects of benzodiazepines are mediated by the central 17and the peripheral 18benzodiazepine receptors. To analyze the involvement of the benzodiazepine receptors in the potentiation of the proopiomelanocortin expression by diazepam and midazolam, we tested the effect of benzodiazepine antagonists on the proopiomelanocortin expression in AtT20PL cells (fig. 2). Flumazenil 19(10 μm) and PK11195 20(10 μm), antagonists for the central and the peripheral benzodiazepine receptor, respectively, do not significantly affect the CRH- or forskolin-induced proopiomelanocortin expression in the presence of benzodiazepines. In this experiment, midazolam (100 μm) induced a small but significant increase in luciferase activity in the absence of CRH or forskolin, which also was not affected by benzodiazepine antagonists.
Involvement of Cyclic AMP/Cyclic AMP-dependent Protein Kinase Pathway
It has been demonstrated that CRH and forskolin induce expression of proopiomelanocortin by increasing intracellular cyclic AMP level and activating cyclic AMP-dependent protein kinase (PKA). 6To explore the possibility that potentiating effect of diazepam and midazolam is mediated by cyclic AMP/PKA pathway, we tested the effect of H89, a specific inhibitor of PKA (fig. 3). As shown previously, 6the stimulatory effect of CRH on proopiomelanocortin expression was completely abolished by H89 (30 μm). Furthermore, figure 3demonstrates that proopiomelanocortin expression by CRH was not significantly enhanced by diazepam and midazolam in the presence of H89.
Effect of Benzodiazepines on CRH-stimulated Proopiomelanocortin Expression under Isobutylmethylxanthine Treatment
To further clarify the mechanism of the enhancing effect of benzodiazepines, we tested the effect of diazepam and midazolam on proopiomelanocortin gene expression stimulated by CRH and forskolin in cells treated with isobutylmethylxanthine (200 μm), a nonselective phosphodiesterase inhibitor 21(fig. 4). Isobutylmethylxanthine itself did not significantly induce proopiomelanocortin expression, but enhanced the response to CRH and forskolin by 7.8 ± 1.9-fold (n = 6) and 9.6 ± 2.2-fold (n = 6), compared with the response in cells without isobutylmethylxanthine treatment, respectively. However, in the presence of isobutylmethylxanthine, neither diazepam (100 μm) nor midazolam (100 μm) exhibited potentiating effect on proopiomelanocortin gene expression. Unexpectedly, the CRH- or forskolin-stimulated proopiomelanocortin expression was significantly reduced by benzodiazepines.
Effect of Benzodiazepines on Cyclic AMP Efflux
To clarify the effect of benzodiazepines on the cyclic AMP regulation in AtT20PL cells, we next estimated the cyclic AMP efflux in the presence and absence of benzodiazepines. As shown previously, 6CRH (1 μm) significantly increased cyclic AMP efflux (fig. 5A). Diazepam (10 and 100 μm) and midazolam (100 μm) significantly enhanced the CRH response (fig. 5B). In the absence of benzodiazepines, forskolin (10 μm) showed marginal increase in cyclic AMP efflux (fig. 5A). However, in the presence of diazepam (100 μm) or midazolam (100 μm), forskolin significantly stimulated the cyclic AMP efflux. Thus, diazepam and midazolam dose-dependently enhance cyclic AMP efflux induced by CRH or forskolin.
Figure 6demonstrates the effect of isobutylmethylxanthine on the cyclic AMP efflux. Isobutylmethylxanthine (200 μm) increased the cyclic AMP efflux by 7.3 ± 1.4-fold (n = 4), compared with the basal level. CRH and forskolin further enhanced the isobutylmethylxanthine-stimulated cyclic AMP efflux by 23.1 ± 2.5-fold (n = 4) and 5.5 ± 1.2-fold (n = 4), respectively. However, diazepam (100 μm) and midazolam (100 μm) did not further increase CRH- or forskolin-stimulated cyclic AMP efflux in the presence of isobutylmethylxanthine. The forskolin-stimulated cyclic AMP efflux was significantly reduced by benzodiazepines.
Although benzodiazepines are frequently used in clinical anesthesia, the effects of benzodiazepines on HPA-mediated stress responses induced by various stimuli, including surgical noxious stimulation, are not completely understood. In this investigation, to elucidate the effects of benzodiazepines on HPA axis, we examined the effects of benzodiazepines on the proopiomelanocortin gene expression in AtT20PL cells. The results show that benzodiazepines potentiate the effect of CRH or forskolin on proopiomelanocortin gene expression.
