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 [dopa- mine /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. mRNA trophoresis and nick-translated with [a-32P]dCTP to a spe- abundance for chromogranins A and B, preproenkephalin, cific radioactivity of 2-3 x 108 cpm/,ug of DNA. A and neuropeptide Y was determined by hybridization of 935-base-pair DNA fragment containing the full coding re- specific probes to S jig of size-fractionated total RNA. These gion of rat mRNAe" was obtained by endonuclease diges- probes hybridized, in accordance with previous determina- tion of plasmid pYSEC-1 (30) with Sac I and Sma I. The tions, with single mRNA species of different size (Fig. 1)- fragment was isolated by gel electrophoresis and labeled to a i.e., 2600 bases for chromogranin B (32), 2100 bases for specific activity of 3-4 x 10' cpm/,ug of DNA by nick- chromogranin A (29), 1450 bases for preproenkephalin (30), translation (31). A 39-residue oligonucleotide based on and 820 bases for neuropeptide Y (35). amino acids 2-14 of rat chromogranin B and the nucleotide Effects of Insulin. Twenty-four hours after a 2-hr period of sequence of human chromogranin B (32) was constructed. insulin stress, levels of mRNAs for preproenkephalin and Its sequence is AGT CAC CAT TTC TTC ATT GTG GTC neuropeptide Y were greatly elevated, whereas chromogra- CCT GlT ATC CAC TGG. This probe allowed detection of nin A and B mRNAs were unchanged (Fig. 1). Values a 2600-base mRNA species in rat adrenal medulla, cerebral obtained by scanning the autoradiograms of the blots, ex- cortex, and pituitary, as well as in rat pheochromocytoma pressed as percent of control + SEM, were 79 ± 4% (n = (PC12), rat insulinoma (RINm), and mouse pituitary ade- 7) for chromogranin A, 79 ± 8% (n = 12) for chromogranin noma (AtT20) cell lines, consistent with the reported distri- B, 627 ± 49% (n = 12) for preproenkephalin and 658 ± 75% bution of chromogranin B protein in endocrine tissues (2, (n = 4) for neuropeptide Y (Fig. 2). Values obtained 48 hr 33). No mRNA was detected with this probe in rat kidney, after insulin-induced hypoglycemia were 99 ± 15% (n = 7) liver, or adrenal cortex. A 48-mer corresponding to bases for chromogranin A and 85 ± 8% (n = 8) for chromogranin 171-218 (amino acids 1-16) of human neuropeptide Y (34), B, and those 96 hr after insulin stress were 97 ± 6% (n = 11) 94% identical with the rat nucleotide sequence (35), was also for chromogranin A and 102 ± 8% (n = 11) for chromogra- prepared. Both oligonucleotides were synthesized on an ABI nin B. The possibility that the insulin-induced increase in 380A automated DNA synthesizer (Applied Biosystems, levels of neuropeptide mRNAs was due to a direct effect of Foster City, CA) according to the manufacturer's instruc- insulin on chromaffin cells was tested by examining mRNA tions and purified by electrophoresis in a preparative poly- levels of mRNAenk and mRNAChA in denervated rats (Table acrylamide gel containing 8 M urea. Oligonucleotide probes 1). Eight days after bilateral splanchnic transection, were labeled with [y-32P]ATP by use of bacteriophage T4 mRNAeIk and mRNAChA levels were not significantly differ- DNA kinase (31). ent from those of control animals that underwent sham Blot Hybridization and Quantitation of mRNA. Total nu- operation. Bilateral splanchnicectomy completely prevented cleic acids were size-fractionated in 1% agarose gels con- the insulin-induced increase in mRNAenklevels (125% vs. taining 2.2 M formaldehyde in electrophoresis buffer (20 mM 634%). These results clearly argue against direct effects of Mops/5 mM NaOAc/1 mM EDTA, pH 7.0; ref. 28). After insulin on chromaffin cells and indicate that neuropeptide electrophoresis for 10 min at 40 V followed by 3 hr at 100 V, biosynthesis is regulated by a neurogenic mechanism. gels were stained for 15 min in ethidium bromide and Effects of Hypophysectomy. Total nucleic acids were ex- destained in electrophoresis buffer overnight in the dark. tracted from adrenal medullae 12 days after hypophysecto- Ethidium bromide staining was used as a measurement of my and hybridized with specific probes. Two additional total nucleic acids applied. RNA was electrophoretically groups of rats were either treated with dexamethasone for 6 transferred to a GeneScreen nylon membrane (New England days starting on day 6 after hypophysectomy (19) or injected Downloaded by guest on September 27, 2021 3242 Neurobiology: Fischer-Colbrie et al. Proc. Natl. Acad. Sci. USA 85 (1988)

