Regulation of Pineal α1B-Adrenergic Receptor mRNA: Day/Night Rhythm and β-Adrenergic Receptor/Cyclic AMP Control

  1. Steven L. Coon1,
  2. Susan K. McCune2,
  3. David Sugden3 and
  4. David C. Klein1
  1. 1Section on Neuroendocrinology, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 (S.L.C., D.C.K.), 2Division of Neonatology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3200 (S.K.M.), and 3Department of Physiology, Kings College London, University of London, London, England W8 7AH (D.S.)
     
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    Abstract

    Mammalian pineal function is regulated by norepinephrine acting through α1B- and β1-adrenergic receptors (ARs). Noradrenergic stimulation of α1B-ARs potentiates the β1-AR-driven increase in cAMP, serotoninN-acetyltransferase, and melatonin production. In the present study, we describe a 3-fold daily rhythm in mRNA-encoding α1B-ARs in the pineal gland, with a peak at midnight. Pharmacological studies indicate that this increase in α1B-AR mRNA is due to activation of β-ARs. Second messenger studies indicate that α1B-AR mRNA is increased by agents that increase cAMP, including dibutyryl cAMP, cholera toxin, forskolin, or vasoactive intestinal peptide. These observations indicate that α1B-AR mRNA can be physiologically regulated by a β-AR-dependent enhancement of cAMP. It also was observed that in vivo and in vitrochanges in α1B-AR mRNA are not accompanied by similar changes in α1B-AR binding, indicating that turnover of α1B-AR protein is significantly slower than that of α1B-AR mRNA and that post-transcriptional mechanisms play an important role in regulating α1B-AR binding.

    The pineal gland contains a high abundance of both α1- and β1-ARs, which play an important role in regulating circadian pineal function, including melatonin synthesis (1-3). Adrenergic stimulation of the gland increases at night in response to central stimulation of the release of norepinephrine from sympathetic nerve processes, terminating in pineal extracellular space.

    α1- and β1-ARs interact in the pineal gland through biochemical “AND” gates (4-6). The receptor-receptor interaction occurs at postreceptor sites and involves α1-AR potentiation of the effects of β1-ARs. Potentiation is evident from analysis of cAMP, cGMP, serotonin N-acetyltransferase, and melatonin (7, 8). β1-ARs positively regulate the activity of both adenylyl and guanylyl cyclases through mechanisms involving G proteins (9, 10). The specific functions of α1-ARs are mediation of adrenergically stimulated increases in [Ca2+]i and activation of PKC and phospholipases (9, 11-13). Pharmacological studies suggest that the α1-ARs in the pineal gland are predominantly of the α1B subtype (14), which is consistent with evidence of a high abundance of α1B-AR mRNA (15).

    Because of the importance of α1B-AR to pineal physiology, it is important to understand the mechanisms regulating α1B-ARs in this neuroendocrine gland. Previously, rat pineal glands have been shown not to exhibit a daily rhythm in α1B-AR binding in vivo despite the large nocturnal increase in NE (16). Additionally, neither acute nor chronic administration of phenylephrine or isoproterenol had any effect on α1B-AR binding (16). In contrast, pineal β1-AR binding has been shown to fluctuate on a daily basis (17). Furthermore, pineal β1-AR mRNA can be up-regulated through β-AR, an effect that can be mimicked by forskolin (18). Because α1B-AR binding and mRNA can be up-regulated in DDT1 MF-2 smooth muscle cells by β2-AR acting through cAMP (19), we investigated whether α1B-AR mRNA might be regulated in the rat pineal gland by an adrenergic → cAMP mechanism.

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    Materials and Methods

    In vivo experiments.

    The animals used in this study were 150–200 g male Sprague-Dawley rats that had been in our facilities for at least 1 week (light:dark 14:10, lights on at 5:00 a.m.). In some cases, animals were kept in light at night to block stimulation of the pineal gland by the SCN (5). Animals were killed by decapitation at the times indicated. Tissues were removed immediately and placed on dry ice. Handling and maintenance of rats were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

    In vitro experiments.

    Adult female Sprague-Dawley rats were decapitated and the pineal glands were placed immediately in Dulbecco’s modified Eagle’s medium containing 50 units/ml penicillin, 50 μg/ml streptomycin, 2 mmglutamine, and 20 mm HEPES, pH 7.4 (4°). After cleaning, the glands were cultured in 200 μl of Biggers-Gwatkin-Judah medium with Fitton-Jackson modification (no serum) supplemented with 0.5 mm ascorbic acid as an antioxidant in 24-well cluster dishes as described previously (2). For treatments, glands were transferred into fresh medium containing freshly prepared ascorbic acid (0.5 mm) and the indicated drugs or vehicle. Drug treatments were performed in 24-well cluster dishes in a top-loading incubator with a humidified atmosphere of 5% CO2/95% O2 at 37° for the indicated times (2).