The AtT20 cells (mouse anterior pituitary tumor cell line) have been widely used to study regulation of secretion of ACTH and β-endorphin, because these cells faithfully model corticotroph cells. 22In the field of anesthesiology, it was shown that propofol suppresses β-endorphin secretion from AtT20 cells and was suggested that propofol inhibits neuropeptide exocytosis. 23We have used AtT20PL cells, a clone of AtT20 cells stably transfected with approximately 0.7 kb of the rat proopiomelanocortin 5′-promoter-luciferase fusion gene. 24AtT20PL cells have enabled us to easily estimate the level of proopiomelanocortin gene expression by measurement of luciferase activity without assessing the amount of proopiomelanocortin messenger RNA by Northern blotting. Amount of proopiomelanocortin messenger RNA and protein may be affected also by a number of factors other than the promoter activity, including regulatory elements of the gene, degradation of messenger RNA, and posttranslational modifications. However, the luciferase assay is appropriate for assessing promoter activity, which determines the speed of gene transcription and is regulated by various factors. By the use of AtT20PL cells, the mechanism of regulation of proopiomelanocortin gene expression by physiologic secretagogues and cytokines have been investigated. 6,7,16
It is known that benzodiazepines, including diazepam and midazolam, bind with the central benzodiazepine receptor, which constitutes a part of the γ-aminobutyric acid type A (GABAA) receptor located in the central nervous system, and produce a facilitatory effect on the GABAAreceptor channel, resulting in their sedative, hypnotic and anxiolytic effects. 17Benzodiazepines also bind with peripheral benzodiazepine receptors, located mainly in peripheral tissues including adrenal gland and kidney, and are suggested to affect mitochondrial cholesterol transport. 18In this study, we demonstrate that the potentiating effect of diazepam and midazolam on the proopiomelanocortin gene expression was affected by neither flumazenil nor PK11195, antagonists for the central and peripheral benzodiazepine receptor, respectively. These results indicate that the effect of diazepam and midazolam on proopiomelanocortin gene expression is mediated by a mechanism that involves neither the central nor the peripheral benzodiazepine receptors. Biologic effects of benzodiazepines mediated by mechanisms other than the central or the peripheral benzodiazepine receptors have been described. Yamakage et al. demonstrated that diazepam and midazolam have inhibitory effects on the activity of voltage-dependent Ca2+channels in canine tracheal smooth muscle cells, which was not affected by flumazenil and PK11195. 25Furthermore, we have recently reported that midazolam, but not diazepam, induces expression of immediate early gene products c-Fos and EGR-1, by activation of extracellular signal-regulated kinases through a mechanism independent of the central or the peripheral benzodiazepine receptors in PC12 cells. 26Thus, it is likely that molecules affected by benzodiazepines other than the benzodiazepine receptors exist.
It was previously reported that a PKA inhibitor, H89, completely abolishes proopiomelanocortin gene expression and ACTH secretion induced by CRH and forskolin, suggesting that the mechanism of action of these secretagogues involves adenylate cyclase activation leading to PKA activation. 6Our data show that potentiation of the proopiomelanocortin expression by diazepam and midazolam was also completely abolished by H89 treatment. Therefore, it is reasonable to speculate that the effect of benzodiazepines on proopiomelanocortin expression involves the cyclic AMP/PKA pathway, consistent with the benzodiazepine-induced increase in CRH- or forskolin-stimulated cyclic AMP efflux. We further demonstrate that diazepam- and midazolam-induced enhancement of the proopiomelanocortin expression and cyclic AMP efflux is not observed in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. This result suggests that diazepam and midazolam increase cyclic AMP content by inhibiting phosphodiesterase, rather than by enhancing production of cyclic AMP, leading to PKA activation and potentiation of proopiomelanocortin expression.
Molecular biologic and pharmacologic studies have clearly established the existence of seven families of phosphodiesterase isozymes, with their differences in their tissue distribution. 27Consistently with our conclusion, it was demonstrated that the type 4 phosphodiesterase inhibitors stimulates the HPA axis at the level of the anterior pituitary. 28,29However, it was reported that diazepam behaves as a selective type 4 phosphodiesterase inhibitor in cardiac tissue, and that this effect is mediated neither by the central nor the peripheral benzodiazepine receptors. 30Taking these findings into consideration, it is probable that type 4 phosphodiesterase exists in anterior pituitary cells or AtT20 cells, but the molecular identity remains to be clarified. Furthermore, the precise mechanism of phosphodiesterase inhibition by benzodiazepines should be elucidated.
Our results may suggest that benzodiazepines facilitate ACTH and cortisol secretion evoked by stress-induced CRH release in vivo . However, effects of benzodiazepines on CRH release from hypothalamus, which is regulated by a number of factors, 31should also be investigated. Furthermore, sedative effects of benzodiazepines might indirectly change HPA activity. In the present study, potentiation of proopiomelanocortin gene expression and increase in cyclic AMP efflux by benzodiazepines were significant at concentrations 10–100 μm, which nearly corresponds with the reported concentration range of benzodiazepines inducing pharmacologic effects through the non-GABAergic mechanism. 25,26,32,33This concentration range seems to be the highest level of the plasma concentration of benzodiazepines obtained when these drugs are systemically administered. 34,35However, it is possible that the effective concentration of these drugs in plasma is lower, if we take into account the fact that benzodiazepines are highly bound to plasma protein. 36Therefore, clinical implications of our findings should be explored.
In conclusion, benzodiazepines potentiate the effect of CRH or forskolin on proopiomelanocortin gene expression by inhibiting phosphodiesterase through a mechanism independent of the benzodiazepine receptors. It is expected that our results may give a clue to elucidation of benzodiazepine effects on the HPA axis and mechanism of neuroendocrine responses to stresses.
The authors thank Etsuko Kobayashi and Naoko Watanabe (Secretaries, Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan) for secretarial assistance.