I c I c I c I c 700 - F -. 28S- 600 - 238 aig. 18S we4 500 - 16S- 0 a 400-

tI- Ch B Ch A Enk NPY Oe 300 - FIG. 1. Blot hybridization analysis of mRNA abundance in adrenal medulla of insulin-treated rats. Total nucleic acids were extracted from adrenal medullae 24 hr after treatment of rats with 200 - insulin (I) or with saline (controls, C). mRNA was size-fractionated in agarose gels and hybridized with 32P-labeled probes specific for chromogranin A (Ch A), chromogranin B (Ch B), preproenkephalin (Enk), and preproneuropeptide Y (NPY). 100 - with insulin on day 11. RNA blot analysis of these experi- Of O I... ments is shown in Fig. 3. Quantitative results obtained by 0- zoTto < < m In-m >- _l scanning of autoradiograms are presented in Fig. 4. In .C c X CL r C was V0L0 Z 0 0 w hypophysectomized rats, mRNAcl" abundance signifi- insulin cantly reduced (32 + 2.3%, n = 19) compared to sham- control operated controls. Insulin-induced hypoglycemia decreased FIG. 2. Insulin increases levels of neuropeptide mRNAs. Rats mRNA levels slightly further (23 ± 4.91%, n = 9). Dexa- were killed 24 hr after stimulation of splanchnic nerve by insulin methasone treatment restored mRNAChA levels to 86 + treatment. mRNA levels of chromogranin A (Ch A), chromogranin 1.2% (n = 14). For chromogranin B, no difference in B (Ch B), preproenkephalin (Enk), and proneuropeptide Y (NPY) mRNAchn levels of sham-operated versus hypophysecto- were determined by blot hybridization (see Fig. 1) with specific = probes and scanning of autoradiograms. Values are plotted as mized rats (95 ± 9.8%, n 19) was seen. Dexamethasone percent of control + SEM. Stars indicate statistical significance (P treatment slightly reduced mRNA levels in controls (74 + < 0.001 by Student's t test). 11.6%, n = 7) and hypophysectomized rats (79 ± 8.5%, n = 13), but analysis by Student's t test revealed no statistically eral studies on biosynthesis of neuropeptides (enkephalins) significant difference. have yielded similar results (20, 41-43) and the term "stimu- For neuropeptide Y and enkephalin, the small amounts of lus-secretion-synthesis coupling" has been proposed (20, mRNA present in sham-operated and hypophysectomized 44). Depolarization by nicotinic agents increases mRNAenk rats did not permit an accurate measurement. However, and [Met5]enkephalin immunoreactivity as well as vasoac- after insulin treatment, no difference in mRNA levels be- tive intestinal polypeptide (VIP) immunoreactivity in iso- tween sham-operated and hypophysectomized rats was lated bovine chromaffin cells (20-22, 28, 45). For rat adrenal found (Fig. 3, lanes k-n). For comparison, RNA blots were medulla, reports are contradictory. LaGamma et al. (24, 46) hybridized first with the chromogranin A probe and then and Kilpatrick et al. (25) found increased levels of enkeph- with the enkephalin or neuropeptide Y probe. These blots alin immunoreactivity and mRNAenk in rats with denervated showed a marked decrease in mRNAC"A of hypophysecto- splanchnic nerve and proposed a tonic inhibition of enkeph- mized rats (Fig. 3, lanes I and n) relative to controls, in alin biosynthesis via neural input. In contrast, Kanamatsu et contrast to neuropeptide (preproenkephalin and preproneu- al. (26), Sietzen et al. (27), and Unsworth et al. (39) found ropeptide Y) mRNAs. Table 1. mRNAcI" and mRNAenk levels of denervated adrenal DISCUSSION glands after insulin treatment Signal, % control (mean ± SEM) In this study we examined the influence of neurogenic and humoral factors on the levels of mRNA encoding the major Sham Denervated secretory proteins (chromogranins A and B) and neuropep- mRNA - Insulin + Insulin - Insulin + Insulin tides (enkephalin and neuropeptide Y) of rat adrenal me- mRNAChA 100 18 80 9 85 10 94 15 dulla. To increase the firing rate of splanchnic nerve, hy- (n =5) (n =3) (n =3) (n =4) poglycemia was induced by injection of insulin (38, 39). To mRNAenk 100 ± 19 634 ± 48* 89 ± 15 125 ± 13 test for an effect ofcortical hormones, rats were subjected to (n =5) (n =5) (n =3) (n =6) hypophysectomy with or without subsequent injection of dexamethasone. Adrenal glands of rats were denervated by bilateral transection of Stimulation of nicotinic splanchnic nerve or underwent sham operation. Eight days after Regulation by Neural Activity. splanchnicectomy and 24 hr after insulin-induced hypoglycemia, receptors after release of acetylcholine from presynaptic adrenal medullary levels of mRNAcl and mRNAenk were deter- terminals leads to secretion of hormones and proteins from mined by blot hybridization with specific probes. Hybridization adrenal medulla. As a further consequence, repletion of signals were quantitated by densitometric scanning of autoradio- hormones (epinephrine) occurs due to increased activity of grams. Values are expressed as percent of control (sham operation, enzymes involved in biosynthesis. This concept has been no insulin). named "trans-synaptic induction" of biosynthesis (40). Sev- *P < 0.001 vs. control without insulin, by Student's t test. Downloaded by guest on September 27, 2021 Neurobiology: Fischer-Colbrie et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3243

