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Proc. Natl. Acad. Sci. USA Vol. 85, pp. 3240-3244, May 1988 Neurobiology Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla R. FISCHER-COLBRIE*, A. IACANGELOt, AND L. E. EIDENtt *Department of Pharmacology, University of Innsbruck, A-6020 Innsbruck, Austria; and tUnit on Molecular and Cellular Neurobiology, Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, MD 20205 Communicated by Julius Axelrod, December 24, 1987 (received for review September 20, 1987) ABSTRACT The influence of neurogenic versus humoral mechanism of adaptation to the environment, allowing in- factors on mRNA levels of several secretory proteins of rat creased sensitivity of response to situations such as stress, adrenal medulla was studieM in vivo. Increased splanchnic physical exercise, hypotension, or shock. Conceivable nerve activity was generated (reflexly) with insulin treatment. mechanisms for regulating the biosynthesis and metabolism Twenty-four hours after insulin injection, levels of mRNAs of adrenomedullary secretory proteins are altered impulse encoding neuropeptides (enkephalin and neuropeptide Y) were activities of the splanchnic nerve or regulation via humoral increased 6.5-fold, whereas those of mRNAs for the major factors, such as hormones of the pituitary-adrenal axis. It is secretory proteins (chromogranins A and B) were unchanged. well established (15-17) that stimulation of presynaptic Bilateral. transection of the splanchnic nerves completely pre- nerves increases synthesis of dopamine ,3-hydroxylase in rat vented this increase. Hypophysectomy decreased levels of adrenal medulla, whereas hypophysectomy decreases those chromogranin A mRNA to 32% of control, suggesting a levels (18). Similarly, maintenance of activity of another dependence on hormones of the pituitary-adrenal axis. Treat- catecholamine-synthesizing enzyme (phenylethanolamine ment of hypophysectomized rats with dexamethasone restored N-methyltransferase) is dependent on glucocorticoids (19). chromogranin A mRNA to basal levels. Chromogranin B Studies on the regulation of preproenkephalin mRNA mRNA levels were not changed by either insulin treatment or (mRNAenk) have given apparently conflicting results. For hypophysectomy. These results demonstrate (i) that different bovine chromaffin cells, an increase in mRNA"nJ was re- classes of secretory proteins present in chromaffin granules ported to follow treatment with depolarizing agents (20-23), are regulated by different mechanisms, (ii-) that this regulation whereas two groups (24, 25) reported increased levels of occurs at a pretranslational site, and (iii) that the relative mRNAe"k in rat adrenal medulla after splanchnic denerva- concentration of secretory constituents of chromaffin granules tion. Kanamatsu et al. (26), however, found that insulin- may vary. The significance of an altered composition of induced hypoglycemia, leading to an increased firing rate of secretory-granule constituents, which may be important in the splanchnic nerve, increased mRNAenk 15-fold. A recent hypotension or stress, is discussed. paper (27) from one of our laboratories (R.F.-C.) showed that nerve stimulation did increase enkephalin immunoreac- tiyity, whereas maintenance of chromogranin A immuno- Secretory vesicles of adrenal medulla store and release a reactivity was dependent on an intact pituitary-adrenal axis. complex mixture of proteins and neuropeptides in addition Here we present evidence for separate regulation of neuro- to the classical hormones epinephrine and norepinephrine peptides (enkephalin and neuropeptide Y) and chromogra- (1-4). These proteins comprise three groups of acidic pro- nins A and B by neural and humoral factors and demonstrate teins of unknown -function [chromogranins A and B and that this regulation occurs at a pretranslational site. secretogranin II (chromogranin C)] and two enzymes [dopamine /3-hydroxylase (5) and carboxypeptidase H (6, 7)]. In MATERIALS AND METHODS addition, several neuropeptides [enkephalin-containing pep- Treatment of Animals. Male Sprague-Dawley rats (200 g) tides (8, 9), neuropeptide Y (10-13), and calcitonin gene- were fasted overnight. Hypoglycemia was induced by an i.p. related peptide (14)] have been found in higher concentra- injection of porcine insulin (8.5 units/kg of body weight; tion. Upon stimulation of the splanchnic nerve these com- Ilentin 1I, Eli Lilly). After 2 hr, insulin shock was terminated ponents are released by exocytosis and transported to their by one or two injections (s.c.) of 1 ml of 20%o (wt/vol) ultimate target tissues by the general circulation. Physiologic glucose. The effectiveness of this procedure was evidenced actions at those sites might require processing of released by recovery from insulin-induced coma within 30 min. protein precursors to smaller peptides of yet-unidentified Control animals received injections of phosphate-buffered neuroendocrine function. saline. Rats with adrenal glands denervated by bilateral The regulation of the biosynthesis of these secretory