    RNA preparation and Northern blot analysis.

    Total RNA was extracted using the guanidine HCl/phenol procedure (20) and poly(A)+ RNA was subsequently purified using oligo-dT latex beads (Qiagen, Santa Clarita, CA). RNA was separated on a 1.5% agarose/0.7 m formaldehyde gel for 5 hr at 2.5 V/cm. Electrophoresed RNA was transferred to a charged nylon membrane by passive capillary transfer and cross-linked to the membrane using UV. The hybridization probe for α1B-AR mRNA was32P-labeled by random-priming a full-length cDNA for rat α1B-AR (21). Blots were also probed for either 18 S ribosomal RNA (22) or G3PDH (Clontech, Palo Alto, CA) mRNA to normalize data for variations in RNA loading; G3PDH mRNA did not vary on a 24-hr basis or as a result of drug treatments. Blots were hybridized at 68° for 1 hr in QuikHyb buffer (Stratagene, LaJolla, CA). The final wash was in 0.1 × SSC (1× = 150 mm NaCl, 15 mm sodium citrate)/0.1% sodium dodecyl sulfate at 60° for 15 min. Blots were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). α1B-AR mRNA was present as three bands of approximately 2.0, 2.3, and 2.5 kb in the pineal gland and the brain regions examined; transcripts of approximately 2.5 and 3.0 kb were detected in the liver. For quantitation, the abundance of α1B-AR mRNA in all bands has been summed and normalized to 18 S ribosomal RNA or G3PDH mRNA expression. Transcript sizes were estimated by comparison with standard RNA markers (BRL RNA ladder; GIBCO BRL, Gaithersburg, MD).

    In situ hybridization.

    In situhybridization was performed as described previously (23). Briefly, brains were dissected from adult male Sprague-Dawley rats immediately after decapitation, frozen, and stored at −80° until used. Frozen sections (20 μm) were mounted on slides and fixed with 4% formaldehyde. After dehydration, the sections were hybridized overnight at 37° in 120 μl of 50% formamide, 4× SSC, 1× Denhardt’s solution, and 10% dextran sulfate with 500 μg/ml salmon sperm DNA and 100 mm fresh dithiothreitol containing 1–3 × 106 cpm of 35S-labeled probe. Oligonucleotide probes for α1B-AR mRNA were 3′ end-labeled using terminal deoxynucleotidyl transferase and 35S-dATP to a specific activity of 5–15 × 108 cpm/μg. After overnight hybridization, the slides were washed in 2× SSC with 50% formamide at 40° for 1 hr with three changes, followed by a 1-hr wash at room temperature in 1× SSC with two changes. The sections were exposed to Hyperfilm-β max film (Amersham Life Science, Clearbrook, IL) for 11–17 days at room temperature.

    Estimation of α1B-AR density.

    α1B-AR density was estimated using radioligand binding methodology (16). Pineal glands were removed from the culture plates after treatment, frozen immediately on dry ice, and then stored frozen (−70°). Individual pineal glands were sonicated (3 × 10 sec) in 100 μl of buffer A (50 mm Tris·HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.1 mmphenylmethylsulfonyl fluoride). The homogenate was diluted to 1.4 ml and centrifuged (100,000 × g, 20 min at 4°). The pellet was dispersed by sonication in 720 μl of buffer A. Protein was measured using a Bradford dye-binding procedure. Duplicate membrane aliquots (150 μl, 3–5 μg protein) were incubated (25°, 20 min) with [125I]HEAT (50 or 200 pm; DuPont-New England Nuclear, Boston, MA) in the presence or absence of phentolamine (10 μm) to define nonspecific binding (16). In saturation experiments, the Kd was determined to be 82 ± 13 pm and the Bmax was found to be 273 ± 19 fmol/mg protein (mean ± standard error,n = 4). α1B-AR density also was measured by a similar method in which pairs of glands were homogenized together and duplicate aliquots of membrane were incubated (31°, 50 min) with [3H]prazosin (DuPont-New England Nuclear) in the presence or absence of 10 μm phentolamine (24).