b c d e f g h j k m n. enkephalin immunoreactivity in rat adrenal medulla follow- ing denervation seems more likely to be an expression of increased activation of postsynaptic receptors due to re- leased acetylcholine than to be an unmasking of a tonic inhibition of an intact nerve (25). For neuropeptide Y, data 28S- obtained with bovine chromaffin cells suggest that this 23S peptide might not be regulated via increased cAMP levels 18S 0 40 00 " 40 a (48). 16S- The present study confirms the results of Kanamatsu et al. (26) for proenkephalin and provides additional data for neuropeptide Y and chromogranins A and B. After insulin treatment mRNA levels of enkephalin and neuropeptide Y were markedly increased. Bilateral adrenal denervation SD H Hi HD S SD H HI HD Si Hi Si Hi completely prevented this increase, excluding direct, non- FIG. 3. Blot hybridization analysis of adrenal medullary mRNA neurogenic effects of insulin or decreased blood glucose of hypophysectomized rats. Total nucleic acids of sham-operated levels on regulation of biosynthesis as proposed for insulin- (S) and hypophysectomized (H) rats treated with insulin (I) or induced catecholamine release (49). Thus it seems justified dexamethasone (D) were extracted and hybridized with probes to conclude that increased intensity of impulse activity of the specific for chromogranin A (lanes a-e), chromogranin B (lanes f-j), splanchnic nerve increases biosynthesis of proenkephalin preproenkephalin (lanes k and 1), and proneuropeptide Y (lanes m and neuropeptide Y in rat adrenal medulla. In contrast to and n). Blots were autoradiographed at - 70'C either overnight (for neuropeptides, however, mRNA levels of chromogranins A chromogranins A and B) or for 6 days (enkephalin and neuropeptide and B were unchanged or even slightly reduced. Further- Y). Lanes k-n were hybridized first with the chromogranin A probe more, treatment of hypophysectomized rats with insulin did and then with the preproenkephalin and proneuropeptide Y probes. not elevate reduced levels of mRNAChA. Thus the concept of In lanes m and n some nonspecific hybridization to the 28S ribo- a trans-synaptic induction as suggested for tyrosine hydrox- somal RNA occurred. ylase, dopamine f3-hydroxylase, and enkephalins does not apply for the regulation of chromogranins A and B. elevated levels of [Met5]enkephalin immunoreactivity and Regulation of Biosynthesis by Humoral Factors. A second mRNAenk after stimulation of splanchnic nerve with insulin. possible factor involved in biosynthesis of adrenomedullary As a possible explanation for this discrepancy, it was sug- secretory proteins is hormonal regulation. To test for the gested (26, 39, 47) that endogenous acetylcholine released influence of the hormones of the pituitary-adrenal axis, we from degenerating nerve terminals might stimulate postsy- probed adrenal medullae of hypophysectomized rats for naptic nicotinic receptors. Thus, increases in mRNAenk and chromogranin A and B mRNA. In hypophysectomized rats, mRNAChA levels were reduced to 32% of control. Treatment with dexamethasone restored mRNAChA levels to 86% of 120 - control, demonstrating involvement of glucocorticoids in regulation of chromogranin A biosynthesis. A similar depen-

,-I- dence of mRNAchA levels on glucocorticoids was also found rL recently for PC12 pheochromocytoma cells (D. Rausch, 80 - A.I., and L.E.E., unpublished data). 80 In contrast to mRNAChA, hypophysectomy did not alter O 60- mRNAChB levels. These results are in agreement with data recently obtained on the protein level for both chromogranin .\O *~ 4040 - A and B (27). Thus, there seems to be a marked difference in regulation of chromogranin A and B biosynthesis. For chro- 20 mogranin B, neither treatment with insulin nor hypophysec- tomy changed mRNA abundance. For enkephalins and