transection of the splanchnic nerve were obtained from proteins is still not fully elucidated. Is biosynthesis of all Zivic-Miller (Zelienople, PA). Insulin shock for this group of secretory proteins concomitantly increased after depolariza- animals was performed 8 days after surgery. All rats were tion-induced secretion, so that depleted secretory vesicles sacrificed 24, 48, or 72 hr after insulin-induced hypoglycem- are equally refilled with neuropeptides after exocytosis, or is ia. Hypophysectomy was performed by Zivic-Miller. The biosynthesis of each secretory component regulated individ- completeness ofoperation was confirmed by the interruption ually, resulting in varying concentrations of secretory pro- of animal growth and physical inspection of the sella turcica. teins in refilled vesicles depending on secretory activity or The weight of rats that underwent sham operation was 298 + humoral state? Different relative amounts ofepinephrine and 4 g (n = 43), compared to 170 + 6 g (n = 42) for rats that neuropeptides secreted from chromaffin granules could be a underwent hypophysectomy. On day 6 after surgery, a group The publication costs of this article were defrayed in part by page charge Abbreviations: mRNAenk, preproenkephalin mRNA; mRNAChA, payment. This article must therefore be hereby marked "advertisement" chromogranin A mRNA; mRNAcha, chromogranin B mRNA. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. Downloaded by guest on September 27, 2021 3240 Neurobiology: Fischer-Colbrie et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3241 of animals was injected (s.c.) with 1 mg of dexamethasone Nuclear) in 25 mM phosphate buffer (pH 6.5) for 1 hr at 12 V, (suspended in 1 ml of phosphate-buffered saline) daily for 6 followed by 2 hr at 36 V. Nylon membranes were baked at consecutive days (19). In another group, hypoglycemia was 80'C under reduced pressure for 90 min and incubated for at induced on day 11 after surgery. The amount of insulin least 3 hr at 420C in 10 ml of prehybridization solution [50% injected was reduced 40% (to 5 units/kg) in these animals to formamide/50 mM sodium phosphate, pH 6.5/0.1% improve survival after insulin stress. Hypophysectomized NaDodSO4/0.1% Ficoll/0.1% polyvinylpyrrolidone/0.1% animals were decapitated 12 days after surgery. bovine serum albumin/5 x SSC (750 mM NaCl/75 mM Extraction of Total Nucleic Acids. Immediately after the rats sodium citrate) containing denatured salmon sperm DNA were decapitated, the adrenal glands were removed and (250 ,kg/ml) and yeast tRNA (250 gg/ml)]. For hybridiza- decapsulated. The adrenal medulla was dissected out with a tion, the membranes were incubated at 420C overnight in scalpel. The two adrenal medullae from each rat were homog- prehybridization solution containing 32P-labeled probe (106 enized in 450 gl of 1% NaDodSO4/10 mM Tris Cl, pH 7.6/5 cpm/ml) and then were washed three times with 100 ml of mM EDTA containing proteinase K at 10 ptg/ml, and the 0.2 x SSC/0.1% NaDodSO4 at 42TC. These hybridization homogenates were incubated for 90 min at 420C (28). After and wash conditions were chosen to eliminate hybridization incubation, the homogenates were extracted first with 1 to mRNA sequences with <80% sequence identity to the volume of phenol and then with 1 volume of phenol/ 39-mer oligonucleotide probe (36). Following exposure of chloroform (1:1, vol/vol). Total nucleic acids were precip- the membranes to Kodak XAR-2 film at - 70'C in the itated from the aqueous phase at - 200C overnight with 2.5 presence of intensifying screens (Lightning Plus, DuPont), volumes of ethanol after addition of 0.1 volume of 4.5 M mRNA was quantitated by densitometric scanning of auto- NaOAc (pH 6.5). Nucleic acids were recovered by centrifu- radiograms of each blot with a Beckman DU-8 spectropho- gation for 60 min at 12,000 x gm., dried under reduced tometer equipped with a slab-gel-scanning unit. It was as- pressure, and resuspended in 75 pul of 2.2 M formalde- sured that signals were in a linear range by scanning serial hyde/50o formamide 20 mM Mops/5 mM NaOAc/1 mM dilutions of samples. EDTA, pH 7.0. Samples were denatured at 650C for 5 min and stored at - 20°C until use. RNA from other rat tissues and cell RESULTS lines was obtained as described (29). Methods for extraction of total nucleic acids were compared Preparation of cDNA and Oligodeoxyribonucleotide Probes to find a method well suited for rat adrenal medulla. A method for Hybridization. Plasmid pCHRG12B, containing a DNA originally described (28, 37) for RNA extraction of isolated insert complementary to rat chromogranin A mRNA cells was found to yield high recovery with no detectable (mRNAChA) (29), was digested with Pst I restriction endo- degradation of RNA. Two adrenal medullae [1.4 ± 0.2 mg, n nuclease. A 995-base-pair Pst I fragment within the trans- = 6) dissected from a pair of adrenal glands (28 ± 2 mg, n = lated region of mRNAChA was purified by agarose gel elec- 6) from one rat yielded =15 ,ug of total nucleic acids.

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