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    Results

    α1B-AR mRNA is highly abundant in the pineal gland compared with the brain and liver. In situ hybridization and Northern blot analysis were used to estimate the relative abundance of α1B-AR mRNA in the pineal gland and other tissues (Fig.1). In situ hybridization indicated that the abundance of mRNA encoding this receptor was higher than in any other brain area examined, including the thalamus, which is believed to contain the highest brain level of α1B-AR mRNA (15, 23,25). Northern blot analysis confirmed this and also indicated that the pineal gland contained a higher abundance of α1B-AR mRNA as compared with liver (Fig. 1, B and C), which is known to express high levels (25, 26).

    Figure 1
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    Figure 1

    α1B-AR mRNA is expressed at high levels in the rat pineal gland. A, Coronal sections from the brain of an adult rat killed during the day were hybridized with a suite of three α1B-AR-specific oligonucleotides. B, Northern blot analysis of α1B-AR mRNA in the pineal gland compared with total brain and liver showing the transcripts detected. The blot contained poly(A)+ RNA from day pineal glands (1 μg), total brain (6 μg), and liver (3 μg). Molecular size standards are indicated to the right and the calculated sizes of the three α1B-AR pineal transcripts are indicated to the left. C, Northern blot analysis comparing the abundance of α1B-AR mRNA in various brain regions and liver. Each lanecontained 20 μg of total RNA. RNA preparation and Northern blot analysis are as described in the text. The histogram is derived from the Northern blots shown in the inset. The abundances of the three α1B-AR transcripts have been summed and normalized to 18 S ribosomal RNA expression.

    Pineal α1B-AR mRNA exhibits a 3-fold day/night rhythm that is under photoneural regulation.

    The abundance of pineal α1B-AR mRNA varies on a 24-hr basis (Fig.2). The lowest levels occur at the beginning of the night and the highest values occur approximately 6 hr after lights are turned off (Fig. 2). The increase at night is approximately 3-fold, according to the results of three independent experiments. The nocturnal increase in α1B-AR mRNA was blocked by continued exposure to light at night (Fig. 2).

    Figure 2
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    Figure 2

    Daily rhythm in pineal α1B-AR mRNA. Northern blot analysis of α1B-AR mRNA in pineal glands taken from rats at the indicated times of the day. Eachlane contained 20 μg of total RNA from a pool of seven pineal glands. Three groups of rats remained in light during the final dark period and were killed at the same time as their lights-off counterparts. Shaded bar, lights off. Similar results were obtained in three additional experiments. RNA preparation and Northern blot analysis are as described in the text.Graph, derived from the Northern blots shown in theinset. The abundances of the three α1B-AR transcripts have been summed and normalized to G3PDH mRNA expression.

    β-Adrenergic and VIP regulation of pineal α1B-AR mRNA..

    Sympathetic stimulation of the pineal gland is believed to be mediated primarily by the release of NE. To determine whether NE regulates pineal α1B-AR mRNA, organ culture was used. NE treatment increased the abundance of α1B-AR mRNA by 3- to 5-fold in a dose-dependent manner (Fig. 3A). The maximum stimulation was observed at approximately 9 hr, and α1B-AR mRNA levels remained elevated above basal levels for as long as 24 hr (Fig. 3B). The abundance of α1B-AR mRNA also was increased by the β-adrenergic agonist isoproterenol but not by the α-adrenergic agonist phenylephrine (Fig.4A), suggesting that NE is acting through pineal β1-ARs. This conclusion was supported by the finding that the β-adrenergic antagonist propranolol blocked the action of NE and that the α-adrenergic antagonist prazosin did not (Fig. 4B). Propranolol alone had a small positive effect on α1B-AR mRNA; the basis of this effect was not pursued.

    Figure 3
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    Figure 3

    Norepinephrine increases the abundance of α1B-AR mRNA. A, Northern blot analysis of the effect of NE concentration on accumulation of α1B-AR mRNA. Cultured pineal glands were exposed to the indicated doses for 6 hr. Eachlane contained 3 μg of total RNA from a pool of 4 pineal glands. Data are means ± range of duplicate determinations. Similar results were obtained in three other experiments. B, Northern blot analysis of α1B-AR mRNA from pineal glands exposed to NE (1 μm) for the indicated times; glands were transferred every 6 hr to fresh media containing NE (arrows). Each lane contained 10 μg of total RNA from a pool of six pineal glands. ▪, Level of α1B-AR mRNA after 6 hr under control conditions. Similar results were obtained in two other experiments. RNA preparation and Northern blot analysis were as described for Fig. 2.