0 Sham yp Hyp X HypX neuropeptide Y, data on the protein level demonstrated that Dex Ins Dex hypophysectomy does not change enkephalin immunoreac- tivity in rat adrenal medulla (27, 50). In addition, neuropep- I IGC) 7 T AcCh tide Y levels in rat PC12 cells are not dependent on dexa- methasone (51). In the present study, no difference in - T 20 enkephalin and neuropeptide Y mRNA levels was seen in ... hypophysectomized versus sham-operated rats treated with - -;z ...... insulin, supporting the data obtained for proteins. Zen 6 :: Regulation of the Biogenesis of Chromaffin Granules. The Za .I influence of insulin treatment and hypophysectomy on mRNA levels of four individual proteins has been discussed above in detail. But what do these changes in mRNA levels mean for the whole secretory vesicle, the chromaffin gran- Sham Hyp X Hyp X Hyp X ule? Is increased biosynthesis of secretory components after Dex Ins Dex neurogenic stimulation due to an increased number of secre- tory vesicles, or is the composition of secretory constituents FIG. 4. Hypophysectomy decreases level of mRNAChA. Hy- within a single chromaffin granule changed? Our data indi- pophysectomized (Hyp X) or sham-operated rats were decapitated cate separate regulation of individual secretory proteins, 12 days after surgery. Two additional groups were treated with since after insulin treatment mRNA levels of neuropep- insulin (Ins) or dexamethasone (Dex). Adrenal medullary mRNA only levels of chromogranin A (Ch A, Upper) and chromogranin B (Ch B, tides were increased, whereas those of chromogranins were Lower) were obtained by scanning of autoradiograms (see Fig. 3). unchanged. Since chromogranin A and B are major constit- Values are plotted as percent of control + SEM. Stars indicate uents of chromaffin granules, it seems unlikely that the statistical significance4.I (P < 0.001 by Student's t test). number of vesicles was increased under these conditions. Downloaded by guest on September 27, 2021 3244 Neurobiology: Fischer-Colbrie et al. Proc. NatL Acad. Sci. USA 85 (1988) Therefore, our data argue for a change in the composition of Affolter, H.-U. (1984) Nature (London) 312, 461-463. vesicles, since a colocalization of the various secretory 21. Waschek, J. A., Pruss, R. M., Siegel, R. E., Eiden, L. E., components seems likely. Recent immunological data at the Bader, M.-F. & Aunis, D. (1987) Ann. N.Y. Acad. Sci. 493, 308-323. ultrastructural level demonstrated that at least chromogra- 22. Kley, N, Loeffler, J.-P., Pittius, C. W. & Hollt, V. (1987) J. nins A and B are present in practically all secretory vesicles Biol. Chem. 262, 4083-4089. of a chromaffin cell and in fact are colocalized in the same 23. Siegel, R. E., Eiden, L. E. & Affolter, H.-U. (1985) Neuro- vesicles (W. Weitz, R.F.-C., and H. Winkler, unpublished peptides 6, 543-552. work). On the other hand, at the light-microscopic level a 24. LaGamma, E. F., White, J. D., Adler, J. E., Krause, J. E., colocalization of neuropeptide Y and enkephalin has been McKelvy, J. F. & Black, I. B. (1985) Proc. Natl. Acad. Sci. USA 82, 8252-8255. shown for epinephrine-containing chromaffin cells (13). 25. Kilpatrick, D. L., Howells, R. D., Fleminger, G. & Uden- Hypophysectomy decreased mRNAChA significantly, friend, S. (1984) Proc. Natl. Acad. Sci. USA 81, 7221-7223. whereas mRNAChB was not influenced. Coupland et al. (52) 26. Kanamatsu, T., Unsworth, C. D., Diliberto, E. J., Viveros, reported that the total number of chromaffin granules pres- 0. H. & Hong, J. S. (1986) Proc. NatI. Acad. Sci. USA 83, ent per single cell was unchanged after hypophysectomy; 9245-9249. therefore, according to our data these vesicles should con- 27. Sietzen, M., Schober, M., Fischer-Colbrie, R., Scherman, D., Sperk, G. & Winkler, H. (1987) Neuroscience 22, 131-139. tain less chromogranin A. Thus it seems safe to conclude 28. Eiden, L. E. & Hotchkiss, A. J. (1983) Neuropeptides 4, 1-9. that changes in mRNA levels after neurogenic stimulation of 29. lacangelo, A., Okayama, H. & Eiden, L. E. (1988) FEBS Lett. adrenal medulla or hypophysectomy yield chromaffin gran- 227, 115-121. ules with different concentrations of secretory proteins (see 30. Yoshikawa, K., Williams, C. & Sabol, S. L. (1984) J. Biol. ref. 53 for further discussion). Chem. 259, 14301-14308. A "secretory cocktail" blended differentially in response 31. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., to different patterns of environmental stimulation may be a Cold Spring Harbor, NY). mechanism for adaptation to situations of emotional stress, 32. Benedum, U. M., Lamouroux, A., Konecki, D. 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