    Figure 4
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    Figure 4

    Regulation of α1B-AR mRNA by β-ARs. A, Northern blot analysis of α1B-AR mRNA from pineal glands exposed to adrenergic agonists. Pineal glands were cultured for 6 hr with NE (1 μm), isoproterenol (ISO; 0.1 μm), or phenylephrine (PE; 1 μm). Each lane contained 5 μg of total RNA from a pool of three pineal glands. Duplicate lanes are independent samples from one experiment that was representative of three experiments. B, Northern blot analysis of α1B-AR mRNA from pineal glands exposed to adrenergic antagonists. Glands were cultured with: prazosin alone (5 μm, 6.5 hr); propranolol alone (10 μm, 6.5 hr); NE alone (0.1 μm, 6 hr); prazosin (0.5 hr) then NE + prazosin (6 hr); propranolol (0.5 hr) then NE + propranolol (6 hr). Each lane contained 3 μg of total RNA from a pool of four pineal glands. Duplicate lanes are independent samples from one experiment. RNA preparation and Northern blot analysis were as described for Fig. 2.

    cAMP regulates pineal α1B-AR mRNA.

    β1-AR activation of the pineal gland and many other tissues increases intracellular cAMP concentrations. This and a report that cAMP elevates α1B-AR mRNA in a transformed cell line [DDT1 MF-2 vas deferens cells (19)] point to the possible involvement of cAMP in the regulation of pineal α1B-AR mRNA. This was examined by treating glands with cAMP protagonists, including dibutyryl cAMP, cholera toxin, and forskolin or with VIP, which has been shown to elevate cAMP in this tissue (27). These agents elevated α1B-AR mRNA, suggesting the existence of a β1-AR → cAMP → α1B-AR mRNA regulatory link (Fig. 5).

    Figure 5
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    Figure 5

    α1B-AR mRNA is regulated via cAMP. Northern blot analysis of α1B-AR mRNA from pineal glands exposed to cAMP protagonists or VIP. Pineal glands were cultured for 6 hr with NE (1 nm), dibutyryl cAMP (DB-cAMP; 0.1 mm or 1.0 mm), cholera toxin (CTX; 50 μg/ml), forskolin (FSK; 100 μg/ml), or VIP (0.1 μm). Each lanecontained 5 μg of total RNA from a pool of two pineal glands. Duplicate lanes are independent samples from one experiment that was representative of four experiments. RNA preparation and Northern blot analysis were as described for Fig. 2.    

    Adrenergic agonists do not alter α1B-AR binding.

    Because the conditions under which we observed a daily rhythm in α1B-AR mRNA in vivo (see above) had been shown previously not to effect α1B-AR binding (16), we investigated in vitro whether changes in α1B-AR mRNA could be correlated to changes in α1B-AR binding. Stimulation with NE for up to 24 hr did not affect α1B-AR binding, whereas direct stimulation of adenylyl cyclase with forskolin resulted in a 70–90% increase in α1B-AR binding after long term exposure (Fig.6, Table 1). Elevation of cAMP using VIP (Table 1) or cholera toxin (data not shown) for 12 hr did not effect α1B-AR binding. Similarly, treatment with selective adrenergic agonists phenylephrine or isoproterenol had no effect on binding (Table 1).

    Figure 6
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    Figure 6

    Forskolin (FSK) increases α1B-AR binding but NE has no effect. Measurement of α1B-AR binding in membranes from cultured pineal glands following exposure to NE (0.1 μm) or FSK (100 μg/ml). Binding data were generated using 200 pm[125I]HEAT as described in the text. Data are mean ± standard error of determinations on seven to eight individual glands.

    View this table:
    Table 1

    Effects of treatments with adrenergic agonists and cAMP protagonists on pineal α1B-AR density

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    Discussion

    Data in this report demonstrate that the abundance of pineal α1B-AR mRNA exhibits a physiological daily rhythm that is abolished by continued light at night. These characteristics are typical for many aspects of pineal biochemistry that are regulated by the circadian stimulation of the pineal gland via a neural circuit that includes the SCN, other central neural structures, and the superior cervical ganglia (5, 6). Light acts through the retina and a retina-to-SCN neural connection to block SCN stimulation of the pineal gland, which probably explains why pineal α1B-AR mRNA does not increase in animals exposed to light at night. A prominent feature of this system is that NE is released at night from the sympathetic fibers innervating the pineal gland.

    The role of NE in regulating α1B-AR mRNA was demonstrated in organ culture, in which NE induced a sustained increase in α1B-AR transcripts. This increase seems to be mediated by a β-AR → cAMP mechanism, because the effect could be mimicked by the β-AR agonist isoproterenol and cAMP protagonists, including dibutyryl cAMP, forskolin, cholera toxin, and VIP.

    Regulation of steady state levels of α1B-AR mRNA has been shown previously to be tissue- and agonist-specific and may involve multiple second messenger and transcriptional mechanisms. For example, in thyroid cells, thyrotropin, and cAMP induce a long term increase in α1B-AR mRNA like that seen here in the pineal gland (28). In contrast, in vascular smooth muscle cells, NE and bradykinin transiently decrease α1B-AR mRNA through a mechanism involving PKC (29). In DDT1 MF-2 cells, exposure to NE results in a transient rise in α1B-AR mRNA followed by a long term decrease; the initial increase is mimicked by cAMP and β-AR stimulation (19). In the same cells, long term activation of PKC, or stimulation by glucocorticoids, can cause a prolonged increase in α1B-AR mRNA (30, 31). Thyroid state may either increase or decrease α1B-AR mRNA in rats, depending on the tissue (32). The diversity of regulatory mechanisms may be explained partially by the presence in the α1B-AR promoter of putative cAMP response elements, glucocorticoid response elements, and thyroid response elements (33-35). Additional sequences have been identified for AP-2, SP-1, and other transcription factor binding sites (33-35). The effect of cAMP in up-regulating the abundance of α1B-AR mRNA could be mediated by phosphorylation of the cAMP response element binding protein, which has been shown to occur in the pineal gland (36).

    It was observed that the severalfold changes in α1B-AR mRNA seen in both in vivo and in vitro studies are not accompanied by similar changes in α1B-AR binding (16, this study). The fact that forskolin could increase α1B-AR binding provides evidence that changes in mRNA can, under limited circumstances, be translated into changes in binding. However, the absence of a day/night rhythm in binding and of the lack of an effect of isoproterenol, cholera toxin, and VIP suggests that α1B-AR protein turns over slower than α1B-AR mRNA, thereby masking the mRNA rhythm. The ability of forskolin to induce changes, whereas other cAMP protagonists did not, may reflect temporal differences in their actions on cAMP.

    The possibility that the constant level of receptor binding observed when glands were stimulated with NE was due to a balance between α1B-AR down-regulation induced by α1B-AR occupancy and α1B-AR mRNA up-regulation by β-AR can be discounted because neither phenylephrine nor isoproterenol changed α1B-AR binding. A lack of an agonist-induced decrease in total adrenergic receptor number also has been reported previously (37,38). The possibility exists that increased receptor synthesis is compensated for by an unknown mechanism that is not receptor mediated. Our findings are consistent with previous evidence that a complex relationship between mRNA and receptor binding exists for many adrenergic receptors (39, 40).

    These studies indicate that the pineal gland is a useful model for studying regulation of α1B-AR mRNA. The discovery that large changes in mRNA are not paralleled by similar changes in binding is of special interest because it indicates that mechanisms may exist which damp out hour-to-hour changes in mRNA, thereby maintaining a relatively constant level of receptors. Future studies on regulation of α1B-AR protein in this system may reveal more about the post-transcriptional processes that are involved in regulating the abundance of this receptor.

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    Acknowledgments

    We thank Carl Johnson for technical assistance, Dr. H. Chin (National Institute of Neurological Disorders and Stroke, Bethesda, MD) for the generous gift of the rat α1B-AR cDNA clone, and Drs. A. Bounanno and P. Roseboom for helpful comments regarding the manuscript.

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    Footnotes

    • Send reprint requests to: Dr. David C. Klein, NIH, Bldg. 49, Room 5A38, Bethesda, MD 20892. E-mail:klein{at}helix.nih.gov

    • Abbreviations:
      AR
      adrenergic receptor
      PKC
      protein kinase C
      NE
      norepinephrine
      SCN
      suprachiasmatic nuclei
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      G3PDH
      glyceraldehyde-3-phosphate dehydrogenase
      SSC
      standard saline citrate
      HEAT
      iodo-2-[β-(4-hydroxyphenyl)-ethylaminomethyl]tetralone
      VIP
      vasoactive intestinal peptide
      • Received October 31, 1996.
      • Accepted December 17, 1996.
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    1. Molecular Pharmacology April 1, 1997 vol. 51 no. 4 551-557
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